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Identification and assessment of alternatives to selected phthalates 4 Human health and environmental assessment of alternative plasticisers
Background data for the environmental and human health assessment of the selected alternative plasticisers are presented in Annex 3, which also includes the references to data sources. 4.1 ASE (alkylsulphonic phenylester)4.1.1 Physico-chemical propertiesASE, a mixture of similar esters of sulfonic acids, phenyl and C10 – C18 alkanes, is a liquid at ambient temperatures. It has low solubility in water (2 mg/L) and low volatility (Vp = 0.01 Pa). It is lipophilic with a log KOW >6. 4.1.2 Human health assessmentResults from pharmacokinetic studies show that a single oral application by gavage of 1000 mg sulfonic acid, C10-21-alkane, Ph esters/kg bw leads to a concentration of 65 μg sulfonic acid, C10-21-alkane, Ph esters/g fat tissue. After 34 days 4μg sulfonic acid, C10-21-alkane, Ph esters/g was still found in the fat tissue. An elimination half-life of 8 days was calculated for the fat tissue. No accumulation was observed in the liver. 20-30% of the dose was excreted in the faeces within 24 hours. When 100 mg of the substance was administered by gavage, the concentration in fat tissue was 22 μg sulfonic acid, C10-21-alkane, Ph esters/g fat tissue after 3 days and 1 μg/g fat tissue after 14 days. No accumulation was observed in the liver. ASE has low acute toxicity by the oral route with LD50 reported in the range of 26,380 - 31,650 mg/kg bw in the rat. LD50 by the dermal route was found to be > 1,055 mg/kg bw in a rat in a study with no indication of toxicity. ASE was not irritating to rabbit skin when applied to the ears for 24 hours and also not to humans exposed to a saturated patch for 8 hours and followed by a 7 days observation period. Rabbit eyes did not show signs of irritation when exposed to ASE and observed for 7 days. Subchronic toxicity is studied in a repeated dose 90-day oral toxicity study in the rat. Rats were dosed at 750, 3.000 and 12.000 ppm and a NOAEL was reported at 3,000 ppm corresponding to 228 mg/kg bw in males and 282.6 mg/kg bw in females in spite of significantly dose related absolute and relative liver weights at all dose levels. Effects at the highest dose level included reduced body weight gain, increased feed (females), increased water consumption (males) and increased kidney weight. No accumulation in the liver was observed in repeated dose toxicity studies of shorter duration (28 to 49 days). Elimination half-life for fat tissue was calculated at 15 days. ASE was negative in Ames test, In vitro Mammalian Cytogenetic Test (OECD 473) and in a HGPRT gene mutation assay, all with and without metabolic activation. No effects on fertility were seen in what appears to be a three generation reproductive toxicity (fertility) study in rats dosed at 530 mg/kg bw for 6 weeks and observed for 3 months. For the F0-generation, no effects on fertility are reported. The F1-generation is reported to have normal weight gain, normal weight of endocrine organs and normal first oestrus. For the F2 and F3-generation, no effects on fertility and body weight gain are reported. The study dates back to 1956 and details are not available. It is also not clear if a control group has been included in the study. However, due to the limited information about the study and the high degree of uncertainty, it is not possible to draw any conclusions regarding reproductive toxicity from these data. No other health data was found. In summary the following profile was identified:
4.1.3 Environmental assessmentAerobic biodegradation of ASE is found to be 31% in 28 days. Thus, ASE is not readily biodegradable and its log KOW (>6) is indicative of significant potential for bioaccumulation. Data on effects of ASE on aquatic organisms are few, however, the data in IUCLID indicate low toxicity to fish in OECD acute test with zebrafish (LC0 ≥100 mg/L and LC50 >10,000 mg/L) and similarly a very low toxicity to crustaceans (D. magna); EC >1,000 mg/L and >10,000 mg/L in the two reported tests (OECD acute method). A test with algae (S. suspicatus) gave the same result. It is noted that these test and effect concentrations are far above the reported water solubility of ASE (2 mg/L). No inhibition of the bacteria Photobacterium phosphoreum was observed at 500 mg/L in one test while in another <20% inhibition occurred at 1.2 g/L. The EC50 for inhibition of activated sludge was >10,000 mg/L (all reported in IUCLID). The main constituents of sulphonic acids, C10-21-alkane, Ph esters are not considered as PBT. They do not meet the P/vP criteria based on screening data but they meet the screening B criteria. Assessment of ecotoxicity (T) was not carried out during this assessment by the PBT Working Group, PBT List No. 82. No other environmental effect data have been found.
ND = No Data 4.2 ATBC (acetyl, tri-N-butyl citrate)4.2.1 Physico-chemical propertiesATBC (CAS No. 77-90-7) consists of citrate with three ester bonded butyl groups and one acetyl group bonded to the fourth available oxygen atom. ATBC is a liquid at ambient temperatures and it has a moderate vapour pressure (6.9 Pa at 20 °C). It is sparingly soluble in water (5 mg/L / < 100 mg/L) and quite lipophilic (log KOW = 4.29). 4.2.2 Human health assessmentATBC is easily absorbed and rapidly metabolised and excreted in the rat. In an absorption study radiolabelled material was recovered at 59 - 70% in urine and cage rinse, 25 - 36% in faeces, 2% expired and 0.36 - 1.26 in tissues and carcass. At least 9 radiolabelled metabolites were found in urine and at least 3 in faeces. ATBC has low acute toxicity by the oral route in rats with LD50 reported to exceed 30 g/kg bw. No data on acute toxicity by other routes have been found. ATCB was not irritating to rabbit skin (OECD 404). A study from 1978 in guinea pigs did also not produce irritation, but in an older study from 1955, ATCB produced slight oedema. Patch tests in humans did not produce irritation. ATBC was not irritating to rabbit eyes in a test according to OECD 405. Older studies show slight to moderate irritation. ATCB was not sensitising in guinea pigs or in human volunteers exposed to the substance. Subchronic toxicity has been studied in several repeated dose toxicity studies of varying quality. In a 90 days study (according to OECD 408) in rats exposed to doses of 100, 300 and 1,000 mg/kg/day, NOAEL was reported at 300 mg/kg bw. All rats survived to scheduled necropsy and no treatment related clinical signs were noted throughout the study. Mean body weights were slightly reduced in both sexes in the high dose group and females in the 300 mg/kg dose group beginning at day 28. These findings were however not statistically significant. Increased liver relative weights for both sexes in the 1,000 mg/kg bw dose group and in the males in the 300 mg/kg bw dose group were not associated with any evidence of hepatotoxicity as evaluated by histopathological examination or clinical chemistry. The only other organ weight change was a slightly increased relative kidney weight for males in the high dose group. NOAEL was based on a few statistically significant differences between the control group and animals administered 1,000 mg/kg bw. Chronic toxicity was studied in a two-year oral feeding study in rats administered 200, 2000 or 20000 ppm in the diet. Transient reduction in body weight gain was observed in all dose groups from week 5 to 15. Because of this unexplained depression in growth rate, two additional groups of 10 rats received ATCB in concentrations of 200 and 2,000 ppm in the diet for one year. As the findings could not be reproduced it was considered to be an artifact. Most other findings were not statistically significant or not considered treatment related. NOAEL was concluded to be 2,000 ppm (100 mg/kg/day) using a conservative approach as the study lacks in detail and is without GLP. In a 13-week toxicity study with an In Utero Exposure phase, sensitive reproductive and developmental endpoints were examined. Wistar rats received ATBC in the diet in concentration levels of 100, 300 and 1,000 mg/kg/day. F0 males and females were treated for four weeks prior to mating. F1 male and female offspring were exposed in utero and from birth until start of the 13 week study. F1 offspring selected for the study were then treated for 13 weeks. Based on the results a NOAEL for males was established at 100 mg/kg/day and the NOAEL for females at 300 mg/kg/day. At the highest dose level a slight reduction in body weight gain was seen in both sexes, liver weights were increased and hepatic hypertrophy (common finding at high doses of xenobiotics) was seen in males and females. Weak peroxisome proliferase was measured in males at 300 mg/kg/day and in both sexes at 1,000 mg/kg/day. Slight, reversible variations in urinary composition and plasma electrolyte concentration were considered to be due to adaptation to excretion of high levels of test material and and/or metabolites and were not considered toxicologically significant. ATBC showed no evidence of mutagenic activity in several Bacterial Reverse Mutation tests (Ames) with and without metabolic activation and also not in Mammalian Cell Gene mutation assays with and without activation. In vitro cytotoxicity was observed in mouse lymphoma cells and less pronounced in HeLa cells. ATCB was not genotoxic in in vivo/in vitrounscheduled DNA synthesis study. ATBC was administered to rats with the diet in a 2-generation reproductive toxicity study in the following doses: 100, 300 and 1,000 mg/kg/day. NOAEL for both parental animals and offspring was found to be 100 mg/kg/day. No treatment-related clinical observations were noted throughout the study in either F0 or F1 parental animals. Body weights the F1 parental males in the 300 and 1,000 mg/kg/day groups were lower that controls and appeared to be related to treatment. Body weights of the F0 females in the 1,000 mg/kg/day group at the end of pregnancy (gestation days 21 or 22) was significantly lower than control values. No reproductive effects were observed in the F0 and F1 generation and no treatment related abnormalities. Slightly lower body weight and slightly higher mortality was observed among pups in the 300 and 1,000 dose groups, which could be a consequence of reduced water consumption. No significant treatment related effects on development and embryotoxic effects were observed in a 12 month study in rats. ATBC showed some signs of neurotoxicity when applied in a 3% acacia to the sciatic nerve in rats and in a 5% suspension of ATBC in 3% gum acacia to the conjunctival sac of the eye of a rabbit. The substance was found to have local anaesthetic action in rabbits and to block neural transmission in rats when placed in contact with a nerve trunk. In summary the following profile was identified:
R: Reproductive toxicity; D: Developmental toxicity 4.2.3 Environmental assessmentA number of studies have been conducted to determine the aerobic biodegradability of ATBC. In the modified MITI test with activated sludge inoculum, 80% of the theoretical BOD was reached in 4 weeks. In the static biometer EPA test (EPA 835.3300), ATBC was characterised as readily biodegradable based >60% ThCO2 observed within a 10-14 day window following the lag period. Also in the ASTM D 5338 test, ATBC was found to readily biodegradable as well as ultimately biodedegradable. A BCF = 250 and a KOC = 1,800 have been calculated for ATBC based on water solubility = 5 mg/L. These values indicate some bioaccumulation potential as well as strong sorption properties i.e. low mobility in soil. The acute toxicity to fish has been studied using a number of species. The most sensitive end point was found for Pimephales promelas larvae (18 hr) in a 7 day static-renewal test (USPEA Method 1000.0). The LC50 (48 h) was 2.8 mg/L and the LC50 (168 hr) was 1.9 mg/L. In an older (1974) non-guideline flow-through study, the 96 h LC50 for Lepomis macrochirus was estimated at 38-60 mg/L and for the mummichog, Fundalus heteroclitus, to 59 mg/L (both nominal). ECOSAR modelling using P. promelas as model species gave LC50 = 1.67 mg/L. The water flea Cerodaphnia dubia was used for testing acute toxicity to daphnia using the USEPA 850.1010 method. The 48 h EC50 was determined to be 7.82 mg/L. ECOSAR modelling using D. magna as the model species gave an LC50 = 0.704 mg/L (48 h). The toxicity to algae has not been tested for ATBC, only estimated by ECOSAR with the green alga Selenastrum capricornutum. The 96 hour EC50 was 0.148 mg/L by this method.
4.3 COMGHA4.3.1 Physico-chemical properties12-(Acetoxy)-stearic acid, 2,3-bis(acetoxy)propyl ester is the main constituent (ca. 84%) in a plasticiser consisting of two castor oil derivatives and commonly known as COMGHA (Soft-n-safe). The other main component (ca. 10%) is octadecanoic acid, 2,3-(bis(acetoxy)propyl ester. The CAS number of the mixture, which is a greasy substance, is 736150-63-3. It has a very low volatility (Vp = 0.00000011 Pa at 25 °C), low water solubility (<0.33 mg/L / 7 mg/L) and is highly lipophilic (log KOW = 6.4). 4.3.2 Human health assessmentToxicokinetic studies on COMGHA show that there is no significant absorption of the material across gastrointestinal epithelium. Based on the results from a 90-days oral toxicity study, it was concluded that there were no marked effects on peroxisomal enzyme activities in liver samples at concentration levels of 0.4%, 1.2% and 3.6% in the diet. Acute toxicity (OECD 402) of COMGHA by the dermal route has been studied in rat and LD50 found to be > 2,000 mg/kg bw. Other acute toxicity data are not available. COMGHA was not irritating to rabbit skin (OECD 404) and rabbit eyes (OECD 405) and also not a skin sensitizer when studied in a local lymph node assay in mice (OECD 429). No signs of toxicity were observed in a 28-day repeated dose oral toxicity study (OECD 407) in rats when administered at 3% and 7.5% of the diet/gavage. No effect on the palatability of the diet was observed during the study. In a 90-day oral toxicity study (OECD 408) with extreme doses: 3, 8.5, 20 ml/kg/day administered by gavage, NOAEL was found to be < 3 ml/kg/day. In a 90-day oral toxicity study (OECD 408) with adequate doses: NOAEL was found to be 5,000 mg/kg/day. COMGHA was not found mutagenic in Ames test (OECD 471) and no clastogenic activity was seen in the In vitro Mammalian Chromosome Aberration Test (OECD 473). COMGHA was also not mutagenic in In vitro Mammalian Cell Gene Mutation Test (OECD 476). More studies are planned to investigate genotoxic effects in vivo as well as chronic toxicity, teratogenicity and reproductive toxicity. In summary the following profile was identified:
4.3.3 Environmental assessmentCOMGHA was found to be readily biodegradable when tested by OECD method 301: 98% degradation occurred in 28 days. The log KOW of 6.4 indicates significant bioaccumulation potential and very low mobility in soil. Acute toxicity to zebrafish was tested using OECD 203 but the LC50 (96 h) could not be determined as it was higher than the solubility of COMGHA. A no observed effect concentration (LC10) after 96 h is stated to be 0.28 mg/L (presumably the highest concentration tested but no detailed information is given). The acute toxicity to daphnia was EC50 = 0.92 mg/L in the OECD 202 test but COMGHA is, according to the manufacturer, not considered to be acutely toxic at the solubility concentration (<0.33 mg/L). The 72 hour growth inhibition EC50 for algae was 106 mg/L, however a 70-95% loss in test concentration over the test period was observed. Regarding inhibition of activated sludge respiration (OECD 209), the EC20 (and EC50) was >143 mg/L. No further data on the ecotoxicity of COMGHA is available.
4.4 DEGD (diethylene glycol dibenzoate)4.4.1 Physico-chemical propertiesDEGD is the esterification product of two benzoate groups with diethylene glycol. It has CAS No. 120-55-8. DEGD becomes liquid at temperatures in the range 24-33 °C and has a very low volatility (Vp = 0,000017 Pa at 25 °C). It has a rather low water solubility of 38.3 mg/L and it is moderately lipophilic with a log KOW of 3.0-3.2. 4.4.2 Human health assessmentMetabolism of DEGD was studied in Sprague-Dawley CD rats after single oral doses of 50 mg/kg (low level) and 750 mg/kg (high level). Almost all of single oral doses of 50 and 750 mg/kg of DEGD administered to the rats were adsorbed, metabolized and excreted in the urine within 24 hours of administration. DEGD was metabolized via hydrolysis of the ester bonds to benzoic acid. The free acid was then conjugated with either glycine (major pathway) or glucuronic acid (minor pathway) prior to excretion. DEGD has low acute toxicity by the oral route in rats with LD50 reported at 4,198 mg/kg bw (OECD 401). Dermal LD50 in rats was found to be > 2,000 mg/kg bw (OECD 402). An acute inhalation toxicity study in the rat was conducted with DEGD resulting in an LC50 > 200 mg/L (4 h). No dermal reaction was reported following a single semi-occlusive application of DEGD to intact rabbit skin for 4 hours. A single instillation of DEGD into the eye of the rabbit elicited transient very slight conjunctival irritation only. No allergic skin reaction was reported in guinea pigs after repeated skin contact (intradermal and topical) using the Magnusson and Kligman method. Subchronic toxicity was studied in a repeated dose 13 week oral toxicity study in the rat (OECD 408). Animals received DEGD in the diet in concentration levels of 250, 1000, 1750 or 2500 mg/kg/day. A NOAEL of 1,000 mg/kg bw was established based on the results of the study. There were no findings of toxicological importance at a dosage of 1,000 mg/kg/day or below. In animals receiving 1,750 or 2,500 mg/kg/day, there was an adverse effect on bodyweight gain, changes in clinical pathology parameters and an increased incidence/degree of haemosiderosis in the spleen. In addition, at 2,500 mg/kg/day, a few treatment-related clinical signs were evident, minimal periportal hepatocyte hypertrophy was noted in both sexes. When selected animals previously receiving 2,500 mg/kg/day were maintained off-dose for 4-weeks, all treatment related changes showed evidence of recovery or recovered completely. No effects were reported in dogs administered up to 300 mg/kg/day of DEGD in their diet for 90 days. DEGD did not demonstrate mutagenic potential in bacterial (Ames test, OECD 471/2) or mammalian cell (mouse lymphoma cells, OECD 476) systems with and without metabolic activation. No reponse considered to be indicative of clastogenic activity was observed in a In-vitro Mammalian Chromosome Aberration Test in CHL cells (OECD 473). Prenatal developmental toxicity of DEGD (purity 97.67%) in rats was studied in a test according to US EPA 870.3700 Harmonized Guideline (corresponding to OECD 414). Animals were administered doses of 250, 500 and 1,000 mg/kg/day in the diet. NOEL for maternal toxicity was found to be 1,000 mg/kg/day. At 1,000 mg/kg/day, there were no detectable signs of maternal toxicity; there were no maternal deaths and all females had a live litter at sacrifice. NOAEL for prenatal development was found to be 500 mg/kg/day. A small number of foetuses with cervical ribs was seen at 1,000 mg/kg/day, but not considered indicative of substantial disturbance of morphological development. NOEL for foetal growth and development was 250 mg/kg/day. At 1,000 mg/kg/day mean foetal weights, and consequently litter weight were slightly lower than the control, combined with foetal weight and female foetal weight attaining statistical significance. At 1,000 mg/kg/day 4 foetuses showed cervical ribs, this incidence being higher than the concurrent control and marginally outside the current background control data. Although the incidence of this finding was relatively low, it is considered that a treatment relationship could not be ruled out. Reproductive toxicity of DEGD (purity 97.67%) in rats was studied in a 2-generation test according to OECD 416 and at dose levels of 1000, 3300 or 10000 ppm in the diet. corresponding to 50, 165 and 500 mg/kg bw/day based on standard conversion factors. There were no obvious toxicological effects of treatment for the two generations on the general condition of the parental animals although a slight disturbance in the pattern of maternal weight change was noted at 10,000 ppm in both generations and at 3,300 ppm in the Fl generation. There was no effect on fertility and reproductive performance at any of the dietary inclusion levels in either generation. Litter parameters at birth of the Fl and F2progeny and their survival to weaning showed no apparent detrimental effects of treatment. However, for the F2 offspring at 10,000 ppm there was a reduction in weight gain from birth to weaning. No abnormal findings were apparent at necropsy of the FO or Fl parental animals, the post weaned unselected Floffspring or the F2 offspring. Organ weight assessment of the FOand Fl parent animals did not suggest any adverse effects on any organs. Assessment of spermatogenesis and histopathology in both parental generations showed that there were no injurious effects on the testes or other reproductive organs. Furthermore, detailed histopathological examination of the tissues from both sexes in both generations did not reveal any adverse effects of treatment. The only possible effect of treatment detected at assessment of organ weights from Fl and F2 offspring was lower absolute and bodyweight relative spleen weights among F2 males and females compared with controls. The evidence from this study suggested that a dietary concentration of 10,000 ppm (500 mg/kg bw/day) should be considered as the NOAEL for the FO and Flparent animals. The NOAEL for the developing offspring is considered to be 3,300 ppm (165 mg/kg bw/day). The NOEL for reproductive parameters is considered to be 10,000 ppm (500 mg/kg bw/day). Evaluation of estrogenic activity at doses of 500, 1000, 1500 or 2000 mg/kg/day for 7 days by oral gavage in ovariectomized adult Spraque-Dawley (CD) rats using vaginal cornification and the uterotrophic response as the endpoints demonstrated that DEGD did not exhibit estrogenic activity up to and including the maximally tolerated dose. In summary the following profile was identified:
4.4.3 Environmental assessmentDEGD is found to be readily biodegradable (93% of ThOD in 28 days) in the modified Sturm test (OECD 301B) while in the Closed Bottle Test (OECD 301D) the BOD5/COD ratio was only 0.32 (>0.5 required for ready biodegradability). A KOC of 540 indicates rather low mobility of DEGD in soil, and a moderately high calculated BCF of 120 indicates some bioaccumulation potential. The aquatic toxicity of DEGD is quite uniform in short term/acute OECD tests between the three main standard groups of test organisms; fish, crustaceans and algae. Thus, the acute (96 h) LC50 to fish (Pimephales promelas) is 3.9 mg/L, while the EC50 (48 h) for daphnia is 6.7 mg/L and the 72 hours growth rate-based EC50 for algae (Seleneastrum capricornutum, now known as Pseudokirchneriella subcapitata) is 11 mg/L. This could indicate a non-specific mode-of-action of DEGD. The acute toxicity to earthworm (Eisenia foetida) (14 days) was found to be >1,000 ppm while the inhibitory effect (IC50) on the bacterium Pseudomonas putida could not be determined specifically but only be stated as higher than the highest testable concentration of 10 mg/L. Activated sludge respiration was not inhibited at 100 mg/L.
4.5 DGD (dipropylene glycol dibenzoate)4.5.1 Physico-chemical propertiesDGD, CAS No. 27138-31-4, is the esterification product of two benzoate groups with dipropylene glycol. DGD is a liquid and is quite similar to DEGD except for two extra methyl groups. It is a substance with low volatility (Vp = 0.00016 Pa at 25 °C), a quite low water solubility of 8.9-15 mg/L and relatively lipophilic character with a log KOW of 3.9. 4.5.2 Human health assessmentStudies show that DGD is rapidly metabolised and excreted from the body and not accumulated in rats. 70% was excreted in the urine within 48 hours of administration as hippuric acid and about 10% was observed in faeces. Half-life of radiocarbon in the blood was 3 hours and for other organs 2-15 hours. DGD has low acute toxicity by the oral route in rats with LD50 reported at 3,914 mg/kg bw (OECD 401). Dermal LD50 in rats was found to be > 2,000 mg/kg bw (OECD 402). An acute inhalation toxicity study in the rat was conducted with DGD resulting in an LC50 > 200 mg/L (4 h). No dermal irritation was reported following a single semi-occlusive application of DGD to intact rabbit skin for 4 hours (OECD 404). A single instillation of DGD into the eye of the rabbit elicited transient very slight conjunctival irritation only (OECD 405). No allergic skin reaction was reported in guinea pigs after repeated skin contact (intradermal and topical) using the Magnusson and Kligman method (OECD 406). Subchronic toxicity was studied in a repeated dose 13 week oral toxicity study in the rat (OECD 408). Animals received DGD in the diet in concentration levels of 250, 1000, 1750 or 2500 mg/kg/day. A NOAEL of 1,000 mg/kg bw (or below) was established based on the results of the study. A few minor intergroup differences were noted at 1,000 mg/kg/day but were insufficient to be of toxicological importance. Higher dosages of 1,750 or 2,500 mg/kg/day were tolerated but the adverse effect on bodyweight was more pronounced, there were increases in circulating enzyme activities, low grade hepatocyte hypertrophy and an increased incidence and degree of hemosiderosis in the spleen in one or both sexes. At 2,500 mg/kg/day, an increased incidence of minimal epithelial hyperplasia was noted in the caecum. When selected animals previously receiving 2,500 mg/kg/day were maintained off dose for 4 weeks, all treatment related effects showed evidence of recovery or recovered completely. DGD did not demonstrate mutagenic potential in bacterial (Ames test, OECD 471/2) or mammalian cell (mouse lymphoma cells, OECD 476) systems with and without metabolic activation. No response considered to be indicative of clastogenic activity was observed in a In-vitro Mammalian Chromosome Aberration Test in CHL cells (OECD 473) with and without activation. Prenatal developmental toxicity of DGD (purity 94.84%) in rats was studied in a test according to US EPA 870.3700 (corresponding to OECD 414). Animals were administered doses of 250, 500 and 1,000 mg/kg/day in the diet. NOEL for maternal toxicity was found to be 1,000 mg/kg/day. At 1,000 mg/kg/day, there were no detectable signs of maternal toxicity; there were no maternal deaths and all females had a live litter at sacrifice. NOAEL for prenatal development was found to be 500 mg/kg/day. A small number of foetuses with cervical ribs was seen at 1,000 mg/kg/day. NOEL for foetal growth and development was 250 mg/kg/day. There were no effects of treatment on prenatal survival or growth. At 1,000 mg/kg/day, treatment was associated with a small but definite increase in the number of foetuses with cervical ribs. Reproductive toxicity of DGD (purity 94.84%) in rats was studied in a 2-generation test according to OECD 416 and at dose levels of 1000, 3300 or 10000 ppm in the diet corresponding to 50, 165 and 500 mg/kg bw/day based on standard conversion factors. There were no obvious toxicological effects of treatment for the two generations on the general condition of the parental animals or on their fertility and reproductive performance. Litter parameters at birth of the Fland F2 progeny and their survival to weaning showed no apparent detrimental effects of treatment. However, in both the Fland F2 offspring at 10,000 ppm there was a slight reduction in weight gain during days 14-21 of age and this finding may be linked to the transition to direct exposure to the test material as the offspring weaned onto solid diet at the same dietary inclusion levels as their parents. No abnormal findings were apparent at necropsy of the FO or Fl parental animals, the post weaned unselected Fl offspring or the F2 offspring. Organ weight assessment of the FO and Fl parent animals did not suggest any adverse effects on any organs. Assessment of spermatogenesis and histopathology in both parental generations showed that there were no injurious effects on these testes or other reproductive organs. Furthermore, detailed histopathological examination of the tissues from both sexes in both generations did not reveal any adverse effects of treatment. Regarding survival and growth of the offspring, there were no unequivocal adverse effects. However, a slight reduction in bodyweight gain during days 14 to 21 (F1 and F2), likely due to the neonatal consumption of the dam’s treated diet, and a slight reduction in spleen weights only observed in the F2 generation are of questionable toxicological relevance. The evidence from this study suggested that a dietary concentration of 10,000 ppm (500 mg/kg bw/day) should be considered as the NOEL for the F0 and F1 parent animals. The NOAEL for survival and growth of the offspring is considered to be 10,000 ppm (500 mg/kg bw/day). Evaluation of estrogenic activity in a uterotrophic Assay at doses of 500, 1000, 1500 or 2000 mg/kg/day for 7 days by oral gavage in ovariectomized (ovaries removed and no natural source of oestrogen) adult Spraque-Dawley (CD) rats using vaginal cornification and the uterotrophic response as the endpoints demonstrated that DGD did not exhibit estrogenic activity up to and including the maximally tolerated dose. In summary the following profile was identified:
D: Developmental 4.5.3 Environmental assessmentDGD is found to be readily biodegradable (85% of ThOD in 28 days) in the modified Sturm test (OECD 301B) while in the Closed Bottle Test (OECD 301D) the BOD5/COD ratio was only 0.29 (>0.5 required for ready biodegradability). 75% was degraded after 120 days in an anaerobic biodegradation test with a pass level of 60% (US EPA 796.3140, corresponding to OECD 311) and therefore considered to be ultimately biodegradable under anaerobic conditions. The log KOW of 3.9 indicates some bioaccumulation potential and, at the same time, a likely low mobility in soil. The LC50 of fish (P. promelas) exposed to DGD for 96 hours was found to be 3.7 mg/L (OECD 203), the 48 hour EC50 for daphnia 19.3 mg/L (OECD 202), and the 72 hours growth rate-based EC50 for algae (Seleneastrum capricornutum, now known as Pseudokirchneriella subcapitata) is 11 mg/L. The corresponding 72 h NOEC was 1.0 mg/L. These quite uniform values across three main taxonomic groups could indicate a non-specific mode-of-action of DGD. The acute toxicity of DGD to earthworm (Eisenia foetida) (14 days) was found to be >1,000 ppm while the inhibitory effect (IC50) on the bacterium Pseudomonas putida could not be determined specifically but only be stated as higher than the highest testable concentration of 10 mg/L. Activated sludge respiration was not inhibited at 100 mg/L.
4.6 DEHT (di-ethylhexyl-terephthalate)4.6.1 Physico-chemical propertiesDEHT, CAS No. 6422-86-2, is a phthalate ester stoekiometrically equal to DEHP, i.e. phthalate ester bound to two ethylhexyl groups, but with a different spatial structure, because one of the carboxylic groups is placed differently on the benzyl ring ("tere" means tertiary, or third, because the carboxylic group is placed on the third carbon atom counted from the first carboxyl group). DEHT is a liquid with low volatility (Vp = 0.0029 Pa) and a very low solubility in water; determined to be 0.4 μg/L in a recent (2002) GLP study using the slow-stir method. Previously reported, higher solubilities (in the low or sub-mg/L range) are now believed to be incorrectly determined. The log KOW of DEHT is as high as 8.39. 4.6.2 Human health assessmentDEHT has been shown in both in vitro and in vivo studies to have the potential to undergo complete hydrolysis to yield terephthalic acid and 2-ethylhexanol (2-EH), which are rapidly eliminated. Results of these metabolism studies also indicate DEHT was not well absorbed within the gastrointestinal tract, with 36% of it recovered in the faeces still intact. A study to assess dermal absorption rate indicated that DEHT has a very low potential to penetrate the skin (0.103 µg/cm²/hr), which further limits systemic exposure potential. DEHT has low acute toxicity by the oral route in rats with LD50 reported at >3,200 mg/kg bw (male rat, no guideline) and >5,000 mg/kg bw (TSCA FHSA Regulations (1979): 16 CFR Part 1500.40). Dermal LD50 in male guinea pigs was found to be > 19,670 mg/kg bw. No deaths occurred following inhalation exposure of mice for 4 hr to "saturated" vapours; however, mucosal irritation, loss of coordination and decreased mobility were noted. Recovery occurred in 24 hours. DEHT was concluded to be a slight dermal irritant in male guinea pigs with no evidence of percutaneous absorption following a single-dose occlusive dermal application and 24 hours exposure. DEHT produces slight irritation to rabbit eyes in a study using a procedure similar to OECD 405. In studies with some limitations, no skin sensitization was observed in humans or guinea pigs. Subchronic toxicity was evaluated in a 90-days repeated dose toxicity study where rats were fed diets containing DEHT in concentrations of 0.1, 0.5 or 1%. Study was conducted in a manner similar to the one described in the U.S. EPA guideline, 799.9310 TSCA. The only significant treatment related difference between controls and treated animals was increased relative liver weight in the 1.0% dose group. NOEL was 0.5% corresponding to approximately 500 mg/kg/day. In a 21-days repeated oral toxicity study in rats NOEL was also 0.5% (≈ 500 mg/kg/day) based on increased relative liver weight in females at 1.0%. DEHT did not produce mutagenicity in Ames tests (procedure similar to OECD 471) and also no response considered to be indicative of clastogenic activity in doses up to 1,000 nL/mL in a In-vitro Mammalian Chromosome Aberration assays in CHO cells (procedure similar to OECD 473) with and without activation. DEHT was evaluated for combined chronic toxicity and carcinogenicity. The test substance was administered in the diets of male and female Fischer-344 inbred rats at concentrations of 20, 142, and 1,000 mg/kg/day. Clinical evaluations revealed no treatment-related signs, however, eye opacities (cataracts) occurred frequently in all groups. At 1,000 mg/kg/day, body weights and female liver weights were reduced. There were no consistent reductions in food consumption. There were no treatment-related effects evident from the gross and histopathologic examinations conducted at 6 and 12 months. At 18 months, two basic lesions of the females in the 1,000 mg/kg/day level appear to be associated with treatment. These were hyperplasia and/or transitional cell adenomas of the urinary bladder and adenomas or adenocarcinomas of the uterus. In another combined chronic toxicity and carcinogenicity study (industry study) in F-344 rats DEHT was administered in doses of 1,500, 6,000 and 12,000 ppm (79, 324 and 666 mg/kg bw/day (m) and 102, 418 and 901 mg/kg bw/day) in the diet. It was concluded that the oral administration of DEHT via the diet was well tolerated at all dose levels. There was no effect upon tumour incidence and therefore the NOEL for tumorigenicity was at least 666 mg/kg bw/day in males and 901 mg bw/day in females. Toxic responses were confined to low weight gain and food conversion efficiency in males and females receiving 6,000 and 12,000 ppm. Consequently the NOEL for chronic toxicity in the study was 1,500 ppm (79 mg bw/day (m) and 102 mg bw/day (f)). Reproductive toxicity of DEHT in Sprague-Dawley rats was studied in a 2-generation test according to OECD 416 and at dose levels of 3000, 6000 or 10000 ppm in the diet. The NOAEL for reproductive toxicity was 1.0% in the diet (500-700 mg/kg bw/day for males and 800-1,000 mg/kg bw/day for females; highest dose tested), and the NOAEL for parental and offspring toxicity based on reduced body weight gains was 0.3% (150-200 mg/kg bw/day for males and 250-300 mg/kg bw/day for females). Mean maternal body weights and body weight gains were reduced for F0 and F1 females in the 1.0% group throughout pregnancy and decreased mean terminal body weights were noted in F1 males and females given 0.6% or 1.0% test material. The results of this study, in conjunction with the 90-day study which also showed no effect of DEHT on histology of reproductive organs indicate that DEHT has a low potential to induce reproductive toxicity. Developmental toxicity was evaluated in a dietary study following OECD Test Guideline 414 and at dose levels of 3000, 6000 or 10000 ppm in the diet. The NOEL for maternal toxicity was 0.6% (458 mg/kg/day) and the NOEL for developmental toxicity was 1.0% (747 mg/kg/day; highest dose tested). The ability of DEHT to induce anti-androgenic like effects in male offspring was assessed by giving pregnant rats 750 mg/kg DEHT by gavage on gestation day 14 until postnatal day (PND) 3. No changes indicative of a feminization effect were induced in male pups. NOEL for maternal toxicity and teratogenicity was 750 mg/kg. Results of an uterotrophic assay in which immature females were given up to 2,000 mg/kg/day DEHT by gavage on PND 19-21 also indicate that DEHT does not possess estrogenic activity. In summary the following profile was identified:
D: Developmental 4.6.3 Environmental assessmentIn a study from 1986, performed according to the EPA aerobic biodegradation guideline, the biodegradability of DEHT was found to be 56% in 28 days, corresponding to a classification as inherently biodegradable. The BCF of 393 (in oysters, determined in an EPA protocol study) indicates a medium potential to bioaccumulate, and the KOC of 2,000 a high sorptivity to soil organic matter. A 7 days flow-through study with Salmo gairdneri using acetone to enhance solubility of DEHT resulted in an LC50 of ≥ 0.25 mg/L (measured), and a 71 days early life-stage study with the same species gave a NOEC ≥ 0.28 mg/L (measured). The acute toxicity (immobilization) to Daphnia magna was determined in a standard 48 hour static test and found to be EC50 ≥ 1.4 mg/L while the 21 days NOEC (reproduction) for D. magna in a flow-through test was 0.76 mg/L (both measured). Inhibition of growth of Seleneastrum capricornutum(now known as Pseudokirchneriella subcapitata) in the standard 72 hours static test did not occur at the possible test concentrations and the EC50 was therefore just stated to be ≥ 0.86 mg/L. In the OECD 218 test on sediment- dwelling organisms (larvae of Chironomus riparius), the EC50 after 28 days was determined to ≥ 950 mg/kg while the NOEC for emergence was 180 mg/kg. In a 3 hour activated sludge inhibition test according to OECD 209 the EC50 was found to be higher than 10 mg/L.
4.7 DINA (diisononyl adipate)4.7.1 Physico-chemical propertiesDINA is formed by an adipate (hexanoic acid) ester bound with two C-9 alkanes. The CAS No. is 33703-08-1. It is a liquid at room temperature and it appears to be moderately volatile (Vp < 10 Pa at 20 °C). It has a low water solubility of < 1 mg/L and is extremely lipophilic with a log KOW = 9.24. 4.7.2 Human health assessmentNo data on toxicokinetics have been identified. DINA has low acute toxicity by the oral and dermal route with oral LD50 in rats reported at > 5,000 mg/kg bw and dermal LD50 in rabbits reported at > 3,160 mg/kg bw. DINA was not irritating to rabbit skin (OECD 404) or rabbit eyes (OECD 405). No allergic skin reaction was reported in guinea pigs after repeated skin contact (intradermal and topical) using the Magnusson and Kligman method (OECD 406). Subchronic toxicity was evaluated in a repeated dose 13 week oral toxicity study in the rat at dose levels up to 500 mg/kg/day. NOAEL in males was 500 mg/kg/day. Increased relative kidney weight was observed at 500 mg/kg/day but no change in absolute kidney weight and histopathological changes was seen. It was concluded that there were no significant findings at any dose level. In a 13 week oral toxicity study in dogs fed at doses up to 3% for 8 weeks and 6% for 5 weeks, NOAEL was 1% in the diet corresponding to approximately 274 mg/kg/day. Adverse effects at the high dose included decreased body weight and food consumption, increased liver weight, elevated enzyme levels, liver and kidney discoloration, and histopathological changes in the liver and kidneys. DINA did not produce mutagenicity in Ames tests or mammalian cell (mouse lymphoma cells) systems with and without metabolic activation. No other data on health effects was found. Bridging data gaps and read across to the structural analogue, adipic acid, bis (2-ethylhexyl) ester (C22) (CAS No. 103-23-1) and three other structurally similar alkyl diesters (C12-C32) was suggested in the US EPA HPV programme for reproductive and developmental toxicity. These diesters show no or low potential for reproductive and developmental toxicity. In summary the following profile was identified:
4.7.3 Environmental assessmentDINA is readily biodegradable, it has been found to degrade by 82% in 28 days in the modified MITI test (OECD 301C) and by > 90% in the EEC manometric respirometric method. No data on mobility in soil have been found, but the extremely high log KOW of 9.24 indicates a very low mobility. The BCF in a 21 day test with D. magna was 1,102-2,031 while in a 35 day test with the blue mussel, Mytilus edulis, a BCF = 11,000 was found. No results for BCF in fish have been identified but an estimate from the USEPA gave a BCF = 3.2. The tests performed with DINA have all been carried out at concentrations exceeding the aqueous solubility i.e. by the use of solubility enhancing solvents such as DMF or acetone. The acute (96 h) toxicity to fish was determined in a test with Leuciscus idus according to DIN 38412. The LC50 was > 500 mg/L (nominal), which corresponded to > 2.6 mg/L (measured). The 79/831/EEC static acute immobilization test was conducted with Daphnia magna, and an EC50 > 100 mg/L was determined. A 21days NOEC > 100 mg/L was found for effects on reproduction of D. magna when using the OECD 202, part 2 test method. The 72 hour EC50 for algae was > 100 mg/L.
4.8 DINCH (di-isononyl- cyclohexane-1,2-dicarboxylate)4.8.1 Physico-chemical propertiesDINCH (CAS No. 166412-78-8) is the hydrogenated parallel to DINP, with the difference that the ring structure is cyclohexane (a cyclic alkane) instead of a benzene ring (an aromate). It is a colourless liquid at 20 °C with a very low water solubility of <0.02 mg/L, low volatily (Vp = 0.000022 Pa) and highly lipophilic character (log KOW >6.2). 4.8.2 Human health assessmentDINCH is rapidly absorbed after oral administration and readily eliminated. After 24 hours approximately 80% of the radioactivity is excreted, after 48 hours more than 90% is excreted via urine and mainly via faeces. There is no indication of bioaccumulation. The characterisation of metabolites after oral and intravenous administration of DINCH indicates two main pathways: the partial hydrolysis of DINCH to the mono-isonyl ester followed by conjugation to glucuronic acid, which is the most abundant metabolite in bile, or the hydrolysis of the remaining ester bond to yield free cyclohexane dicarboxylic acid, the predominant urinary metabolite. DINCH has low acute toxicity by the oral (OECD 423) with LD50 in rats reported at > 5,000 mg/kg bw. Dermal LD50 in rabbits (OECD 402) was reported at > 2,000 mg/kg bw. DINCH was slightly irritating to rabbit skin (OECD 404) with mean scores for erythema of 2.0 in one animal and 1.7 in two animals. DINCH was not irritating to rabbit eyes (OECD 405). There was no evidence of skin sensitisation in guinea pigs in a study according to OECD 406. DINCH was studied in a 28-days oral repeated dose toxicity study (OECD 407) in rats at concentrations of 600, 3000 and 15000 ppm in the diet. Doses of 15,000 ppm caused changes in clinical chemistry parameters in animals of both sexes. Indications of mild renal function impairment (urinary epithelial cells, elevated serum Na+/K+) were observed in male rats. Female rats showed signs that may be associated with hepatic microsomal enzyme induction, characterised by stimulation of γ-glutamyltransferase synthesis and by increased excretion of bilirubin due to stimulation of phase II reactions. NOAEL was established as 3,000 ppm (318 mg/kg bw/day (males) and 342 mg/kg bw/day (females)) in this study, based on the absence of effects on clinical chemistry parameters at this intake level. In a 90-days oral repeated dose toxicity study (OECD 408) in rats DINCH was administered in the diet at concentrations of 1500, 4500 and 15000 ppm. NOAEL was established at 107.1 mg/kg bw/day (males) and 389.4 mg/kg bw/day (females) in this study, based on kidney weight changes in both sexes and the appearance of degenerated epithelial cells in the urine of males. In a combined chronic toxicity/carcinogenicity study (OECD 453) rats were administered doses of 40, 200 or 1,000 mg/kg bw/day in the diet. After 24 months of treatment, dose-related follicular cell hyperplasia and increased number of follicular adenomas were observed in the thyroid glands of male rats administered 200 mg/kg bw/day and in both genders administered 1,000 mg/kg bw/day. The thyroid glands are a target organ for the effects of the notified substance in rats. There was a dose-related increased incidence of follicular adenomas in the thyroid gland of mid and high dose male rats and high dose female rats. However, thyroid effects in rats are potentially secondary effects associated with liver enzyme induction and of limited relevance to humans. Such an indirect mechanism is plausible based on the findings of increased GGT activity and lower serum bilirubin levels in this study, and supported by further studies (see special studies below) on enzyme induction and cell proliferation. NOAEL was established at 40 mg/kg bw/day (males) and 200 mg/kg bw/day (females) based on liver weight changes (both sexes) and kidney weight changes (males). DINCH did not produce mutagenicity in Ames tests (OECD 471) or in in vitro mammalian CHO cells with and without metabolic activation. No clastogenic activity was seen in a chromosome aberration assay (OECD 473) with and without activation. DINCH was also not found to be clastogenic or aneuploidogenic in a in vivo mouse nucleus test (OECD 474). Developmental toxicity was examined in rabbits (OECD 414) at doses of 100, 300 or 1,000 mg/kg bw/day in the diet. DINCH did not have any adverse effects on maternal toxicity, gestational parameters or developmental toxicity up to 1,000 mg/kg bw/day. NOAEL was established at 1,000 mg/kg bw/day. In a similar study in rats (OECD 414) with animals dosed at 200, 600 or 1,200 mg/kg bw/day, NOAEL was established at 1,200 mg/kg bw/day due to absence of adverse effects on maternal toxicity and prenatal development. Same conclusions were obtained in a study following elements of OECD 414 and OECD 415 (one-generation reproduction toxicity test) with rats dosed at 750 and 1,000 mg/kg bw/day. NOAEL for maternal and development toxicity was established at 1,000 mg/kg bw/day. Toxicity to reproduction was studied in a two-generation study with animals dosed at 100, 300 or 1,000 mg/kg bw/day. Under the conditions of this reproduction study, the NOAEL for fertility and reproductive performance was established at 1,000 mg/kg bw/day for F0 and F1generation rats of both genders. The NOAEL for general toxicity was 1,000 mg/kg bw/day (F0 rats of both genders) and 100 mg/kg bw/day for the F1 male and female rats (based on tubular vacuolisation and flaky thyroid follicular colloid). The NOAEL for developmental toxicity (growth and development of offspring) was 1,000 mg/kg bw/day for the F1 and F2 pups. In summary the following profile was identified:
4.8.3 Environmental assessmentWith only 41% degradation in the CO2 evolution test (OECD 301B), DINCH cannot be classified as readily biodegradable. The BCF of 189 indicates a moderate bioaccumulation potential but 90% depuration of the substance occurred within 1.6 days. The log KOW of >6.2 indicates high sorption potential. The acute toxicity to fish was tested with zebrafish using the 96 hour static EC-test method and found to be LC50 >100 mg/L. Similarly, the acute EC50 for daphnia was found to be higher than the highest test concentration in OECD 202 of 100 mg/L. In a 21 days reproduction test (OECD 211) no effects occurred at the highest test level of 0.021 mg/L (measured) and the NOEC was therefore determined to be ≥0.021 mg/L. The rate based 72 hour EC50 for algae (Scenedesmus subspicatus) was found to be >100 mg/L and the corresponding NOEC ≥100 mg/L. DINCH was found to be virtually non-toxic to activated sludge (EC50 >1,000 mg/L) and to earthworms in the 14 days acute artificial soil test (LC50 >1,000 mg/kg).
4.9 GTA (glycerol triacetate)4.9.1 Physico-chemical propertiesGTA ("Triacetin") is an ester of glycerol and three acetate groups. Its CAS no. is 102-76-1. It is a liquid at room temperature with a relatively low vapour pressure of 0.33 Pa at 25 °C. It has high solubility in water (58,000-70,000 mg/L) and a correspondingly low log KOW of 0.21-0.36. 4.9.2 Human health assessmentGTA is rapidly absorbed following ingestion and metabolised like other shorter-chain triglycerides. Several studies confirmed that GTA is hydrolysed to glycerol and acetic acid by digestive enzymes, particularly lipases, liver or plasma carboesterases. GTA is readily hydrolyzed to free glycerol and acetic acid, when incubated with rat intestine in vitro. The chemical infused in dogs undergoes intravascular hydrolysis and the majority of the resulting acetate is oxidized nearly quantitatively. The substance has been shown to be a source of liver glycogen and when fed in amounts equal in caloric value to 15% glucose it was utilised as efficiently as glucose. The acute oral and dermal toxicity of GTA is low. In an oral acute toxicity study in rats (OECD 401), a limit dose of 2,000 mg/kg bw caused no mortality and no signs of systemic toxicity during the 14-day observation period. The LD50 in rats by gavage is determined to be >2,000 mg/kg bw for both sexes, and dermal LD50 in rabbits and guinea pigs were >2,000 mg/kg bw. Acute inhalation toxicity is considered to be very low, since the LC50 in an acute inhalation toxicity study in rats was >1,721 mg/m³ for both sexes (OECD 403) and repeated daily exposure of rats to 73,700 mg/m³ produced no sign of toxicity after 5 days. GTA was not found irritating to rabbit skin and eyes in studies following OECD 404 and 405. GTA did not induce sensitisation in guinea pigs. In a combined repeat dose and reproductive/developmental screening toxicity test (OECD 422), rats were exposed to 40, 200 or 1,000 mg/kg/day by oral gavage for 44 days from 2 weeks prior to mating for males and for 41 - 48 days from 14 days before mating to day 3 postpartum for females. GTA had no effects on clinical signs, body weight, food consumption, and organ weight or necropsy findings. No histopathological changes ascribable to the compound were observed in either sex. There were no abnormalities in haematological or blood chemical parameters in males. The NOAEL for repeated dose oral toxicity is thus considered to be 1,000 mg/kg bw/day for both sexes. An inhalation study was conducted in rats given GTA for 90 days at a dose of 249 ppm (2,220 mg/m³). No signs of toxicity were noted during the exposure. The NOAEL is considered to be 249 ppm (2,220 mg/m³) for 90 days. Although the inhalation study is considered to be useful, it does not fully comply with the current testing protocol. GTA did not induce gene mutation in Ames test at concentrations up to 5,000 ug /plate (OECD 471 and 472). Induction of chromosome aberrations was observed in the Chinese hamster lung cells at the highest concentration (2.2 mg/mL, 10 mM) in the presence of metabolic activation (OECD 473). Because of high toxicity (75%) that might be caused by low pH (4.9) at the end of the treatment, the chromosomal aberration observed might not be biological relevant. Under un-physiological culture condition, such as low pH, it was reported that the frequency of chromosomal aberrations could be increased. Polyploidy was not induced under any of the conditions tested. Results were equivocal, but taking all data into consideration, GTA could be considered to be non-genotoxic. The combined repeated dose and reproductive/developmental toxicity study in rats at doses of 40, 200 or 1,000 mg/kg bw/day (OECD 422) showed no statistically significant adverse effects on reproductive parameters ( mating index, fertility index, gestation length, numbers of corpora lutea and implantations, implantation index, gestation index, delivery index, parturition and maternal behaviour at delivery and lactation). In addition, there were no significant differences in numbers of offspring or live offspring, the sex ratio, the live birth index, the viability index or body weight. Developmental toxicity, clinical signs of toxicity, and change in necropsy findings were not found in offspring. Therefore, the NOAEL is considered to be 1,000 mg/kg bw/day for parental animals and offspring. In summary the following profile was identified:
4.9.3 Environmental assessmentThe bulk of data on aerobic biodegradability of GTA suggests that the substance is readily biodegradable, e.g. 77% degradation after 14 days based on BOD, 93% after 28 days based on ThCO2 and 94% after 28 days based on TOC (OECD methods 301B, 301C and 301 D). The KOC of 10.5 indicates high mobility in soil (corresponds well with high water solubility and low log KOW), and the calculated BCF = 1.3 implies an insignificant bioaccumulation potential. The acute toxicity of GTA to fish has been studied on a number of species such as Pimephales promelas, Oryzias latipes, Cyprinus carpio, Brachydanio rerio and Leuciscus idus. In the test with O. letipes, the lethal level was not reached at the highest test concentration of 100 mg/L, and among the other species the LC50 ranged from 165 to 300 mg/L with P. promelas being the most sensitive species (tested with OECD 203, DIN38412 or ISO 7346/2 (conforming to OECD 203)). In a prolonged test with O. latipes the 14 days LC50 was > 100 mg/L (nomimal) (OECD 204). EC50'ies for acute toxicity to D. magna range from 380 to 811 mg/L with the most sensitive result being obtained with the DIN 39412 Teil 11 test, which conforms to OECD 202. The 21 days NOEC on reproduction of D. magna was 100 mg/L in the OECD 211 test. Inhibition of growth of Seleneastrum capricornutum (now known as Pseudokirchneriella subcapitata) in the standard 72 hours static test (OECD 201) could not be determined but was > 1,000 mg/L. The NOEC (72 h) was determined to be 556 mg/L. The toxicity to bacteria, Pseudemonas putida, was determined using the EN ISO 10712 guideline. A 16 hours NOEC = 3,000 mg/L was determined.
4.10 TXIB (trimethyl pentanyl diisobutyrate)4.10.1 Physico-chemical propertiesTXIB is an ester of the branched alkane trimethyl pentanyl with two butyrate groups. Its CAS no. is 6846-50-0. The vapour pressure is only 0.089 Pa, the water solubility low (1-2 mg/L; 15 mg/L) and the lipophilicity quite high (log KOW is 4.1 or more). 4.10.2 Human health assessmentTXIB was rapidly adsorped, metabolized and excreted. The major route of elimination was urine (47 – 72% total dose) within 5 - 10 days and the majority of this occurring in the first 72 hours. Radioactivity in faeces accounted for 14 – 31% of the dose with elimination being essentially complete by 7 days with the majority isolated after 48 hours. Radiolabeled CO2 was not detected. In total, excretions accounted for 95-99% of the dose. Residual radioactivity of treated animals approached control by two weeks. Identification of metabolites showed the faeces to contain both 2,2,4-trimethyl pentanediol (TMPD) and TXIB-3-14C indicating esterase cleavage of the two isobutyrates. A small potion of the absorbed material in the urine was unchanged TXIB-3-14C while the majority consisted of metabolites consistent with complete cleavage to the glycol (TMPD) parent molecule. Although much of the urinary metabolite was unidentified it does, nonetheless, represent rapidly cleared material. TXIB has low acute toxicity by the oral route with LD50 in rats reported at > 3,200 mg/kg bw. Dermal LD50 in rabbit (OECD 402) was reported at > 2,000 mg/kg bw. LC50 in rats exposed to 0.12 mg/L or 5.3 mg/L for 6 hours was > 5.3 mg/L. Skin irritation was studied in guinea pigs and rabbits. In guinea pigs TXIB was slightly irritating to the skin and in rabbits (OECD 404) no irritation was observed. TXIB was not found irritating to rabbit eyes (OECD 405). TXIB was not found sensitising to skin in guinea pigs in a test following a protocol similar to OECD 406. In a study on human volunteers using a modified Draize procedure , TXIB was found non-irritating ad did not induce any evidence of sensitisation. TXIB was studied in a 103 days oral repeated dose toxicity study in rats receiving concentrations of 1% and 1.0% in the diet. There was a slight, significant increase in the relative liver weights in the 1.0% group and in the absolute liver weights in the 1.0% male group when compared to controls. NOAEL was established at 0.1%. In another study rats were exposed to the same concentrations for 52 or 99 days (I: 52 days TXIB diet, II: 99 days TXIB diet, III: 52 days TXIB + 47 days control diet or 52 days control diet + 47 days TXIB diet). From the study it appeared that high doses of TXIB cause significant adaptive changes in the rat liver, and these changes are reversible if the animal is returned to normal diet. NOAEL was 0.1%. In dogs (beagles) fed a diet with 0.1, 0.35 or 1.0% TXIB for 13 weeks, NOAEL was established at 1.0% because of no toxicological significant findings related to treatment. In a combined repeat dose and reproductive/developmental screening toxicity test (OECD 422), rats were exposed to 30, 150 or 750 mg/kg/day. A NOEL at 30 mg/kg/day was established based on effects on liver and kidneys in the higher dose groups. When evaluating the reproductive toxicity, NOEL (Parental) and NOEL (F1 offspring) was established at 750 mg/kg/day, as no effects on reproduction (mating, fertility and oestrus cycle, dams during pregnancy and lactation, pubs after birth) related to treatment were observed. In another combined study (OECD 421 with additional sperm motility asssessment) TXIB was administered in doses of 91, 276 or 905 mg/kg /day in males and 120, 359 and 1,135 mg/kg/day in females. Statistically significant reproductive effects observed in the high dose group include reduced number of implantation sites, reduced mean litter weights on postnatal (PND) 0, reduced mean number of live pubs on PND 4, decreased mean absolute epididymal sperm counts, and reduced absolute and relative testicular sperm counts. The mean number of live pubs per litter was also reduced on PND 0. NOAEL for reproductive and developmental toxicity was 276 mg/kg/day on males and 359 mg/kg/day in females based on reduced number of implantation sites and reduced mean number of live pubs on PND 0. TXIB did not produce mutagenicity in Ames tests (Japanese guideline) or in in vitro mammalian CHL cells (Japanese guideline) with and without metabolic activation. In summary the following profile was identified:
4.10.3 Environmental assessmentTXIB was found to be inherently biodegradable in the OECD 301C test and the BCF (carp) determined to be in the range 5.2-31 (OECD 305C), i.e. a low bioaccumulation potential. The BCF of 4.1 (or more) indicates a low mobility of TXIB in soil. Acute toxicity of TXIB to fish: LC50 (96 h) = 18 mg/L (OECD 203; Oryzias latipes). In one test (OECD 202) on Daphnia magna the EC50 was found to be 300 mg/L while in another EC50 >1.46 mg/L. The 14 days NOEC (reproduction) for D. magna was determined to 3.2 mg/L in a test basically performed according to OECD principles but apparently not fulfilling all test requirements. The 72 hours biomass-based EC50 for algae (Seleneastrum capricornutum, now known as Pseudokirchneriella subcapitata) has been determined to 8.0 mg/L using the OECD 201 method. The corresponding 72 h NOEC was 5.3 mg/L.
4.11 Summary of human health and environmental assessment of alternative plasticisers4.11.1 Human health assessment summaryTable 4.11 provides an overview of the toxicological properties of the selected alternatives. All substances have been tested for acute toxicity for at least one exposure route, sensitisation (except ASE), subchronic toxicity and mutagenicity. All substances except ASE, COMGHA and DINA have been tested for both reproductive and developmental toxicity. With regard to carcinogenicity only ATBC, DEHT and DINCH have been tested in combined chronic toxicity and carcinogenicity studies. For DEGD, DGD and DEHT estrogenic activity has been tested in a uterotrophic assay without positive response. Most data used for the evaluation are considered of good quality, i.e. studies following accepted guidelines (OECD or US EPA) or studies considered acceptable at the time they were carried out. For some of the studies little information is available to evaluate the quality. However, key information is obtained from IUCLID data sheets, USEPA or OECD HPV robust summaries. Studies evaluated under the USEPA HPV programme are considered to be reliable without restrictions or reliable with restrictions (Klimisch codes 1 and 2, and restrictions in cat. 2 generally not severe). From the overview it can be seen that all ten substances have low acute toxicity. With regard to local effects most substances are non-irritating to skin and eyes or only produce slight irritation which would not lead to classification. None of the tested substances show any sensitising potential. Effects from repeated dose toxicity studies mainly include reduced body weight gain, increased organ weights (liver and/or kidney) and for some substances also changes in clinical chemistry or clinical pathology parameters. However, more serious pathological effects were not observed. Only GTA did not show any adverse effects in repeat dose toxicity studies. Studies to evaluate the potential for reproductive/developmental toxicity primarily show toxic effects on parents and offspring resulting in effects on body weight or relative organ weights. Effects on foetal growth and development of cervical ribs were observed in prenatal development studies with DEGD and DGD. With regard to DEHT it was concluded that DEHT has a low potential to induce reproductive toxicity. TXIB showed statistically significant reproductive and developmental toxicity in a combined study (repeat dose/reproductive-developmental screening). Effects included reduced number of implantation sites, reduced mean litter weights and reduced mean number of live pubs. Carcinogenicity has only been evaluated for three substances in combined studies with negative outcome. Click here to see: Table 4.11 Overview of toxicological properties of selected alternatives 4.11.2 Environmental assessment summaryTable 4.12 summarises the main data on environmental fate (biodegradation, bioaccumulation and mobility) and ecotoxicological effects (fish, daphnia and algae) of the 10 studied phthalate alternatives. The data on effects on bacteria have been omitted in the summary table because the effects were generally negligible at relevant exposure levels, while terrestrial data were so sparse that a comparative evaluation cannot be made for these organisms anyway. Useful fate data regarding biodegradability (in water) and bioaccumulative properties (either as BCF or log KOW) are available for all alternatives while other fate data are quite variable and incomplete. With regard to ecotoxicological effect data, results from short-term tests with the base-set of organisms - fish, crustaceans and algae - exist for all 10 substances although the duration of some studies deviate from the current OECD standard. The low solubility of many of the phthalate alternatives has rendered it necessary to enhance solubility by means of organic solvents in order to be able to carry out the tests. Overall, the data obtained are of good quality i.e. they are mostly based on studies performed according to accepted guideline procedures, and the studies have been evaluated (e.g. in the USEPA HPV robust summaries) to be reliable without restrictions or reliable with restrictions (Klimisch codes 1 and 2, and restrictions in cat. 2 generally not severe). None of the 10 studied alternatives fulfil the criteria for being PBT or vPvB substances.
GTA (triacetin) appears to be easily biodegradable; it does not have bioaccumulative properties and has very moderate toxicity in the aquatic environment. DEGD, DGD and DINA also come out rather favourable (if using the estimated BCF of 3.2 for DINA), while ATBC and COMGHA come out negatively despite their degradability because of their aquatic toxicities and bioaccumulative properties. ASE and DINCH both have low acute toxicities to aquatic organisms but are not easily degradable and have high log KOW values. DEHT is also not easily biodegradable and is bioaccumulative but its aquatic toxicity cannot be fully evaluated based on the data available. 4.11.3 Health and environmental assessment overviewA simplified overview of the main toxicological and ecotoxicological properties of the evaluated substances is shown in Table 4.13. In the table a rough overview of the quality and completeness of data is presented using the scoring system indicated in note 4 to the table. Click here to see: Table 4.13 Overview of main toxicological and ecotoxicological properties
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