Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Study

Result

Comment

DEGEE

Continuous breeding multi-generation study by drinking water (NTP, 1984) in mice

NOAEL=2200mg/kgbw/day

Equivalent to a TEGEE dose of 2920mg/kgbw/day

DEGBE

Single generation study by oral gavage on rats (Nolen, 1985)

No effects on reproductive end points at maximum tested dose of 1000mg/kgbw/day

Equivalent to a TEGEE dose of 1100mg/kgbw/day

Single generation limit dose study by dermal exposure on rats (Auletta, 1993)

No effects on reproductive end points at tested dose of 2000mg/kgbw/day

Equivalent to a TEGEE dose of 2200mg/kgbw/day

Single generation limit dose study by oral gavage on rats plus parallel group to examine effects on oestrus cycle (Sitarek, 2012)

No effects on reproductive end points at maximum tested dose of 1000mg/kgbw/day. Maternal toxicity NOAEL 500mg/kgbw/day

Equivalent to a TEGEE dose of 1100mg/kgbw/day for reproductive NOAEL

Repeat dose toxicity drinking water in rat (90 day) sperm end points (Johnson, 2005)

No effects seen at maximum tested dose of 1000mg/kgbw/day

Equivalent to a TEGEE dose of 1100mg/kgbw/day

TEGME

Repeat dose toxicity oral in rat (90 day) sperm and oestrus cycle end points (Dow, 1990)

NOAEL for Sperm effects 1200mg/kgbw/day.
LOAEL for Sperm effects 4000mg/kgbw/day.

NOAEL for Oestrus cycle end points 4000mg/kgbw/day (max dose tested)

Equivalent to a TEGEE dose of 1300 and 4340mg/kgbw/day

Sub-chronic neurotoxicity oral test with oestrus cycle end points in rat (Dow, 1990)

NOAEL 4000mg/kgbw/day (max dose tested)

Equivalent to a TEGEE dose of 4340mg/kgbw/day

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 200 mg/kg bw/day
Additional information

Data for DEGEE

A two-generational continuous breeding study investigated the effects on reproduction and fertility of 0%, 0.25%, 1.25%, and 2.5% DEGEE in drinking water (99% pure, NTP 1984; Williams 1990). (Assuming an average daily water consumption of 7 ml per 40-g mouse, drinking water concentrations of 0.25, 1.25 and 2.5% diethylene glycol monoethyl ether (DEGEE) represented average daily intakes of approximately 440, 2200 and 4400 mg DEGEE/kg/day, respectively.) Male and female CD-1 mice were continuously treated for 1 week prior to mating and for a 14 week breeding period followed by a 21 day holding period when they were separated and housed individually. The fifth or final litters from mice treated with 0% or 2.5% DEGEE were reared and weaned at Day 21 (all others were euthanized). Because reproductive effects were not observed during the 126-day exposure period, the F0 parents were not necropsied. The day of delivery of each litter, the number of litters per breeding pair, the number and percentage of fertile pairs, the number, percentage and sex of live pups per litter, and the mean body weights of live offspring were recorded. The F1 parents were euthanized and necropsied. Body, liver, brain and pituitary weights were recorded. Selected reproductive tissues from males (left testis with epididymis attached, right testis, right epididymis, prostate and seminal vesicles) and females (ovary with oviduct attached, and uterus) were weighed, fixed and embedded in paraffin, stained and evaluated by light microscopy. The sperm concentration, percentage of motile sperm, and percentage of abnormal sperm in the right cauda epididymis also were evaluated.

There were two deaths among the male F0 animals treated at high dose and small decreases in the mean body weights. The body weights of the F1 offspring exposed to 2.5% level were slightly depressed at birth, at weaning and at 74 +/-10 days. The only significant changes observed in high dose F1 animals at necropsy were decreases in the absolute and relative brain weights of both sexes, and an increase in the absolute and relative liver weights of females and relative liver weight of males. The highest dose also increased liver weight and decreased brain weight in both sexes of the F1 generation. No histopathological changes were seen in any of the organs that were examined, including the testes. There was a 3% reduction in adjusted live pup weight for males from the 0.25% dose group, and a 5% reduction in this parameter for females in the high dose group. The effects on pup weights were considered by the investigators to have questionable biological significance since they were not dose-related in males and were small in females. DEGEE did not have adverse effects on fertility and reproductive performance despite a 34% decrease in caudal epididymal sperm motility in the F1 males at 2.5% compared to controls. There was no effect on fertility or reproduction of the F1 generation mice despite this effect on sperm. Fertility index: 100% (at 0%) and 84% (at 2.5%).

According to the FDA ICH Guidelines for Industry, Detection of Toxicity to Reproduction for Medicinal Products: Addendum on Toxicity to Male Fertility (April 1996), histopathology of the testis has been shown to be the most sensitive method for the detection of effects on spermatogenesis. The document also states that "sperm analysis (counts, motility and morphology) can be used as an optional method to confirm findings by other methods and to further characterize effects". Although there was a significant decrease (34%) in the percentage of motile sperm from the cauda epididymis in high dose F1 males exposed to DEGEE, there was no effect of treatment on histopathology of the testes. A study by Chapin et al. (1997) (which examined the database of continuous breeding mouse studies of 72 chemicals), indicates that the motility of sperm (sperm moving/total sperm) in untreated male CD-1 Swiss mice (the same species used in the Williams study) ranges from 47 - 95%, and that this number has to fall below 37% before a reduction in fertility of Swiss CD-1 mice is observed. In the current study, the percentages of motile sperm in control and high dose F1 animals were 64% +/- 5 and 42% +/- 6, respectively, ie with the motility of the high dose animals a little outside the normal range but above that which would be required for a reduction in fertility to be manifest. Furthermore, there was no effect of treatment on epididymal sperm counts, which according to Chapin et al. correlate so well with fertility that a 20% reduction in count results in reduced fertility. It is not, however, clear whether the decreased sperm motility would be associated with the same lack of effects in humans. According to EPA's reproductive toxicity risk assessment guidance, "human fertility is generally lower than that of test species and may be more susceptible to damage from toxic agents. Therefore, the conservative approach should be taken that, within the limits indicated for these parameters, statistically significant changes in measures of sperm count, morphology, or motility as well as number of normal sperm should be considered adverse effects" (U.S. EPA, 1996). Therefore, based on a reduction in sperm motility at 2.5%, a NOAEL of 1.25% (equivalent to 2200 mg/kg/day) is assigned for reproductive toxicity to the F1 generation.

Data for DEGBE  

In a well conducted single generation fertility study which conformed to the basic requirements of an OECD guideline study, DEGBE (purity 93-95%) was given to rats by oral gavage at doses of 250, 500 and 1000mg/kgbw/day (Nolen, 1985). A cross-over design was used in which either males or females were treated, with the corresponding males or females untreated. The premating exposure period for males was 60 days and for females 14 days and treatment of the latter was continued until either GD13 sacrifice for half the group or until weaning of offspring for the second half of the group.

Fertility of males and females was not affected. There were no effects on resorptions, live/dead embryo ratios, viability, corpora lutea or implants.  The only statistically significant finding was a reduction in pup weight gain at the highest dosed tested in the treated females/untreated males group, suggesting slight toxicity to the neonate. This was only seen at GD14 and was not observed at the time points either side (GD4 and GD21). The minor and transient nature of this finding is not deemed biologically significant. The overall conclusion of the study was that DEGBE is not toxic to the fertility of male or female rats at doses up to 1000mg/kg.

Nolen (1985) – gavage study in CD rats

Parameter              \              Dose

Control

250mg/kg

500mg/kg

1000mg/kg

Live pups born to cohort where males treated for 60 days prior to mating

13.4 (1.7)

12.8 (3.4)

13.8 (2.0)

12.0 (4.0)

Live pups born to cohort where females treated for 14 days prior to mating

13.6 (1.7)

13.1 (3.2)

13.2 (3.4)

12.3 (1.9)

An apparent decrease in the number of live born pups can be seen from the data, as shown in the table above. The same apparent effect is produced according to whether the males or females are dosed, but in neither case is the ‘effect’ statistically significant, and for treated males in particular there does not appear to be a clear dose response relationship (and it is only really apparent at the top dose level). The significance of this as a relevant observation is dubious. It should be noted that the authors reported that DEGBE had “no adverse effect on fertility in either sex and no adverse effects on embryos, foetuses, or neonates, except that the mean pup weights were reduced slightly during the later stages of lactation among the offspring of the females dosed with 1000 mg/kg/day”. It should be noted that the latter was seen on day 14 post partum, but not on days 0, 4 or 21 and was not seen at all in the dosed male cohort; so, whilst a statistically significant single observation, it is not likely to be a biologically significant one.

In a well conducted single generation fertility study, DEGBE produced no signs of toxicity to reproduction in either male or female rats when tested with a single dermally applied limit dose of 2000mg/kg bw/day (Auletta, 1993).  The premating exposure period was 90 days for both males and females, with treatment for the latter continued throughout the gestation and lactation period. Litters were checked for number and sex of pups, presence of gross anomalies, survival, and weight gain throughout lactation. Litters were standardised at PP4. Reproductive parameters assessed included Mating indices, pregnancy rates and male fertility rates.

There were no effects on male and female mating indices, pregnancy rates, male fertility indices. Histopathology showed no effects on the testes. The only finding that was dermal irritation resulting from repeated application of the test substance to the same application site. A no effect dose for reproductive effects was 2000mg/kgbw/day from dermal exposure.

A single generation reproductive toxicity study was undertaken using DEGBE (purity >98%) in drinking water in rats following the OECD 415 guideline with additional satellite groups for the analysis of clinical chemistry, haematology and oxidative stress markers (Sitarek, 2012). The main study was a limit dose study with a target dose of 1000mg/kgbw/day, whilst the satellite dose groups were dosed at 250, 500 and 1000mg/kgbw/day for females and 250 and 500mg/kgbw/day for males.  Males were dosed for 10 weeks (9 weeks before mating and 1 week during mating) and females for 3 weeks prior to mating, during pregnancy and lactation (total 9 weeks). Parameters examined included testis weight, epididymis weight, and for satellite groups haematology, clinical chemistry, markers of oxidative stress in plasma, liver and testes. Reproductive parameters measured included fertility (inseminated:pregnant), viability and survival rates.

Parameters used to assess the general toxicity showed an increase in relative spleen weight and absolute kidney weight at 500mg/kg for males, and in females an increase in absolute spleen, kidney, thyroid and pituitary gland weight at 250mg/kg. Females only in the 1000 mg/kg group showed cell proliferation in the spleen red pulp and the partial disappearance of the lymph nodules. Males showed statistically significant changes seen in Hematocrit, Hemoglobin, RBC and Lymphocytes from 250mg/kg; no changes deviated more than 15% from control values. No significant changes were seen in females. In the clinical chemistry and oxidative stress markers experiment, males showed statistically significant changes in multiple parameters (Ceruloplasmin, blood urea nitrogen, and thiobarbituric acid reactive substances (TBARS) (testes) at 250mg/kg and also HDL-cholesterol and TBARS (liver) from 500mg/kg, but not always with clear dose response relationships. Females showed statistically significant changes and dose response reduction in superoxide dismutase activity activity, but this finding was not seen in males. For the clinical chemistry measurements, no values deviated more than 25-30% from control values. No adverse histopathology was observed. Overall, there was no effect seen on any reproductive parameters up to the maximum tested dose of 1000mg/kg.

In a separate study by the same author to assess the impact of exposure to the substance on estrous cycle of females (Sitarek, 2012), female rats were given doses of 250, 500 and 1000mg/kgbw/day by oral gavage for 8 weeks. Mortality, body weight gain, feed and water intake of all the females was monitored during the exposure period and after cessation of the exposure. Vaginal smears were taken every morning for 12 days, after 2 and 6 weeks of exposure and 4 weeks after the cessation of the exposure. Microscopic specimens were prepared and stained with hematoxylin and Shorr’s stain. On the basis of the cytological image, four phases of the estrous cycle were distinguished. The time of the estrous cycle’s duration was assumed to be the period starting on the date on which the estrous phase was diagnosed in the vaginal smear and ending on the date on which the proestrous phase was observed.

Adverse clinical observations were seen in the high dose group. Some were less active, and showed bristling hair, dyspnea, and blood around the nose and mouth. There were also deaths in this high dose group: two females died in the third week of exposure, while one fatal case was recorded in each the fifth and eighth week of exposure and the first week post-exposure. Gross necropsy of these animals revealed dark liver. Microscopic examination revealed hyperkeratosis in forestomach in all of the dead females, while one of them, had also, pneumonia. Body weights were consistently lower throughout study in the high dose group and food intake was reduced in mid and high dose groups. However, there was no effect seen on any reproductive parameters examined up to the maximum tested dose of 1000mg/kg. Toxic effects were seen at the highest tested dose (reduced body weight gain and adverse clinical observations.)

A well reported 90 day sub-chronic OECD guideline and GLP drinking water study using DEGBE (purity 99.2%) also examined for the control and high dose (1000mg/kgbw/day) animals, total spermatid count per testis, total sperm counts per epididymidis, total counts per gram of tissue, sperm motility in addition to the standard toxicological end points. 200 sperm per animal assessed for morphology. The overall NOAEL for the study was 250mg/kgbw/day (mid dose used) due to overall effects on water consumption, haematology, clinical chemistry, organ weights, histopathology and liver enzyme changes. However, there were no adverse effects seen on sperm at a maximum tested dose of 1000mg/kgbw/day (Johnson, 2005).

Data for TEGME   

There are no specific reproductive toxicity studies available on TEGME. However, there is some useful data that pertains to the reproductive toxicity end point from extensions used in repeat dose studies.

In a guideline (OECD 408) and GLP study, SD rats were exposed for 90 days to TEGME in drinking water at concentrations of 400, 1200 and 4000mg/kgbw/day (Dow, 1990a). There were no gross or microscopic lesions found in the nervous system of any rats that were attributed to TEGME ingestion. Treatment-related degeneration and/or atrophy of seminiferous tubules were observed for most males in the high dose treatment group. The cell types affected in the tubules were spermatocytes and developing spermatids. Similar lesions were not observed for males in the low-dose or mid-dose treatment groups. At the Sponsor's request, testes from all males on the study were examined histologically. Significant seminiferous tubule degeneration and/or atrophy were observed in 12 of 15 males in the high dose treatment group. The target cells in the tubules appeared to be spermatocytes and developing spermatids. Treatment-related lesions were not observed in the low-dose and mid-dose groups. No other potential treatment-related alterations in organ weights, gross pathology, or histopathology were observed in this study. No treatment related effects were observed on the oestrous cycle. On the basis of these findings 4000 mg/kg bw/day l is considered to be a subchronic LOAEL for TEGME and 1200mg/kg bw/day a NOAEL under the conditions of this study for these reproductive toxicity related end points.

In a guideline (US EPA 40 CFR 799.5000) and GLP neurotoxicity study, SD rats were exposed for 90 days to TEGME in drinking water at concentrations of 400, 1200 and 4000mg/kgbw/day (Dow, 1990b). Parameters assessed included a functional observation battery and locomotor activity. The study showed no significant clinical signs of toxicity, alterations in the functional observational battery, or gross or microscopic lesions in the nervous system at any dose level. Minor decreases in motor activity were observed in the high dose group at the Day 60 (males only) and Day 90 (males and females) evaluation periods. These decreases in motor activity were not considered to be neurotoxicologically significant based on the small magnitude of the changes, the parallel changes observed in body weights at these evaluation periods, and the lack of corroborative behavioural effects from the FOB evaluations or histological changes in central or peripheral nervous system tissues. Based on these findings 4000mg/kg/day is a sub-chronic NOAEL for TEGME neurotoxicity.

None of the above source substances exhibit any significant toxicity towards reproduction. The read across used with the above data is as shown below in the context of the matrix of E series glycol ethers.

READ ACROSS  FROM SOURCE TO TARGET SUBSTANCES

Number of EO units

Alcohol chain length

Methyl

Ethyl

Propyl

Butyl

Pentyl

Hexyl

Mono

Di

Source

Source

Tri

Source

 Target

Tetra

 

 

 

 

 

 

 

 

Substance with no suitable or useful data available

Based on the justification shown attached to the read across record in this end point, it is reasonable to assume that the target substance will share the same lack of reprotoxic properties as the source substances.

 

Effects on developmental toxicity

Description of key information

Study

Result

Comment

TEGME

US EPA guideline developmental toxicity Klimisch 1 study in rats

NOAEL (maternal): 625mg/kgbw/day

NOAEL (devtox): 625mg/kgbw/day

LOAEL (maternal): 1250mg/kgbw/day

LOAEL (devtox): 1250mg/kgbw/day

Equivalent to TEGEE doses of 680 and 1360 mg/kgbw/day respectively

US EPA guideline developmental toxicity Klimisch 1 study in rabbits

NOAEL (maternal): 500mg/kgbw/day

NOAEL (devtox): 1000mg/kgbw/day

LOAEL (maternal): 1000mg/kgbw/day

LOAEL (devtox): 1500mg/kgbw/day

Equivalent to a TEGEE doses of 540, 1090 and 1630 mg/kgbw/day respectively

Chernoff Kavlock reproductive and developmental toxicity oral screening study

NOAEL (devtox): 1000mg/kgbw/day

No statistically significant effects at maximum tested dose

Equivalent to TEGEE dose of 1090mg/kgbw/day

DEGEE

Chernoff Kavlock reproductive and developmental toxicity oral screening study

NOAEL (devtox): 5500mg/kgbw/day

No significant effects at only tested dose

Equivalent to a TEGEE dose of 7260mg/kgbw/day

DEGBE

Dietary study in rats, treatment GD0-20.

NOAEL (maternal): 633mg/kgbw/day

NOAEL (devtox): 633mg/kgbw/day

No statistically significant effects at maximum tested dose

Equivalent to a TEGEE dose of 695mg/kgbw/day.

Dermal study in rabbits, treatment GD8-19, 4hrs/day

[NOAEL (maternal-local irritation): 100mg/kgbw/day]

NOAEL (maternal-all other): 1000mg/kgbw/day

NOAEL (devtox): 1000mg/kgbw/day

No statistically significant effects at maximum tested dose

Equivalent to a TEGEE dose of 1100mg/kgbw/day

TEGBE

 

 

Chernoff Kavlock reproductive and developmental toxicity oral screening study

NOAEL (devtox): 1000mg/kgbw/day

No statistically significant effects at maximum tested dose

 

TEGEE

Chernoff Kavlock reproductive and developmental toxicity oral screening study

NOAEL (devtox): 1000mg/kgbw/day

No statistically significant effects at maximum tested dose

.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
800 mg/kg bw/day
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Additional information

Data on TEGME

In a guideline (US EPA OTS 798.4900) and GLP study, the developmental toxicity of TEGME was evaluated in rats following oral (gavage) administration at doses of 625, 1250, 2500 and 5000 mg/kg bw/day on Days 6-15 of gestation. 25 animals were used per dose level and for control (sham dosed) animals. Ovaries and uterine content was examined after termination and fetal observations included both soft tissue and skeletal examinations (Hoberman, 1990a).

The 5000 mg/kg/day dosage of TEGME caused significant numbers of rats to have decreased motor activity, excess salivation, ataxia, and impaired righting reflex, as compared with the control group numbers. Lost righting reflex, urine-stained abdominal fur, and rales occurred for a few high dosage group rats. Statistically significant reductions in maternal body weight gains occurred for the high dosage group during the dosage period. As a result, the mean maternal body weights were statistically significantly reduced for the high dosage group on days 9, 12 and 16 of gestation, as well as during the post-treatment period, on days 18 and 20 of gestation. The mean gravid uterine weight was significantly lower for the high dosage group, and the mean maternal body weight on day 20 tended to be lower for the high dosage group compared with the control. Maternal body weights were not affected by treatment with TEGME for any of the other dosage groups. Groups given 2500 and 5000 mg/kg/day of TEGME had statistically significantly reduced feed consumption for the entire dosage period (calculated as days 6 to 16 of gestation), as compared with the control group. The 2500 mg/kg/day dosage group had statistically significantly reduced relative maternal feed consumption from days 6 to 16, 6 to 18, and 12 to 16 of gestation, and the 5000 mg/kg/day dosage group had significantly reduced mean absolute and relative maternal feed consumption throughout the entire dosage period. The 1250 mg/kg/day dose level slightly reduced relative feed consumption during the dosing period. Thus, 1250 mg/kg/day was considered a LOAEL, and 625 mg/kg/day is considered a NOEL for maternal toxicity.

Small, significant increases in embryo-fetal lethality occurred in the 5000 mg/kg/day dosage group (litter averages for total resorptions, late resorptions, percentage of resorbed conceptuses and dams with at least one resorption), as compared with the control group values. None of these effects were seen for the other dosage groups. Dosages of 1250 mg/kg/day and above reduced fetal body weights. However, this effect was statistically significant for only the 2500 and 5000 mg/kg/day dosage groups. Dosages of TEGME as high as 5000 mg/kg/day did not cause gross external, internal soft tissue or skeletal malformations in the fetuses. These dosages also did not result in increased incidences of gross external or soft tissue variations in the fetuses. There were no dosage-dependent, statistically significant differences in the litter or fetal incidences of these parameters. Groups given 1250 mg/kg/day and higher dosages of TEGME had significant increases in the litter and/or fetal incidences of reversible delays in fetal ossification, common observations in fetuses with reduced body weights. The 2500 and 5000 mg/kg/day dosage groups also had significant increases in the litter and/or fetal incidences of cervical ribs, a common variation in rats. Thus, 1250 mg/kg/day is considered very near the LOAEL (borderline NOAEL) for TEGME developmental toxicity, and 625 mg/kg/day is considered the NOEL for developmental toxicity.

In a guideline (US EPA OTS 798.4900) and GLP study, the developmental toxicity TEGME was evaluated in rabbits following oral (gavage) administration at doses of 250, 500, 1000 and 1500 mg/kg bw/day on Days 6-18 of gestation. 20 animals were used per dose level and for control (sham dosed) animals. Ovaries and uterine content was examined after termination and fetal observations included both soft tissue and skeletal examinations (Hoberman, 1990b).

At the 1500 mg/kg/day dosage, TEGME caused the deaths of eight does. These showed clinical signs of treatment, including weight loss, decreased feed consumption, and four of these does had gastric ulcerations. Two other does given the 1500 mg/kg/day dosage of TEGME aborted: these also showed treatment-related clinical signs: weight loss and reduced feed consumption.  Dosages of 500 mg/kg/day and higher caused increased body weight gain and feed consumption during the postdosage period, a common phenomenon seen in developmental toxicity studies after the termination of dosing. Based on the data in this study, the maternal NOEL for TEGME was considered to be 250 mg/kg/day, and 500 mg/kg/day is considered the NOAEL for maternal toxicity.

Administration of TEGME to pregnant rabbits at dosages as high as 1500 mg/kg/day did not cause statistically significant differences in the numbers of pregnant does, the averages for corpora lutea, implantations, live fetuses, resorptions, fetal sex rations, or fetal body weights. Dosages of TEGME as high as 1500 mg/kg/day did not cause gross external, internal soft tissues, or skeletal malformations in the fetuses. These dosages also did not result in increased incidences of gross external or internal soft tissue variations in the fetuses. There were no dosage-dependent, statistically significant differences in the litter or fetal incidences of these parameters. The 1500 mg/kg/day dosage significantly increased the fetal and/or litter incidences of two common skeletal variations (fetal ossification - angulated hyoid alae and reversible delays in ossification of the xiphoid). Based on these findings the NOEL for developmental toxicity is considered to be 1000 mg/kg/day and TEGME should not be classified as a developmental toxicant since it does not present a unique hazard to the development of rabbit conceptuses.

In a concurrent probe study, doses of 1250 mg/kg/day resulted in clinical signs, gross necropsy findings, death or moribund sacrifice.  One of 5 does in the 1250 mg/kg/day dosage group and 5 of 5 does in the 2500 and 5000 mg/kg/day dosage group did not survive to scheduled sacrifice. Average body weight gain was not effected by dosages as high as 1250mg/kg/day. Feed consumption was decreased in the 1250 mg/kg/day and above dosage groups. In the does that survived, no effects of TEGME were observed on implantation, embryo-fetal viability, fetal sex ratio, fetal body weights, and gross morphology.

In a developmental toxicity screening test on TEGME supplied as bridging study for this end point (Leber, 1990), Wistar derived rats were exposed GD7-16 to TEGBE (250, 1000 mg/kg by oral gavage in a procedure similar to that developed by Chernoff and Kavlock (1982). Negative and positive controls (latter methoxyethanol at 50 and 250 mg/kg) were used. 10 animals were used per dose level and in concurrent controls. The criteria for evaluation were that for a substance to pass the screen and show no potential for reprotoxicity, there should be:

  • No effect on litter size, pup weight, survival or pup weight gain.
  • No reduction in litter size (mean less than 8) or reduced post natal survival (screening criteria for teratogenicity)
  • No reduction on post natal weight gain (<30%) with no reduction in survival (screening criteria for foetal toxicity)

According to the above criteria, there were no significant findings attributable to TEGME treatment whereas the positive control (methoxyethanol) showed signs of severe reproductive toxicity. According to the screening criteria, TEGME does not show evidence of reproductive toxicity in this screening assay.

Data on DEGEE    

In a screening study using the Chernoff Kavlock assay, DEGEE was administered to pregnant mice during the period GD7-14 at a single dose level of 5500mg/kgbw/day. No significant embryotoxicity was observed (Schuler 1984; Hardin 1987). Parameters unaffected compared to control animals included viability, pups per litter, number of dead pups, pup survival and pup weight gain. Pup weight at birth was statistically significantly reduced but only by 6%. On the basis of this study, it was concluded that the substance was a low priority for further work and that developmental toxicity was not an important characteristic of the substance.

Data on DEGBE    

In a study that conformed to the basic requirements of the relevant OECD guideline, Wistar rats (20 per dose group) were exposed to DEGBE in the diet at doses equivalent to 25, 115 and 633mg/kg/day over the period GD 0-20 (Ema, 1988). 5 -6 dams from each dose group were allowed to litter spontaneously. Numbers of live and dead pups recorded, sexed, weighed, checked for anomalies (external and in oral cavity) and numbers adjusted to 4 males/4 females per litter on PP0. Offspring were weighed daily and weaned on day 21. Dams killed at this time point and the numbers of implantation remnants were recorded. Offspring were reared until 10 weeks old - weighed weekly. Statistical treatment as described except after weaning, analysis on the basis of individual animals. There was no treatment with the substance after GD20. Parameters assessed included gestation period, number of live born and live birth index, survival rate before and after weaning and body weight.

In dams, body weight gain was significantly lower than control group at all doses (12-18% reduction) but with no dose response relationship. No other significant effects were noted. No effects on any implantation parameters, corpora lutea, live fetus numbers, sex ratios, fetal or placental body weight were seen. No external or internal anomalies were found. One fetus showed skeletal defects (fused sternebrae) in each of the mid and high dose groups. A low incidence of skeletal variations were seen in all dose groups; the high dose group rate was higher than the control group but was not statistically significant. Delayed sternebrae ossification was seen in all dose groups with some evidence for a dose response but this was again not statistically significant from controls. The degree of ossification of the low dose group was significantly lower than controls but not from the other dose groups; the lack of this effect in higher dose groups means it is unlikely to be biologically significant. In the satellite groups allowed to litter, there was no significant effect on any of the parameters measured. It should however be borne in mind that the statistical power of this part of the study is low with only 5 or 6 animals per dose group. There was slight evidence for a fall off in survival rate, number of implantation remnants per litter and live newborns per litter but this was far from clear and numbers in the low dose group were higher than controls. This data is shown in the table below:

 

Ema (1988) – Dietary study in Wistar rats

Parameter              \              Dose

Control

25mg/kg

115mg/kg

633mg/kg

Maternal body weight gain during pregnancy

114 (12)

98 (15)*

93 (24)*

100 (16)*

No. implantations/litter

11.3 (1.6)

10.8 (2.0)

12.0 (1.5)

11.0 (2.1)

No. live foetuses/litter

10.9 (1.5)

9.8 (2.6)

11.1 (1.8)

10.6 (1.9)

No. implantation remnants/litter

10.4 (1.1)

10.7 (1.4)

9.4 (0.5)

8.8 (1.3)

No. live newborns/litter

9.6 (1.5)

10.3 (1.6)

8.6 (0.9)

8.2 (0.8)

Live birth index

92.4

96.6

91.6

94

Figures in parenthesis are standard deviations. *Significantly different from control value (p<0.05). Live birth index is ratio of live newborns at birth to no. implantation remnants, expressed as a percentage.

The changes seen above were not statistically significant. In contrast, the number of implantations per litter and the number of live foetuses per litterer show no sign of a correlation with dose. It should also be noted that maternal body weight gain at all doses was statistically significant from controls (but not in a dose dependent manner) and that the authors of the study made no comment on the changes described above, concluding that “DEGBE has no adverse effects on the pre and post-natal development of the offspring in rats.”

In a well conducted teratology study which conformed to basic guideline requirements, New Zealand White rabbits were exposed by the dermal route to DEGBE at doses of 100, 300 and 1000mg/kg bw/day (Nolen, 1985). Exposure was during GD 8-19 for 4 hours/day. Ovaries and uterine content was examined after termination and fetal observations included both soft tissue and skeletal examinations (Nolen, 1985). All treated dams showed reduced weight gain but only the mid dose group reached statistical significance and there was clearly no dose response relationship. These effects were not thought to be related to the amount of dose absorbed. The only finding that was clearly attributed to treatment was significant irritation at the site of application manifest at doses from 300mg/kg bw/day upwards. There were no significant differences seen in the mean numbers of corpora lutea, implants, resorptions or viable foetuses or in the mean foetal body weight and incidence of skeletal anomalies or of gross or visceral malformations.

Hermsen et al (2011) used the Zebrafish Embryotoxicity test to examine the developmental toxicity potential of a number of glycol ethers. This group used a novel quantitative evaluation method to assess the development of the zebrafish embryo based on specific endpoints in time, the general morphology score (GMS) system. For teratogenic effects a separate scoring list was developed and used. They assessed the acid metabolites methoxyacetic acid (MAA), ethoxyacetic acid (EAA), phenoxyacetic acid, butoxyethoxyacetic acid, methoxyethoxyacetic acid and the parent glycol ethers of the first two, i.e. methoxyethanol and ethoxyethanol. Only MAA and EAA resulted in a concentration dependent decrease in GMS. The other glycol ether metabolites did not reduce the GMS as compared to the controls up to the highest concentration that could be tested without causing general toxicity. Embryos exposed to MAA and EAA showed comparable dysmorphology after exposure; several teratogenic effects were observed following exposure to these substances, among which heart, head and tail malformations (characteristics seen in mammalian developmental toxicity tests), were the most pronounced. Unlike their metabolites, the parent compounds EGME and EGEE did not show any effect on general morphology and teratogenicity. No effects were seen with the other glycol ethers. This data leads to the conclusion that butoxyethoxyacetic acid (the main metabolite of DEGBE) does not have developmental toxicity potential.

Data on TEGBE    

There is data available from a developmental toxicity screening test on TEGBE supplied as supporting evidence for this end point (Leber, 1990). Whilst this does not meet the requirements for this end point on its own, it has value as a bridging study to the developmental toxicity studies of the source substances, since TEGEE and TEGME were also evaluated. In this screening study, Wistar derived rats were exposed GD7-16 to TEGBE (250, 1000 mg/kg by oral gavage in a procedure similar to that developed by Chernoff and Kavlock (1982). Negative and positive controls (latter methoxyethanol at 50 and 250 mg/kg) were also used. 10 animals were used per dose level and in concurrent controls. The criteria for evaluation were that for a substance to pass the screen and show no potential for reprotoxicity, there should be:

  • No effect on litter size, pup weight, survival or pup weight gain.
  • No reduction in litter size (mean less than 8) or reduced post natal survival (screening criteria for teratogenicity)
  • No reduction on post natal weight gain (<30%) with no reduction in survival (screening criteria for foetal toxicity)
        

According to the above criteria, there were no significant findings attributable to TEGBE treatment whereas the positive control (methoxyethanol) showed signs of severe reproductive toxicity. According to the screening criteria, TEGBE does not show evidence of reproductive toxicity in this screening assay.

Data on TEGEE    

In the same study by Leber referred to above also examined, TEGEE was also examined at the same doses. According to the above criteria, there were no significant findings attributable to TEGEE treatment and therefore TEGEE does not show evidence of reproductive toxicity in this screening assay. This result provides useful information to increase the confidence in bridging across from TEGME to TEGEE.

Overall summary

The three source substances do not shown any significant evidence of reproductive toxicity at levels that are not maternally toxic and at doses at or below 1000mg/kgbw/day – the maximum test dose now recommended in current test guidelines. According to the hypothesis of this read across justification, these results can be read across to predict the reproductive toxicity of TEGBE. The screening study available with TEGBE, TEGEE and TEGME, which is negative for all three, and which provides bridging information to provide confidence in the reliability of the read across predictions.

 The read across used with the above data is as shown below.

READ ACROSS FROM SOURCE TO TARGET SUBSTANCES

Number of EO units

Alcohol chain length

Methyl

Ethyl

Propyl

Butyl

Pentyl

Hexyl

Mono

Di

Source

Source

Tri

Source

 Target

Tetra

 

 

 

 

 

 

 

 

Substance with no suitable data available

Based on the justification shown attached to the read across record in this end point, it is reasonable to assume that the target substance will share the same lack of developmental toxicity properties as the source substances.

Justification for classification or non-classification

No effects are seen on reproductive parameters at doses <1000mg/kgbw/day. The LOAELs for all developmental effects in rats and rabbits are predicted to be above 1000mg/kgbqw/day and the more sensitive species, rabbit, shows clear evidence of maternal toxicity in the surrogate substance before developmental effects are manifest. There is no evidence for a unique hazard to conceptuses. On this basis, the substance does not meet the criteria for classification either as a developmental or reprotoxic substance.