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The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
accepted calculation method
Justification for type of information:
The basis for this read-across approach is that the target substance is expected to undergo transformation into terephthalic acid (202-830-0; 100-21-0) and ethane-1,2-diol (203-473-3; 107-21-1). The toxicity of the metabolites will accurately predict the toxicity of the bis(2-hydroxyethyl)terephthalate (BHET; 959-26-2; 213-497-6). Refer to the JUSTIFICATION FOR READ-ACROSS OF TOXICOLOGICAL INFORMATION in Section 13 of this dossier for further details.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
not specified
Remarks:
The published information does not report if CLP compliance was followed or not
Limit test:
no
Species:
rat
Sex:
male/female
Route of administration:
oral: feed
Duration of treatment / exposure:
two generations, including 10-week pre-mating periods
Dose / conc.:
20 000 ppm
Remarks:
2011-5072 mg/kg bw/day depending on body weight and gestational status
Dose / conc.:
5 000 ppm
Remarks:
492-1218 mg/kg bw/day depending on body weight and gestational status
Dose / conc.:
1 000 ppm
Remarks:
98-261 mg/kg bw/day depending on body weight and gestational status
No. of animals per sex per dose:
26 rats per dose and sex
Body weight and weight changes:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
High-dose parental males of both generations had reduced mean body weights. Also at the high dose, all treated parental male groups had decreased absolute and relative kidney weights, and males and females of both generations had increased relative liver weights. Urinary bladder effects at the high dose in parental animals of both generations, occurring at higher incidences in the F1-generation, included hematuria, masses, firm deposits, thickening of the bladder wall and/or prominent blood vessels. Microscopic findings were limited to two parental males of each generation at the high dose with renal papillary necrosis, graded as minimal to slight, and occurring in animals that also had abnormal macroscopic findings.
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
There were no treatment-related effects on smear cycle and pattern, pre-coital interval, gestation length, proportion of successful matings, pups born live, pup survival, sex ration, sperm motility, sperm number or sperm morphology.
Dose descriptor:
NOAEL
Effect level:
20 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of significant findings at the highest concentration tested
Remarks on result:
other: 2011-5072 mg/kg bw/day
Critical effects observed:
no
Body weight and weight changes:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Details on results:
High-dose parental males of both generations had reduced mean body weights. Also at the high dose, all treated parental male groups had decreased absolute and relative kidney weights, and males and females of both generations had increased relative liver weights. Urinary bladder effects at the high dose in parental animals of both generations, occurring at higher incidences in the F1-generation, included hematuria, masses, firm deposits, thickening of the bladder wall and/or prominent blood vessels. Microscopic findings were limited to two parental males of each generation at the high dose with renal papillary necrosis, graded as minimal to slight, and occurring in animals that also had abnormal macroscopic findings.
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
There were no treatment-related effects on smear cycle and pattern, pre-coital interval, gestation length, proportion of successful matings, pups born live, pup survival, sex ration, sperm motility, sperm number or sperm morphology.
Dose descriptor:
NOAEL
Effect level:
20 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of significant findings at the highest concentration tested
Remarks on result:
other: 2011-5072 mg/kg bw/day
Critical effects observed:
no
Anogenital distance (AGD):
effects observed, treatment-related
Description (incidence and severity):
A 1.6-day delay in F1 vaginal opening and reduced anogenital distance in high-dose females, and respective 0.8 and 1..6-days in preputial separation in F1 males at the mid and high doses were all associated with reduced body weight.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
20 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of significant findings at the highest concentration tested
Remarks on result:
other: 2011-5072 mg/kg bw/day
Critical effects observed:
no
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A reduction in high-dose F2 pup body weight from PND 1-15 was attributed to a larger litter size. Pub body weights were reduced from PND 15 onward, corresponding to the age at which pups began to ingest TPA.
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
20 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of significant findings at the highest concentration tested
Remarks on result:
other: 2011-5072 mg/kg bw/day
Critical effects observed:
no
Reproductive effects observed:
no
Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
multi-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Guideline:
other: "Fertility Assessment by Continuous Breeding (FACB)" assay.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Ethylene glycol obtained from Ashland Chemical Company
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age: CD-1 mice were purchased at 6 weeks of age
- Housing: Two animals were housed per cage in polycarbonate shoebox type cages with stainless steel wire bar lids. Cages were rotated (relative placement) at least once a week.
- Diet: Purina certified rodent chow animal diet, ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C
- Humidity (%): 20 to 70%
- Photoperiod (hrs dark / hrs light): 14-hour light cycle
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

WATER PREPARATION
- Rate of preparation of water: fresh preparation once every 2 weeks
- Storage temperature of water: room temperature, under yellow light

VEHICLE
- distilled water
Details on mating procedure:
Animals were randomly paired within each treatment group. Treatment was initiated at 11 weeks of age and was continued for 18 weeks (1 week of premating, 14 weeks of cohabitation, and 3 weeks thereafter). During this period, the following parameters were evaluated: body weight, number of litters produced, number of live pups per litter, minimum number of dead pups per litter, group body weight of live pups (males and females recorded separately), percent of infertile pairs and abnormal pups and a brief description of the deformity, if any.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Aliquots of various dosage formulations were sent to Midwest Research Institute (MRI), Kansas City, MO, for analysis at week 1 of Task 1 and weeks 1, 5, 11, and 17 of Task 2.
Duration of treatment / exposure:
Task 1: 14 consecutive days
Task 2: 18 weeks (1 week prior to cohabitation, 14 weeks of cohabitation, and 3 weeks thereafter)
Task 4: 10 weeks
Frequency of treatment:
dosed/undosed water ad libitum, daily
Details on study schedule:
Fertility Assessment by Continuous Breeding (FACB). It consists of four related tasks, not all of which are necessarily performed for a given compound. These tasks include Task 1 - dose finding; Task 2 - cohabitation phase; Task 3 - identification of the affected sex and Task 4 - offspring assessment. This test protocol is designed to provide an alternative to multigeneration studies which produce similar comprehensive reproductive data but in a considerably shorter time and at a lower cost.
Task 1 is conducted to determine suitable doses for the continuous breeding phase. The test chemical is administered for 14 consecutive days and them maximum tolerated dose (MTD) is computed. Task 2 is designed to determine the effect of the MTD and two lower dose levels on fertility and reproduction. In this phase, treatment is continued for 18 weeks (1 week prior to cohabitation, 14 weeks of cohabitation, and 3 weeks thereafter). If the fertility is significantly affected, Task 3 is conducted to determine whether the male, female or both sexes are affected. If the overall response in Task 2 is negative, Task 4 is conducted. It is designed to evaluate reproductive performance in the offspring from the final and generally the fifth litter of the control and high dose groups. If the fertility in the first generation offspring is significantly affected, a Task 3 may be performed using these animals to determine the affected sex. At the conclusion of either Task 3 or 4, experimental animals may be necropsied.
During necropsy the liver, brain, pituitary, female reproductive tract (ovaries, oviduct, uterus, and vagina), testes, epididymis, prostate, and seminal vesicles with coagulating glands are weighed and fixed for histopathology. Based on the overall response during Task 3 or 4, vaginal smears are prepared to check the effect on oestrous cycle and sperm studied in detail to evaluate the effect on sperm density, sperm motility, and sperm head morphology.
Dose / conc.:
0 other: % in water (w/v)
Remarks:
dose range-finding, main study
Dose / conc.:
0.25 other: % in water (w/v)
Remarks:
dose range-finding, main study; corresponding to a dose of approx. 410 mg/kg bw
Dose / conc.:
0.5 other: % in water (w/v)
Remarks:
dose range-finding, main study; corresponding to a dose of approx. 840 mg/kg bw
Dose / conc.:
1 other: % in water (w/v)
Remarks:
dose range-finding, main study; corresponding to a dose of approx. 1640 mg/kg bw
Dose / conc.:
2.5 other: % in water (w/v)
Remarks:
dose range-finding
Dose / conc.:
5 other: % in water (w/v)
Remarks:
dose range-finding
No. of animals per sex per dose:
Task 1: 8/8 male/female
Task 2: 20/20 male/female treatment; 40/40 male/female vehicle control
Task 4: 20/20 male/female treatment and control
Control animals:
yes, concurrent vehicle
Details on study design:
Based on the information available in the literature, the following dose levels were selected: 0, 0.25, 0.5, 1.0, 2.5, and 5.0% administered in drinking water.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- time schedule: twice daily

BODY WEIGHT: Yes
- time schedule for examinations: weekly

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- time schedule for examinations: weekly
- estimate of substance intake by multiplying water consumption by chemical concentration divided by the sum weight of the pair
Litter observations:
The following parameters were examined in [F1 / F2 / F3] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS: yes
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals, after litter was weaned

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Postmortem examinations (offspring):
SACRIFICE
- the F1 offspring was sacrificed after mating trial was completed
- these animals were subjected to postmortem examinations as follows:
body weight,
organ weight: liver, brain, pituitary
reproductive tract: females: cranial half of the vagina, cervix, uterus, and ovaries; males: testes, epididymis, seminal vesicles and prostate
Statistics:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
Dose range-finding study: severe respiratory distress; at 2.5 and 5 % treatment groups
Main study: no clinical signs in all treatment groups
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Dose range-finding study: treatment related deaths in the 2.5 (2/8 males) and 5 % (3/8 males; 1/8 females) groups
Main study: 2 females died in 0.5% treatment group; 1 males and 2 females died in control group
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Dose range-finding study: dilated renal tubules, tubular nephrosis related to oxalate crystal accumulation at 2.5 and 5 % treatment groups
Main study: moderate number of oxalate crystals in renal tubuli of one animal (0.5% treatment group)
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Main study: statistically significant decrease in number of litters per fertile pair, mean number of live pups, and mean live pup weight in 1 % treatment group. As well as significant reduced average total litter size; average number of male pups per litter; and both male and female pup weights.
Key result
Dose descriptor:
NOEL
Effect level:
0.5 other: percent in drinking water (w/v)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: equivalent to approx. 840 mg/kg bw/d
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
1 % treatment group: facial defects, pattern of skeletal defects, affected skull, cleft lip, abnormally shaped or missing sternbrae, fused ribs and abnormally shaped vertebrae, twisting of spine.
Neither the 0.25 nor 0.5% dose groups were significantly affected.
Histopathological findings:
no effects observed
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Exposure to ethylene glycol resulted in a small but significant decrease in the number of litters per breeding pair, in the number of live pups per pair and in the live pup weight. A significant number of pups in the 1.0% dose group were born with distinct facial deformities. In the retained litters at this dose, the facial deformities were more obvious with age. These malformed animals also exhibited fused ribs and shortened nasal, parietal, and/or frontal bones of the skull. When pups from the high dose group were raised to adulthood (with continued exposure to ethylene glycol) and mated, they exhibited decreased mating and fertility indices relative to controls handled in the same manner, but there were no effects on litter size, pup weight or sex ratio.
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
0.5 other: percent in drinking water (w/v)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
Remarks on result:
other: equivalent to approx. 840 mg/kg bw/d
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 other: percent in drinking water (w/v); equivalent to approx. 1640 mg/kg bw/d
System:
musculoskeletal system
Organ:
other: facial deformities
Treatment related:
yes
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
gross malformations; eight animals with distinct facial deformities in the 1% EG group were not necropsied and later utilized for skeletal examinations.

Skeletal Examination: A series of skeletal deformities were apparent in offspring delivered by the high dose (1% EG) group. Briefly, (a) frontal and nasal passages were significantly shortened and sometimes curved; (b) one or more pairs of ribs were fused; (c) one or more ribs were branched; (d) one or more centra were abnormal; and (e) parietals were smaller than the normal width. None of these abnormalities were noted in the untreated CD-1 mice.
Histopathological findings:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
facial and skeletal defects
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Fertility was 80% for the control group compared to 61% to those animals receiving 1% EG, but the difference was not statistically significant. The number of live pups per litter and the live pup weight were lower in the EG-treated group, as they were in task 2, but differences were not statistically significant in either case. Although no clinical signs of EG toxicity were reported in the study, unusual facial features were noticed in some of the offspring of the treated mice but not in the controls. The affected offspring generally had a shorter snout with wide-set eyes compared to the control CD- 1 mice. The examination revealed a pattern of skeletal defects in the treated mice affecting the skull, sternebrae, ribs, and vertebrae in both males and females. The defects included shortened frontal, nasal, and parietal bones; one or more pairs of fused ribs; abnormally shaped or missing sternebrae; abnormally shaped vertebrae; and twisting of the spine. The untreated mice showed no such defects. It was apparent from low-magnification examination of the histological sections that the size and shape of the bones in treated mice differed from the controls. Bones from treated mice were smaller and had altered shapes. However, histologic alterations were not evident when bones from treated mice were examined by light microscopy. Formation of lamellar bone, numbers and activity of osteoblasts and osteoclasts, and cartilage structure were identical in treated and control tissues. Tooth structure was normal in all treated mice. No alterations were seen in nasal turbinates, skeletal muscle, various salivary glands, eyes, or in those portions of the olfactory lobes of the brain which were frequently present in the sections. Deposition of calcium oxalate crystals were not found during careful examination of the microvasculature of bony or adjacent soft tissues. No other treatment-related effects were noted in the EG treated mice.
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
0.5 other: percent in drinking water (w/v)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: skeletal deformities
Remarks on result:
other: equivalent to approx. 840 mg/kg bw/d
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 other: percent in drinking water (w/v); equivalent to approx. 1640 mg/kg bw/d
System:
other: facial deformities
Organ:
other: gross malformations
Treatment related:
yes
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 other: percent in drinking water (w/v); equivalent to approx. 1640 mg/kg bw/d
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
not specified

TASK 1:


EG administered in drinking water for 2 weeks at 0.25, 0.5, 1.0, and 2.5% (w/v) dose levels (equivalent to approx. 410, 840, and 1640 mg/kg bw/d) had no significant effect on body weights of both male and female mice. Mice exposed to 5% EG lost weight. The group mean body weights for control male mice at the beginning and the end of Task 1 were 34.4 and 34.6 g, respectively. Corresponding values for the 5% EG group were 34.9 and 31.4 g.


Certain animals in the 2.5 and 5% EG groups were sluggish and appeared to have respiratory problems within one week of treatment. Hair coat changed from normal to rough. During the later part of the first week or the early part of the second week, a number of animals in these two dose groups were lethargic and hunched. A significant number of these mice died. No such symptoms were noted in the control group or in animals in the 0.25, 0.5, and 1% EG groups.


Two of the eight male mice exposed to 2.5% EG died during Task 1. None of the female mice died in this dose group. One female and three male mice died in the 5% EG group. These animals exhibited severe respiratory distress prior to death. Pathologic examination showed distinct lesions in the kidneys and dilated tubule with moderate numbers of oxalate crystals in the lumina. The lungs were moderately congested. The cause of death was diagnosed as tubular nephrosis due to oxalate crystals.


Daily consumption of distilled water by the control mice or dosed water by the animals in the different treatment groups was essentially the same except for the male mice in the 5% EG group (p<0 .001).


 


TASK 2:


EG administered in drinking water at 0.25, 0.5, and 1% (w/v) dose level (equivalent to approx. 410, 840, and 1640 mg/kg bw/d) had no apparent effect on male or female body weights. The group mean body weights of male mice in the control and different treatment groups varied between 36.8 to 37.3 g at the beginning of Task 2. After 18 weeks of treatment, the group mean body weights for animals exposed to 0, 0.25, 0.5, and 1% EG were 40.3, 40.6, 40.2, and 40.3 g, respectively. Body weights for female mice varied considerably during Task 2 and the weight depended on the gestation phase.


Only five (5) mice died during 18 weeks of Task 2; one male and two females in the control group and two females in the 0.5% EG group. The cause of death for the control male mouse was mild myodegeneration of the heart and pulmonary congestion. One of the two control female mice revealed gravid uterus. Only one of the two female mice in the 0.5% EG group was necropsied. Pathologic examination revealed dilated tubule with moderate numbers of oxalate crystals in the lumina. The cause of death was suspected due to tubular nephrosis due to oxalate crystals.


The presence of ethylene glycol at a concentration of 1% or less did not significantly interfere with daily water consumption during Task 2 for both male and female mice.


Reproductive Performance and Fertility
Ethylene glycol administered continuously in drinking water at 0.25, 0 .5, and 1% dose levels had no effect on fertility in CD-1 mice. The fertility index for control and all three dose levels was 100%, i.e. every experimental pair delivered at least one litter. EG treatment at 1% dose level (approx. 1640 mg/kg bw/d) significantly reduced (p <0.05): (1) the average number of litters, 4.45 vs. 4.89 in the control group; (2) the average total litter size; (3) the average number of male pups per litter; and (4) both male and female pup weights. This is regarded as a sequel of developmental toxicity. No significant (p >0.05) differences existed between the 1% EG and the control group with respect to: (1 ) the proportion of live pups; and (2) sex ratio. EG treatment at 0.25 and 0.5% dose levels did not significantly (p >0.05) affect reproductive performance of CD-1 mice with respect to any of the above parameters. Interestingly, a significant number of pups delivered by breeding pairs in the 1% EG group showed distinct facial deformities. Six pups representing three different litters revealed a possible cleft lip/palate.


Gestation Period: EG exposure had no apparent effect on the gestation period. Cumulative days to 1st, 2nd, 3rd, 4th, and 5th litters were 28, 51, 69, 91, and 112 (days of the study), respectively. Corresponding values for the 1% EG group were 30, 55, 75, 96, and 111, respectively. It must be added that these values (days of the study) include 7 days of the premating period.


 


TASK 4:


The average pup weight at weaning ranged between 14.0 to 18.4 g. Offspring from both the control and 1% EG (approx. 1640 mg/kg bw/d) groups gained weight at essentially the same rate.


At least four male and four female mice among the offspring selected for Task 4 matings showed distinct facial deformities. These mice were later used for detailed skeletal examinations.


There was no mortality among the first generation offspring in the control group. Three offspring died in the 1% EG group; one male and two females. The cause of death was not treatment related.


Water consumption by first generation offspring from the control and 1% EG group was monitored on a weekly basis. There were no apparent difference in the average intake of distilled/dosed water by the control/1% EG group offspring.


 


Reproductive Performance and Fertility:
Twenty (20) pairs of first generation offspring were randomly selected from the control as well as 1% EG groups to assess their reproductive performance. The breeding pairs were mated until a copulatory plug was detected or for a maximum of seven days. The percent of plug positive/No. cohabited (mating index) for the control and treated pups was 90 and 74, respectively. Fertility was also affected by EG treatment. The percent of No. fertile/No. cohabited (fertility index) in the control and EG treated pups was 80 and 61, respectively. Other reproductive parameters were not significantly different (p>0.05) from the control values, i.e. the number of live pups per litter (males, females, or combined), proportion of pups born alive, and sex ratio.


 


Gross Necropsy:


No significant differences existed in terms of body weight, reproductive tract, and pituitary weight (p>0.05). Average brain weight of treated animals was lower than the control value (p<0.05). Organ weights were then adjusted for body weight by analysis of covariance. Male offspring body and organ weights for control and the treated animals were essentially the same except for the brain and right cauda. The brain weight was decreased by approximately 8 percent of the control value and right cauda weight by 14 percent. Male organ weights were also adjusted for body weight by analysis of covariance. It must be added here that eight animals with distinct facial deformities in the 1% EG group were not necropsied and later utilized for skeletal examinations.


 


Skeletal Examination:


A series of skeletal deformities were apparent in offspring delivered by the high dose (1% EG) group. Briefly, (a) frontal and nasal passages were significantly shortened and sometimes curved; (b) one or more pairs of ribs were fused; (c) one or more ribs were branched; (d) one or more centra were abnormal; and (e) parietals were smaller than the normal width. None of these abnormalities were noted in the untreated CD-1 mice.

Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
three-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Guideline:
other: Assessing the effect of EG on fertility and general reproductive performance in male and female rats.
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
Ethylene glycol was received from Union Carbide Corporation
Purity: 99.82% monoethylene glycol + 0.18% diethylene glycol
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: young adult nulliparous Fischer 344 rats
- Housing: two per cage in stainless-steel wire cages; during mating, each male was housed with 2 females; after mating and during lactation, the females were housed individually in plastic showbox cages with hardwood chips for nesting.
- Diet: Purina Formulab, ad libitum
- Water: city water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Photoperiod (hrs dark / hrs light): 12 /12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Administration of EG to the F0 rats of both sexes started at approximately 7 weeks of age.
Fresh diet was prepared every 2 weeks with the percentage of test item (EG) adjusted, based on the group mean body weight and food consumption, so as to maintain a relatively constant dosage level. However, the concentration of EG in the diet was not changed during gestation or during the first week of lactation, but was reduced two- and three-fold during the second and third weeks of lactation, respectively, to adjust for increased food consumption by the dams. This change in concentration was based on earlier unpublished results from the laboratory. Increased food consumption during lactation has since been reported in another study performed at the laboratory.
Details on mating procedure:
At approximately 100 days of age, 10 males were added to 20 females in each dosage group. The F1 and F2 rats were treated as described for the F0 animals until approximately 100 days of age, at which time the animals were cohabited. Brother and sister matings were avoided for each generation.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
3 generations
Frequency of treatment:
daily
Details on study schedule:
The date of parturition and the number of live and dead newborn were recorded for each litter. The appearance and behavior of dams and pups were observed daily. Litter size was randomly reduced to 10, if necessary, on Day 4 postpartum. Offspring were weighed as litters at 4 and 14 days and individually at 21 days postpartum, the day they were weaned. F1 rats were randomly selected within each dosage group for the next mating. Each litter was represented except for those conceived very late in the mating period.
Dose / conc.:
40 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
30
Control animals:
yes, concurrent no treatment
Details on study design:
Two untreated diet control groups, designated 0.0A and 0.0B, were included to estimate the variation between 2 groups treated alike.
Parental animals: Observations and examinations:
Body weights and diet consumption were recorded weekly except during gestation and lactation.
Litter observations:
Offspring were weighed as litters at 4 and 14 days and individually at 21 days post partum, the day they were weaned. F1 rats were randomly selected within each dosage group for the next mating.
Postmortem examinations (parental animals):
Necropsies were performed on five males and five females randomly selected from each dosage level of the F2 parents and the F3 weanlings. Microscopic examinations were performed on sections of liver, kidneys, lung, heart, adrenals, thyroid, trachea, accessory sex glands, adipose tissue, lymph nodes, pituitary, thymus, and testes and epididymis, or uterus and ovaries.
Statistics:
Continuous data such as body weights were compared by analysis of variance validated by Bartlett's test for homogeneity of variance. Duncan's multiple range test was used to identify individual mean differences when indicated by a significant F value. Where Bartlett's test indicated heterogeneous variances, t tests for equal or unequal variances were used to delineate differences between groups. Pup weights were compared by the method of Weil (Weil, 1970). Discontinuous data such as implantations and reproductive indices were compared by a multiple sum of ranks test. Frequency data were compared by the X2 test and by Fisher's exact test. The following reproductive indices were calculated and evaluated statistically by the previously described non parametric methods: fertility index (male and female), days from first mating to parturition, gestation index (fraction of pregnancies that resulted in litters with live pups), gestation survival index (fraction of newborn pups alive at birth), 0 to 4-day survival index, 4 to 14-day survival index, 4 to 21day survival index. The last four indices are summarized in the tables as means for ease of understanding and presentation, although the nonparametric statistical methods did not include a comparison of means.
Reproductive indices:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
Throughout the study there was no effect of EG treatment on body weight gain or diet consumption, nor was there any mortality among parental rats.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no reproductive effects associated with EG treatment doses up to 1000 mg/kg bw/d via diet.
Key result
Critical effects observed:
no
Reproductive performance:
no effects observed
Description (incidence and severity):
No treatment-related effect was observed for any of the indices. Also, EG treatment did not affect neonatal body weight at days 4, 14, or 21 post partum.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related histopathologic findings in F2 parents or in F3 weanlings. Although the kidney has been shown to be the primary target organ for EG-induced toxicity, there was no increase in the incidence or severity of kidney lesions in this study. One high dose F2 animal of each sex had mild focal interstitial nephritis. However, this condition was also seen in a control male and a control female. Unilateral hydronephrosis occurred in another high-dose F2 male. In addition, mild focal tubular hyperplasia was observed in one high-dose male F3 pup but was also diagnosed in two control male pups.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: there were no treatment-related effects observed
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Reproductive Indices

     1.0 g/kg bw/d  0.2 g/kg bw/d  0.04 g/kg bw/d  0.0A  0.0B
 F0 -> F1  Fertility index (%)  100  90  100  90  90
   Male  95  90  90  75  90
   Female  100  100  100  100  100
 F1 -> F2  Fertility index (%)  100  100  90  90  90
   Male  85  95  85  90  85
   Female  100  100  100  100  94
 F2 -> F3  Fertility index (%)  100  90  100  80  80
   Male  90  75  85  80  70
   Female  100  100  100  100  100

Neonatal body weight at day 21

     1.0 g/kg bw/d  0.2 g/kg bw/d  0.04 g/kg bw/d  0.0A  0.0B
 F1 pups  males  30.6 +/- 4.5  30.9 +/- 4.9  30.7 +/- 6.4  30.6 +/- 3.6  27.9 +/- 4.3
   females  29.0 +/- 4.5  29.2 +/- 4.5  29.5 +/- 4.7  27.9 +/- 3.3  27.0 +/- 3.5
 F2 pups  males  32.8 +/- 3.5  30.9 +/- 5.8  29.3 +/- 4.7  30.0 +/- 4.0  28.8 +/- 4.3
   females  30.8 +/- 3.4  30.2 +/- 4.9  28.8 +/- 3.8  28.5 +/- 3.1  27.5 +/- 3.4
 F3 pups  males  30.2 +/- 4.0  30.9 +/- 4.0  30.9 +/- 4.0  32.0 +/- 3.9  30.2 +/- 4.6
   females  28.6 +/- 3.8  28.2 +/- 3.4  29.7 +/- 4.0  30.1 +/- 3.5  27.7 +/- 3.9
Conclusions:
In conclusion, there were no reproductive effects associated with the inclusion of as much as 1000 mg/kg bw/d of test item in the diet.

Data source

Materials and methods

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(hydroxyethyl) terephthalate
EC Number:
213-497-6
EC Name:
Bis(hydroxyethyl) terephthalate
Cas Number:
959-26-2
Molecular formula:
C12H14O6
IUPAC Name:
bis(hydroxyethyl) terephthalate
Test material form:
solid
Specific details on test material used for the study:
Bis(2-hydroxyethyl) terephthalate value is read-across from supporting terephthalic acid (202-830-0; 100-21-0) and ethane-1,2-diol (203-473-3; 107-21-1) data.

Test animals

Species:
rat

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of significant findings at the highest concentration assessed for the transformation products

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: P1 (second parental generation)

Effect levels (P1)

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of significant findings at the highest concentration assessed for the transformation products

Target system / organ toxicity (P1)

Critical effects observed:
no

Results: F1 generation

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of significant findings at the highest concentration assessed for the transformation products

Target system / organ toxicity (F1)

Critical effects observed:
no

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of significant findings at the highest concentration assessed for the transformation products

Target system / organ toxicity (F2)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Executive summary:

A two-generation reproduction study was conducted with rats fed 1,000, 5,000, or 20,000 ppm terephthalic acid (Ball, et al., 2012). The delay in vaginal opening and reduced anogenital distance at the high dose in F1 female offspring, and the delay in preputial separation at the mid and high doses in F1 male offspring were attributed to reduced body weight. There is no evidence of terephthalic acid reproductive toxicity.  No reproductive or dominant lethal effects were associated with the inclusion of as much as 1.0 g/kg bw/day of ethane-1,2-diol in the diet of Fischer 344 rats (DePass, et al., 1986). Ethane-1,2-diol administered continuously in drinking water at 0.25, 0 .5, and 1% dose levels had no effect on fertility in CD-1 mice (Lamb, et al., 1985).  Information on the source substances is considered to be directly applicable to an equivalent molar amount of the target substance; therefore, BHET is not reproductively toxic. BHET is predicted to have a NOAEL greater than 1,000 mg/kg bw/day.