Opinion on diethylene glycol monoethyl ether (DEGEE)
`
`SCCS/1316/10
`
`I 3.3.6.2 Mutagenicity/Genotoxicity in vivo ]
`
`Unscheduled DNA assay in rat liver
`
`Guideline:
`Species/strain :
`Group size:
`Main study:
`Test substance:
`Batch:
`Dose levels:
`
`Exposure:
`Route:
`Application volume:
`Vehicle:
`Sacrifice Times:
`
`Positive control:
`
`GLP:
`Date:
`
`Comparable to OECD 486
`Rat/Wistar
`Range-finding study: 3 male and 3 female rats per group
`5 male rats per dose group/sacrifice time
`Transcutol® (purity: >99%)
`9600544
`Range-finding study: 0, 2000 mg/kg bw
`UDS: 0, 800, 2000 mg/kg bw
`Single application
`Oral (gavage)
`10 ml/kg body weight
`Purified water for Transcutol® and DMN, corn oil for 2-AAF,
`Range-finding assays: 48 hours after application
`UDS: 2 - 4 hours and 12 - 14 hours after application
`2-Acetamidofluorene (2-AAF): 75 mg/kg bw (for the 12-14 h
`experiments); Dimethylnitrosamine (DMN): 10 mg/kg bw (for the 2-
`4 h experiments)
`In compliance
`August 1996
`
`Transcutol was assessed for its potential to induce DNA damage and repair in the in vivo/in
`vitro UDS test using rat hepatocytes. In the dose range finding phase, each 3 male Wistar
`rats received doses of 2000 mg/kg bw to determine the maximum tolerated dose to be used
`in the definitive UDS study. The dosing volume was 10 ml/kg bw. All rats were observed for
`4 days for clinical signs of toxicity. Following the last observation the animals were
`sacrificed and not further examined.
`
`In the UDS test, 5 male Wistar rats received a single oral administration of Transcutol at
`800 and 2000 mg/kg bw. Five male rats were used as controls receiving the vehicle
`(purified water) while 5 positive control rats received a single oral application of either 10
`mg DMN/kg bw (2-4 h experiments) or 75 mg 2-AAF/kg bw (12-14 h experiments). Dosing
`was carried out in an interval of 2 hours in each 3 and 2 rats per dose group. All animals
`were observed for clinical signs of intoxication. The animals were exposed for 2 - 4 hours or
`12 - 14 hours. After the exposure periods, the animals were sacrificed and liver perfusion
`was carried out in 3/5 rats per dose group. From each 3/5 animals at least two primary
`hepatocyte cultures were established and exposed for 4 hours to 3H-thymidine, which is
`incorporated into the DNA, if UDS occurs. Following the 3H -thymidine exposure period, cells
`were washed and mounted on cover slips, coated and stored in darkness for 14 days at
`refrigerator temperature. Thereafter, the slides were developed at room temperature, fixed
`and stained. In total, six slides from each animal were prepared. The net nuclear grain
`counts were determined by counting 2/3 slides per animal and 50 cells per slide (100 nuclei
`in total/animal). Appropriate reference mutagens (DMN and 2-AAF) were used as positive
`controls.
`
`In the range-finding segment, the animals did not show any mortalities or clinical findings.
`In the UDS study, no significant increases in the group mean or net nuclear grain (NG)
`count for animals treated with Transcutol were observed at any dose level or at harvest
`time.
`The group mean NG count for the vehicle control was within historical control values, while
`the positive controls induced a significant increase in NG count demonstrating the sensitivity
`and validity of the test system.
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`

`

`Opinion on diethylene glycol monoethyl ether (DEGEE)
`
`SCCS/1316/10
`
`The authors concluded that Transcutol did not induce DNA-damage, i.e. no increased repair
`synthesis in hepatocytes of treated rats under the experimental conditions reported.
`N Ref.: 19
`
`In a micronucleus assay with Swiss CD-1 mice, the test material was administered i.p. to 2
`groups of 4 male mice in two subsequent daily doses of 2 ml/kg (1980 mg/kg bw). Two
`groups of 4 male mice (positive controls) were treated with 100 mg/kg benzoapyrene (BP)
`dissolved in DMSO. One group of 4 rats (control) was left untreated. One group of animals
`treated with test material and BP was killed at 48 hours, and the other was killed at 72
`hours. Negative controls were killed at 72 hours. Bone marrow smears were made and
`stained with Giemsa. One thousand polychromatic erythrocytes (PCE) from each animal
`were scored for micronuclei. The PCE/normochromatic erythrocyte ratio (NCE) was also
`scored to evaluate any toxic effect. The tested material had no effect on the number of NCE,
`ratio of PCE/NCE or on the number of micronuclei. The number of NCE was increased by
`treatment with BP at 72 hours and the number of micronuclei observed in animals treated
`with BP was increased at both time points showing the sensitivity of the assay.
`
`Ref. : 1
`
`Comment
`The study is from 1986 and is poorly reported. Therefore the reliability is considered as
`limited.
`
`General comment
`In one purely reported study from 1986, DEGEE displayed a weak mutagenic activity at high
`concentrations in some tested Salmonella typhimurium strains (TA1535, TA1537, TA1538)
`and in Saccharomyces cerevisiae (D7) while no mutagenic activity were reported in another
`Salmonella test performed according to GLP. DEGEE did not induce unscheduled DNA
`synthesis (UDS) test in primary rat hepatocytes in vivo after exposure of rats up to 2000
`mg/kg bw by gavage. In one poorly reported study from 1986, DEGEE did not induce
`micronuclei in CD-1 mouse bone marrow following 2 daily i.p. injections at 1980 mg/kg bw.
`
`I
`
`I
`
`[3.3.7.
`
`Carcinogenicity
`
`No adequate data available
`
`[3.3.8.
`
`Reproductive toxicity
`
`Oral route
`
`Mice
`
`Guideline:
`Species/strain:
`Group size:
`Test substance:
`Batch:
`Purity:
`Dose levels:
`Route:
`Exposures:
`GLP:
`
`/
`Swiss CD-1 mice
`50 pregnant mice
`DEGEE
`/
`>99%
`5500 mg/kg bw/day
`Oral, gavage
`Pregnant mice, days 7 through 14 of gestation
`In compliance
`
`Fifty mated CD1 mice were orally administered DEGEE (>99% purity) by gavage at 5500
`mg/kg/day (calculated LD10 based on a non-pregnant mouse pilot study) in corn oil from
`GD7-14 (GDl=vaginal sperm plug), then allowed to litter and to rear pups to PND3. 14% of
`the dams died, maternal weight gain was reduced and, of 33 surviving pregnant females,
`there were 32 viable litters (97%) compared with 100% control litter viability. No external
`
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`

`

`Opinion on diethylene glycol monoethyl ether (DEGEE)
`
`SCCS/1316/10
`
`malformations were seen, pup survival to PND was unaffected and no other indication of
`specific developmental toxicity was found.
`
`Ref.: 39, 40
`
`Guideline:
`Species/strain:
`Group size:
`Test substance:
`Batch:
`Purity:
`Dose levels:
`Route:
`Exposures:
`GLP:
`
`/
`CD-1 outbred Swiss albino mice
`20 males and 20 females; control group 40 males and 40 females
`DEGEE
`/
`>99%
`0, 0.25, 1.25, and 2.5% (440, 2200, and 4400 mg/kg bw/d)
`Oral, in drinking water
`See below
`/
`
`Continuous breedinq
`During the first 7 days of treatment (premating exposure) the sexes were housed
`separately. Subsequently, females and males from the same dose group were paired and
`cohabited for 98 days while being continuously exposed to DEGEE. The pairs were then
`separated and exposed for further 3 weeks. The animals received DEGEE in drinking water
`at concentrations of 0, 0.25, 1.25, and 2.5% (440, 2200, and 4400 mg/kg bw/d). During
`the 119 day period, different reproduction parameters were recorded. There was a small
`significant decrease in the mean body weights of the males during weeks 1 and 5 in the
`high dose group. DEGEE had only minimal effects on fertility or reproductive performance.
`
`Offspring assessment. The F1 generation from the final litters was reared and continuously
`treated with 0 or 2.5% DEGEE (4400 mg/kg bw/day) and at 74+10 days of age paired with
`nonsiblings from the same dose group. A significant decrease (34%) in motile sperm from
`de cauda epididymis in males exposed to 2.5% DEGEE was seen. In addition the relative
`liver weights were increased (16% in males and 10% in females).
`
`Ref.: 41, 42
`
`Rats
`
`Prenatal developmental toxicity study in rats
`
`Guideline:
`Species/strain:
`Group size:
`Test substance:
`Batch No:
`Dose level:
`Vehicle:
`Application volume:
`
`Route:
`Exposure period:
`GLP:
`Date:
`
`Comparable to OECD 414
`Rat/Sprague-Dawley
`25 pregnant females per group
`Transcutol HP (purity: 99.98%)
`D 4089
`0, 300, 1000, 2000 mg/kg bw
`Sterile water
`2 or 4 ml/kg bw (0 and 2000 mg/kg bw or 300 and 1000 mg/kg bw,
`respectively)
`Oral (gavage)
`Day 6 - 17 post coitum
`In compliance
`June 2002
`
`The prenatal developmental toxicity was investigated in Sprague-Dawley rats. The test
`substance was administered to 25 mated female rats per group by gavage as an aqueous
`solution in sterile water at doses of 300, 1000 and 2000 mg/kg bw on day 6 through day 17
`post coitum (p.c.). The control group, consisting of 25 females, was dosed with the vehicle
`(sterile water) in parallel. At terminal sacrifice 24 - 25 females/group had implantation sites
`and were considered as pregnant. The dams were examined for clinical condition and
`
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`
`

`

`Opinion on diethylene glycol monoethyl ether (DEGEE)
`
`SCCS/1316/10
`
`reaction to treatment at least once daily. Body weights were reported for days 0, 6, 11, 15,
`18 and 20 of gestation. Food consumption was calculated for the periods (days) 0 to 6, 6 to
`11, 11 to 15, 15 to 18 and 18 to 20 during gestation. All females were killed on day 20 of
`gestation for examination of their uterine contents including examination of the placenta. At
`necropsy the females were examined macroscopically and all foetuses were weighed, sexed
`and examined for external abnormalities. Half of the foetuses were examined internally
`prior to processing for skeletal examination. The remaining foetuses were preserved for
`fixed-visceral examination by the modified Wilson-Barrow technique.
`
`Findinqs in the dams:
`There was no mortality or treatment-related effects on clinical condition in any of the
`groups. Maternal body weight gain and food consumption were statistically significantly
`reduced in the
`2000 mg/kg bw group during the first five days of treatment. No effect on body
`weight/body weight gain or food consumption was observed at 300 and 1000 mg/kg bw.
`Necropsy examination of the adult females did not reveal any treatment-related
`abnormalities.
`
`Reproduction data of dams:
`Pregnancy was confirmed for 24-25 rats/group. One dam of the mid dose group had a
`single early resorption and no live foetuses. The mean number of uterine implantation was
`slightly lower in the 300 mg/kg bw group due to an incidental increase in pre-implantation
`loss as this period was prior to the start of treatment. Pre-implantation data were
`comparable in the other treated and control groups. No substance-related differences were
`seen with regard to conception rate, mean number of corpora lutea, or placental weights.
`
`Examination of foetuses:
`There were no adverse influences of treatment on embryo-foetal survival. Mean foetal
`weights and sex ratio were comparable in all groups. There were no foetal malformations in
`any group and no effect on foetal morphology was noted with regards to external or visceral
`findings. The study authors reported that there was a minor effect on the foetal skeleton in
`form of an increase in the incidence of foetuses with reduced ossification, principally of the
`cranial bones, in the 1000 and 2000 mg/kg bw groups. These effects were considered by
`the study authors as an indication of a retarded skeletal development but clearly no
`indication of teratogenicity.
`
`Conclusion
`The authors concluded that the oral administration of Transcutol to pregnant Sprague-
`Dawley rats from implantation to day 17 of gestation resulted in maternal toxicity at 2000
`mg/kg bw in form of retarded body weight gain and reduced food consumption. Gestation
`was not affected at any dose level.
`
`Prenatal developmental toxicity occurred at 2000 mg/kg bw in form of minor skeletal
`findings predominantly in form of a clear and statistically significant increase in the
`incidence of reduced ossification of cranial bones as an indication of transiently retarded
`development. There was also some variation in the spontaneous increased incidence of
`delayed ossification in some cranial bones at 1000 mg/kg bw but these were partly not
`dose-related.
`
`The authors concluded that the NOAEL for maternal toxicity as well as for prenatal
`developmental toxicity was 1000 mg/kg bw. There was no indication of teratogenicity up to
`the highest dose level tested and therefore the respective NOAEL was >2000 mg/kg bw.
`N Ref.: 26
`
`Comment
`The SCCS concluded in the previous opinions on DEGEE (SCCP/1044/06, SCCP/1200/08) a
`NOAEL of 300 mg/kg bw/day for embryo-foetal toxicity. However, in the recent submission,
`
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`
`

`

`Opinion on diethylene glycol monoethyl ether (DEGEE)
`
`SCCS/1316/10
`
`additional argumentation for the NOAEL of 1000 mg/kg bw/day was provided and the SCCS
`considers 1000 mg/kg bw/day being the NOAEL for maternal and embryo-foetal toxicity. It
`was concluded that there was no indication of teratogenicity at any dose level used in the
`study.
`
`Fertility and general reproductive performance in rats
`
`Elements comparable to OECD 415
`Guideline:
`Rat/Sprague-Dawley
`Species/strain:
`24 males and 24 females per group
`Group size:
`Transcutol HP (purity: 99.9%)
`Test substance:
`0025005
`Batch No:
`0, 300, 1000, 2000 mg/kg bw
`Dose level:
`Sterile water
`Vehicle:
`Application volume: 2 or 4 ml/kg bw (0 and 2000 mg/kg bw or 300 and 1000 mg/kg bw,
`respectively)
`Oral (gavage)
`Males: 63 days before mating, throughout mating and up to one day
`prior to necropsy
`Females: 14: days before mating, throughout mating until day 7 of
`gestation
`In compliance
`November 2001
`
`Route:
`Exposure period:
`
`GLP:
`Date:
`
`Transcutol was investigated for its effects on fertility and general reproductive performance
`in and female Sprague-Dawley rats. Three groups each consisting of 24 male and 24 female
`rats received Transcutol by the oral route (gavage) at dose levels of 300, 1000 and 2000
`mg/kg bw) for a pre-mating period (63 days for males, 14 days for females) and during
`mating. Treatment continued until day 7 of gestation for the females and up to the day
`before necropsy for the males. A similar group of rats received the vehicle (sterile water)
`only over the same periods and served as a control.
`
`Clinical condition and reaction to treatment were recorded daily. Body weights of males
`were recorded twice weekly. Body weights of females were recorded twice weekly during
`pre-mating and mating periods (only pre-mating data are reported) and on days 0, 4, 8 and
`13 of gestation. Food consumption was measured weekly during the pre-mating period and
`for the periods (days) 0 to 8 and 8 to 13 of gestation. All surviving females were killed,
`where possible, on day 13 of gestation for examination of their uterine contents, including
`examination of the placentae. At necropsy, all animals were examined macroscopically and
`the uterine status, number of corpora lutea and numbers and type of uterine implantations
`were determined for females. Testes and epididymides were weighed and used for
`automated sperm analysis. The kidneys of all rats and the ovaries of all females were
`weighed. Selected organs were fixed and preserved for all animals and histopathological
`examinations were performed for control and high dose males. Similar examinations were
`performed for any male animal of the low and mid dose group that had abnormalities
`associated with the sperm analysis.
`
`Results
`There was no treatment-related effect in any of the groups on gonadal function, fertility and
`reproductive performance in any group. The predominant finding was related to a reduction
`in body weight gain and transient clinical findings following test substance application
`(gavage) at 1000 and 2000 mg/kg bw/day. During the premating period these signs
`included salivation and subdued behaviour and were mainly restricted to the males of the
`mid and high dose groups, while in females, these signs could only be noted at 2000 mg/kg
`bw. A body weight reduction was mostly apparent in males at 2000 mg/kg bw, with females
`less affected. Food consumption was not impaired in any treated group.
`
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`
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`
`

`

`Opinion on diethylene glycol monoethyl ether (DEGEE)
`
`SCCS/1316/10
`
`Conclusion
`The authors concluded that the oral administration of Transcutol within the fertility and
`general reproductive performance study in female Sprague-Dawley rats, showed that all
`doses levels, up to 2000 mg/kg bw/day, were well tolerated, although minor effects on
`clinical condition and body weight were observed at the higher dose levels (mainly in
`males). There were no effects of the test article or gonadal function, fertility and
`reproductive performance in any group. Finally, the no observed adverse effects level
`(NOAEL) for fertility and general reproductive performance was 2000 mg/kg bw, while the
`NOAEL for systemic toxicity was 1000 mg/kg bw in
`male and female Sprague-Dawley rats under the condition of this study.
`
`N Ref.: 23
`
`Dermal route
`
`Rats
`
`Guideline:
`Species/strain:
`Group size:
`Test substance:
`Batch:
`Purity:
`Dose levels:
`Route:
`Exposures:
`GLP:
`
`/
`Sprague-Dawley rats
`13 rats, control 17 rats
`DEGEE
`Lot 792796
`/
`0.35 ml x 4 per day from GD 7 - 16
`Skin
`10 days
`/
`
`DEGEE was applied to the skin (unoccluded) of 13 pregnant SD rats to investigate its
`potential for developmental toxicity. Four doses each 2.5 hours apart of 350 mg DEGEE
`(total daily dose of 1400 mg, 5600 mg/kg bw/day) were applied daily to shaved
`interscapular skin of rats on GD 7 - 16 (GD0 = sperm positive). Extragestational weight
`gain in the DEGEE rats was significantly less than in the water controls. Thus, DEGEE
`caused a slight maternal toxicity. No embryotoxic, foetotoxic, or teratogenic effects were,
`however, detected with DEGEE treatment at the concentration of approximately 5600
`mg/kg bw/day.
`
`Ref. : 45
`
`Comment
`No clear conclusion can be drawn from the findings of this study since DEGEE was applied to
`the skin without occlusion, which would potentially enable evaporative loss from the site of
`application.
`
`Inhalation
`
`Rats
`Sprague-Dawley rats, a group of 20 pregnant female rats were exposed to 0, 102 ppm
`DEGEE for 7 h/day from day 7 to day 15 of gestation. The animals were killed for necropsy
`at day 20. No selective developmental toxicity was seen under the treatment conditions. It
`was concluded that DEGEE was not a developmental toxicant.
`
`Ref. : 46
`
`General conclusion on reproductive toxicity
`DEGEE has low toxicity on reproductive performance and development. Evidence of embryo-
`foetal toxicity was restricted to minor skeletal findings which principally included an increase
`
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`
`

`

`Opinion on diethylene glycol monoethyl ether (DEGEE)
`
`SCCS/1316/10
`
`in the incidence of reduced ossification of cranial bones. These minor skeleton findings were
`not considered to be indicative of a teratogenic potential and was not considered an adverse
`effect on the developing foetuses. In a rat study the dose of 1000 mg/kg bw/day was
`considered a NOAEL for maternal and embryo-foetal toxicity.
`
`[3.3.9.
`
`Toxicokinetics
`
`[3.3.9.
`
`1. In vitro metabolism
`
`I
`
`I
`
`Guideline:
`Test system:
`Test substance:
`
`Batch:
`
`Concentrations:
`
`Analysis:
`GLP:
`Date:
`
`/
`Hepatocytes from rats and human
`Non-labelled: Transcutol HP (DEGEE), ethylene glycol mono ethyl
`ether (EGEE)
`Labelled: [14C]-Diethylene glycol monoethyl ether ([14C]-DEGEE),
`[14C]-Ethylene glycol monoethyl ether ([14C]-EGEE)
`Non-labelled: DEGEE: 0025005, EGEE: 049H1248
`Labelled: [14C]-DEGEE: 209-201-053 (radiochemical purity: 100%),
`[14C]-EGEE: 209-209-053 (radiochemical purity: 97%, both
`supplied by Moravek Biochemicals Inc., USA)
`DEGEE: 0, 15, 150, 1500 pM
`EGEE: 150 pM
`Reverse phase HPLC
`/
`June 2001
`
`The objective of this study was to determine the in vitro metabolism profile of diethylene
`glycol monoethyl ether (DEGEE = Transcutol HP) and ethylene glycol mono ethyl ether
`(EGEE) formed by rat and human hepatocytes. The rate and extent of formation of major
`metabolites was used to make predictions concerning the metabolism of DEGEE and EGEE
`by rats compared to humans.
`
`Rat hepatocytes were isolated from 2 different rat livers, R1 and R2. Human hepatocytes
`were also isolated from two different human liver specimens, H1 and H2. Hepatocyte
`suspensions were incubated with 15, 150, and 1500 pM [14C]-DEGEE or 150 pM [14C]-
`EGEE. Incubation medium aliquots were removed at 0, 1, and 4 h after the addition of
`[14C]-DEGEE or [~4C]-EGEE. Control incubations without hepatocytes were included with the
`first experiment using 1500 pM [14C]-DEGEE or 150 pM [14C]-EGEE. Incubation medium
`aliquots were removed from the controls at 0 and 4 h. Samples from the ~4C-DEGEE
`incubations were analyzed for DEGEE, EGEE, and ethoxyacetic acid (EAA), and samples
`from the [~4C]-EGEE incubation were analyzed for EGEE and EAA. HPLC separation with
`detection by an in-line radiochemical detector was used for sample analysis. The hepatocyte
`preparations used in this study exhibited 7-ethoxycoumarin O-deethylation activity, a
`cytochrome P450 associated activity.
`
`Results
`Total ECOD activity for the rat hepatocytes was 17630 and 21549 pmol 7-
`hydroxycoumarin/mg protein/hr, for R1 and R2, respectively. The total ECOD activity of the
`human hepatocyte preparations was 5384 and 5528 7-hydroxycoumarin/mg protein/h, for
`H1 and H2, respectively. At the end of the 4 h incubation period, over 88% of the
`radioactivity was associated with DEGEE in rat hepatocyte incubations with 150 and 1500
`pM [14C]-DEGEE and 70% of the radioactivity was [14C]-DEGEE in cells incubated with the
`lowest concentration (15 pM [~4C]-DEGEE). The rest of the radioactivity was associated with
`several peaks that were not identified. Rat hepatocyte incubations with [~4C]-EGEE (150
`pM) for 4 h contained 9.65% of the radioactivity as Unknown 1 (ethylene glycol; EG),
`52.18% as EAA, and 38.18% of the radioactivity as EGEE. EGEE was metabolized by rat
`hepatocytes in the current study similarly to previous studies. Approximately 98 to 99% of
`the radioactivity remained as [14C]-DEGEE in the H1 and H2 hepatocyte preparations. When
`
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`
`

`

`Opinion on diethylene glycol monoethyl ether (DEGEE)
`
`SCCS/1316/10
`
`H1 hepatocytes were incubated for 4 h with 150 pM [14C]-EGEE, 25.37% of the radioactivity
`identified as Unknown 1 (EG), 69.50% was identified as EAA, and 5.14% was EGEE. H2
`hepatocytes were less active in the metabolism of EGEE than the H1 preparation and the
`metabolite profile included 7.66% of the radioactivity as EG, 17.57% as EAA, and 74.77%
`as EGEE. The results obtained with H2 were comparable to those reported in an earlier
`study.
`
`Conclusion
`The authors concluded that EGEE was readily metabolized by both rat and human
`hepatocytes to ethoxy acetic acid (EAA) and ethylene glycol (EG), and the rat liver cells
`metabolized EGEE at a higher rate than human liver cells, in agreement with published in
`vitro metabolism data. In contrast, DEGEE was slowly metabolized by rat hepatocytes to
`several different unidentified metabolite peaks that accounted for approximately 1-17% of
`the total radioactivity. Human hepatocytes did not metabolize DEGEE significantly.
`N Ref.: 21
`
`[3.3.9.
`
`2. In vivo toxicokinetics or metabolism
`
`]
`
`An anecdotal report of rabbits treated orally or by s.c. injection indicated degradation of
`DEGEE and elimination in the urine as glucuronic conjugates.
`
`Ref. : 47
`
`DEGEE given orally to an adult human at a dose of about 20 mg/kg bw resulted in formation
`of 2-(2-ethoxyethoxy)acetic acid as a major (68% of the dose) metabolite in the urine.
`Ref. : 48
`
`Guideline:
`Species/strain:
`Group size:
`
`Test substance:
`
`Batch:
`
`Comparable to OECD 417
`Rat/Sprague-Dawley and BDIX
`Sprague-Dawley rats:
`3 males and 3 females/time point (blood and plasma kinetics)
`3 males and 3 females/time point (balance of excretion)
`3 males and 3 females/time point (tissue distribution, oral route)
`BDIX Rats:
`3 males and 3 females/time point (blood and plasma kinetics)
`3 males and 3 females/time point (tissue distribution, oral route)
`Non-labelled: Transcutol® HP (DEGEE)
`Labelled: [14C]-Diethylene glycol monoethyl ether ([14C]-DEGEE)
`Non-labelled: DEGEE: 0025005
`Labelled: [14C]-DEGEE: 104-272-053 (radiochemical purity:
`100%, supplied by Moravek Biochemicals Inc., USA)
`Oral and intravenous: 20 mg/kg bw (50 pCi/kg bw)
`Dose level:
`Single application
`Exposure:
`Oral (gavage) and intravenous
`Route:
`Oral: 5 ml/kg bw, intravenous: 2 ml/kg bw
`Application volume:
`Oral: water for injection
`Vehicle:
`Physiological saline solution (0.9% NaCI)
`Intravenous:
`Sampling time-points: Sprague-Dawley rats: 0.25 up to 168 hours after oral or
`intravenous application
`BDIX rats: 0.25 up to 6 hours after oral or intravenous application
`In compliance
`March 2002
`
`GLP:
`Date:
`
`The absorption, distribution and excretion of Transcutol® HP was investigated comparably
`in male and female Sprague-Dawley or BDIX rats after a single oral (gavage) administration
`or intravenous injection at a dose level of 20 mg [14C]-DEGEE/kg bw each.
`
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`
`

`

`Opinion on diethylene glycol monoethyl ether (DEGEE)
`
`SCCS/1316/10
`
`Results and conclusion
`After administration of 20 mg/kg of [14C]-Diethylene glycol monoethyl ether in male and
`female Sprague Dawley rats, the radioactivity was rapidly excreted in urine, irrespectively
`on sex and route of administration (85 % to 90 % within 24 hours post dose).
`
`After intravenous injection, the maximum plasma concentration of the radioactivity was
`observed 0.25 hours post dose and the plasma concentrations corresponded to about 32-35
`mg eq/kg. The maximum plasma concentration of the radioactivity after oral administration
`was observed 0.25 - 0.50 hours post dose and the maximum concentrations corresponded
`to about 23-27 mg eq/kg. The plasma half-life corresponded to 37 to 84 hours and
`measurable concentrations were observed in almost of the tissues 168 hours post dose. The
`absolute bioavailability of the radioactivity is very high (79 -- 95 %). The tissue distribution
`of the radioactivity was characterised by high concentrations observed in pituitary, thyroid,
`adrenals and bone marrow with regards to the concentrations observed in blood / plasma
`(100 to 1000 times less) at the same sampling time. The radioactivity measured in tissues
`was significantly decreased at 48 hours. No biologically relevant difference has been
`observed with BDIX rats.
`
`N Ref.: 25
`
`Guideline:
`Species/strain :
`Group size:
`
`Test substance:
`
`Comparable to OECD 417
`Rat/Sprague-Dawley
`4 male rats in total (2 for plasma samples, 0.75 h; 2 for plasma (24
`h), urine and faeces samples
`Non-labelled: Transcutol HP (DEGEE)
`Labelled: [14C]-Diethylene glycol monoethyl ether ([14C]-DEGEE)
`Non-labelled: DEGEE: 0025005
`Labelled: [14C]-DEGEE: 104-272-053 (radiochemical purity: 100%
`supplied by Moravek Biochemicals Inc., USA)
`1000 mg/kg bw including 1.85 MBq (50 pCi) of [14C]-DEGEE
`Dose level:
`Single application
`Exposure:
`Oral (gavage)
`Route:
`5 ml/kg bw
`Application volume:
`Water for injection
`Vehicle:
`Sampling time-points: Blood: 0.75 and 24 h
`Urine: pre-dose, 0 - 8 h, 8 - 24 h
`Feces: pre-dose, 0 - 24 h
`Liquid scintillation counting
`/
`September 2003
`
`Batch:
`
`Analysis:
`GLP:
`Data:
`
`The metabolic fate and excretion of Transcutol HP was investigated in 4 male Sprague-
`Dawley rats after a single oral administration of 1000 mg [14C]-DEGEE /kg bw by gavage.
`Blood samples were collected at 0.75 h and at 24 h. Urine samples were collected before
`administration and between 0 - 8 hours and 8 - 24 hours and faeces were sampled prior to
`treatment and during 0 - 24 hours..
`
`Results and conclusion
`After administration of [~4C]-DEGEE, 90% of the administrated radioactivity was excreted in
`the urine within the first 24 hours. [~4C]-DEGEE was intensively metabolised, only 3% of the
`urinary excreted radioactivity correspond to unchanged compound. The two major urinary
`metabolites were identified as Ethoxyethoxyacetic acid and Diethylene glycol, which
`represented 83% and 5.4% of the excreted urinary radioactivity, respectively. In plasma,
`only Ethoxyethoxyacetic acid and unchanged [~4C]-DEGEE were detected, which was
`consistent with urinary results.
`
`N Ref.: 27
`
`39
`
`39 of 53
`
`

`

`Opinion on diethylene glycol monoethyl ether (DEGEE)
`
`SCCS/1316/10
`
`Comparable to OECD 417
`Guideline:
`Rat/Sprague-Dawley
`Species/strain:
`30 male rats in total
`Group size:
`Test substance:
`Transcutol HP (DEGEE)
`D 4089
`Batch:
`20, 100 mg/kg bw
`Dose levels:
`Single application
`Exposure:
`Oral (gavage)
`Route:
`5 ml/kg bw
`Application volume:
`Water for injection
`Vehicle:
`Sampling time-points: Blood: 0.5, 1, 3, 6, 24 h
`Urine: pre-dose, 0 - 8 h, 8 - 24 h
`LC/MS/MS.
`In compliance
`December 2003
`
`Analysis:
`GLP:
`Date:
`
`The metabolic fate at a doses of Transcutol® HP was investigated in 30 male Sprague-
`Dawley rats after a single oral administration of 20 mg/kg bw (15 males) or 100 mg/kg bw
`(15 males) by gavage. Blood samples were collected at 0.5 1, 3, 6 and at 24 h. Urinary
`samples were collected before administration and between 0 - 8 h and 8 - 24 h. The
`validated analytical method applied consisted in LC/MS/MS analysis after protein
`precipitation.
`Results and conclusion
`The results obtained confirmed the presence of unchanged DEGEE and ethoxyethoxy acetic
`acid as major metabolites for the 0.5 hour plasma sampling times. However after 3 hours
`the difference observed between the total radioactivity (N Ref.: 27) and the specific analysis
`show a difference probably due to others metabolites. In urine, the amount recovered by
`the analysis of ethoxyethoxyacetic acid was low: about 17% of the administrated dose for
`the rats treated with 20 mg/kg of DEGEE and about 40% of the administrated dose for the
`rats treated with 100 mg/kg of DEGEE. As no satisfactory result was observed for the
`recovery of ethoxyethoxy acetic acid in plasma after precipitation of protein, it was not
`possible to conclude about the presence or not of this metabolite. In urine, a discrepancy
`was observed between these results and radioactivity study mentioned above (N Ref.: 27)
`N Ref.: 28
`
`Conclusion on toxicokinetics and metabolism by the applicant
`An in vitro metabolism study to determine the metabolism profile of Transcutol showed that
`DEGEE was slowly metabolized by rat hepatocytes to several different unidentified
`metabolite peaks that accounted for approximately 1-17% of the total radioactivity. Human
`hepatocytes did not metabolize DEGEE significantly.
`
`In vivo, the absorption, distribution and excretion of Transcutol® was investigated
`comparably in two strains of rats after a single oral or intravenous dose of 20 mg [14C]-
`DEGEE /kg bw each. It was demonstrated that the radioactivity was rapidly excreted in
`urine, irrespectively of sex and route of administration. After intravenous injection, the
`maximum plasma concentration of the radioactivity was observed 0.25 hours post dose,
`while after oral administration it was observed at 0.25 - 0.50 hours post dose. The plasma
`half-life corresponded to 37 to 84 hours and measurable concentrations were observed in
`almost of the tissues 168 hours post dose. The absolute bioavailability of the radioactivity is
`very high (79 -- 95%). The tissue distribution of the radioactivity was characterised by high
`concentrations observed in pituitary, thyroid, adrenals and bone marrow with regards to the
`concentrations observed in blood / plasma (100 to 1000 times less) at the same sampling
`time. The radioactivity measured in tissues was significantly decreased at 48 hours. No
`biologically relevant differences were observed within both strains of rats. In studies on the
`metabolic fate and excretion of Transcutol it could be shown that after a single oral
`administration, 90% of the administered radioactivity was excreted in the urine within the
`first 24 hours and [14C]-DEGEE was intensively metabolised as only 3% of the urinary
`
`4O
`
`40 of 53
`
`

`

`Opinion on diethylene glycol monoethyl ether (DEGEE)
`
`SCCS/1316/10
`
`excreted radioactivity correspond to unchanged compound. The two major urinary
`metabolites were identified as ethoxyethoxyacetic acid and diethylene glycol, which
`represented 83% and 5.4% of the excreted urinary radioactivity, res