2-hexyloxyethanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Full study report from NTP , supported by a well documented publication which meets basic scientific principles.

Justification for type of information:

Read Across to an analogue based on structural similarity. An analogue justification is attached to section 13 of the dataset.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

other: National Toxicology Programme Continuous Breeding Protocol

Deviations:

yes

Remarks:

, F1 tests were only carried out with the control and low dose groups

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Kingston NY
- Age at study initiation: (P) x 11wks; (F1): 74+/-10 days
- Housing: by sex in solid bottom polypropylene or polycarbonate cages with stainless steel wire lids, then subsequently in breeding pairs. Ad-Sorb-Dri bedding used.
- Diet (ad libitum): NIH-07
- Water (ad libitum): tap
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Photoperiod (hrs dark / hrs light): 10/14

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Each dosing solution formulated directly. No loss of compound after 3 days at room temperature and only 0.9% after 21 days lost. Consequently, fresh solutions prepared every 2 weeks.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 7-day pre-mating period and a 98-day cohabitation period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis on days 1, 6, 10 and 14. All doses found to be within 97-104% of expected values.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

continuous

Details on study schedule:

At the completion of the continuous breeding phase, the F0 breeding pairs were separated and housed individually and exposure to 2-butoxyethanol continued. When the last litter was weaned from the continous breeding phase F0 males and females from the 1 % dose group were mated with male and female control animals in a one-week crossover mating study to determine any sex-related reproductive effects of 2-butoxyethanol.

Remarks:

Doses / Concentrations:
0 0.5, 1.0 or 2.0 %
Basis:
nominal in water

Remarks:

Doses / Concentrations:
0, 720, 1340 and 2050 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

20. 40 in control

Control animals:

other: Yes. Control males and females were also mated for comparative purposes.

Details on study design:

-The study itself consisted of three phases:
Phase 1 = continous breeding phase
Phase 2 = F0 males and females mated with control animals
Phase 3 = F1 males and females mated

Exposure to 2-butoxyethanol was discontinued during the one-week mating period and then reintroduced at 1 % dose level (estimated daily intake 1830 mg/kg bw). Control males and females were also mated for comparative purposes. The proportion of successful copulations from the breeding pairs was similar in all groups. However, the number of fertile females was significantly reduced in the group where treated females were mated with control males.

A final phase was conducted to assess the fertility and reproductive effects of 2-butoxyethanol in second generation (F1) pups. The pups selected were those born after the CBP and when the maternal animals were individually housed. As there were insufficient pups in the 1 and 2 % dose groups, only the pups from the 0.5 % dose group were used. The F1 generation pups were nursed, weaned and reared to sexual maturity. After weaning, the mice received 0.5 % 2-butoxyethanol in their drinking water (estimated daily intake 950 mg/kg bw). At 74 ± 10 days of age, the F1 animals from different litters were mated. The animals were necropsied after delivery.

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes

Oestrous cyclicity (parental animals):

F0 Females Mated with Control Males
-When evaluated over the 7 day period prior to necropsy, proportionally more females (7/13) in the 1 % treated group than controls (9/38) had oestrus cycles longer than 7 days.

Sperm parameters (parental animals):

Parameters examined in P and F1 male parental generations

Litter observations:

Parameters examined in P and F1 generations

Postmortem examinations (parental animals):

Parameters examined in P and F1 generations

Postmortem examinations (offspring):

Parameters examined in F1 and F2 generations

Statistics:

Cochran-Armitage test for dose related trends in fertility. Chi-square test for differences in fertility among groups. Pairwise comparisons between dose groups and control using Fisher's exact test. Reproductive indices between groups compared using Kruskal-Wallis test and ordered differences using Jonckheere's test. Pairwise comparisons of treatment groups perfomed using Wilcoxon-Mann-Whitney U test. Pup number per litter corrected for when calculating average pup weight.

Reproductive indices:

The numbers of fertile pairs from the surviving pairs of the continuous breeding phase were 38/39, 19/19, 13/14 and 5/7 at 0, 0.5, 1.0 and 2.0 % dose levels, respectively.

Offspring viability indices:

no further information

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
-During the 98 day cohabitation period, deaths occurred in the female mice: 13/20 in the 2 % group, 6/20 in the 1 % dose group, 1/20 in the 0.5 % dose group and 1/40 in the control group. In the male mice, no deaths occurred.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
-The average body weights in the female 2 % dose group were consistently lower than the controls. Male groups experienced weight loss (1-2 % of initial body weight) in the two highest doses.
-At necropsy, male and female mice from the 1 % dose group had significantly lower body weights.

ORGAN WEIGHTS
-The 1% dose group exhibited increased relative kidney weights, and female also had significant increases in relative liver weight.
-A significant increase in relative kidney weight in the females, and a significant increase in relative liver weight in both the males and females were observed in the F1 generation.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
-Reduced fluid consumption was observed at all dose levels in both sexes (22 %, 18 % and 36 % reduction relative to controls at 0.5 %, 1.0 % and 2.0 %, respectively after 14 weeks of dosing).

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
-In the 1% group no significant differences were observed between the control and treated animals for the average oestrous cycle length and frequency.
-No treatment-related changes in the oestrous cycle length and frequency were noted in the F1 generation.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
-In the 1% group no significant differences were observed between the control and treated animals for the weights of reproductive organs, sperm motility or morphology.
-No treatment-related changes in the weights of reproductive organs, sperm motility or morphology were noted in the F1 generation.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
-Significant reduction in reproductive performance occurred at 1 and 2 % dose levels as indicated by dose-related decrease in number of litters per fertile pair, litter sizes, pup viability and live pup weight.
-No significant fertility and reproductive effects were observed in the F1 animals as indicated by the proportions of successful copulation and fertile females, litter size, pup viability and live pup weights.

HISTOPATHOLOGY (PARENTAL ANIMALS)
-In the only histopathological examination carried out on the treated females, no treatment related kidneys lesions were observed.

Dose descriptor:

NOAEL

Effect level:

720 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Body weight reduction, mortality, reproductive performance

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

BODY WEIGHT (OFFSPRING)
A small but significant reduction (by 3 %) of live F1 pup weight was also observed in the 0.5 % dose group without other significant reproductive effects but no effect was seen in the pups born to the F1 generation.

On the F1 generation (low dose and control only tested) , there were no effects on mating and fertility indices or any other reproductive parameter. There were no significant differences in F2 litter size, pups numbers or weights. There were no effects on sperm parameters in males or estrous cyclicity in females but relative liver and kidney weights were increased in the dose group receiving 2-butoxyethanol (0.5%)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

720 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: pup weight

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

720 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: No reproductive effects observed at single dose tested

Reproductive effects observed:

not specified

The continuous breeding first generation study showed that treatment significantly affected fertility at the mid and high dose levels and marginally affected pup weight at the lowest level. The cross-breeding results suggest that the fertility effects were only due to effects on the female mice. In the cross-over study there were no effecting on mating index (number with copulatory plugs/number co-habited), however for females that co-habited with control males the fertility index (number fertile/number with copulatory plugs) ws significantly reduced compared to the controls and other cross-over group. Similarly, the number of live pups/litter of the 1% treated females/control males fell. These effects may have been an indirect consequence of the severe general systemic toxicity rather than a direct effect of 2-butoxyethanol on the reproductive organs.

Conclusions:

In this study, effects were seen on fertility only at doses which were severely toxic to the animals (1340 and 2050 mg/kg bw/d). A NOAEL of 720 mg/kg bw/day can be identified for fertility effects by oral route in mice. Marginal pup weight reductions at this dose were not repeated in the second generation and therefore not regarded as a significant finding.

Executive summary:

In a continuous breeding study carried out to an NTP protocol, mice were exposed for a period of 14 weeks to the oral drinking water routes at doses from 720 to 2050mg/kg. Significant adverse reproductive effects were observed in the females at very high dose levels (1340 mg/kg and above) which also caused severe general toxicity, including death. Under the conditions of the study, the NOAEL for reproductive toxicity of 2-butoxyethanol (fertility) can be set as 720 mg/kg/day. For developmental toxicity, no NOAEL can be derived. A conservative LOAEL of 720 mg/kg/day can be taken as only a very slight decrease in pup weight was observed at this dose.

At the lowest dose studied, the only adverse finding was a marginal statistically significant reduction in pup weight. However, since this reduction was only 3% compared to controls and was not repeated in the F1 generation, it was not considered a significant adverse finding.

No NOAEL or LOAEL can be determined for systemic parental toxicity because this kind of study is not designed to assess systemic toxicity although it is of note that there were effects (reduced fluid consumption) even at the lowest dosage of 720 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

720 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Additional information

No reproductive toxicity test has been performed with EGHE.

In the absence of reproductive toxicity data on EGHE, data available for structural analogues Ethylene Glycol Butyl Ether, Diethylene glycol Butyl Ether and Di-ethylene glycol Hexyl Ether are used to predict the reproductive toxicity potential of EGHE. This is supported by repeated dose toxicity data on EGHE (absence of effects on reproductive organs).

The justification for using data on structural analogues is provided in the document attached to Section 13 of the IUCLID dossier.

Repeated dose toxicity data on EGHE:

In a repeated dose inhalation study, four groups, each consisting of 20 Fischer 344 rats per sex, were exposed for 6 hours per day, 5 days per week, for 14 weeks to the vapors of ethylene glycol hexyl ether at target concentrations of 0 (control), 20, 40, or 85 ppm and followed by 4 week recovery period. Actual mean concentrations obtained for this study were 20, 41, and 71 ppm ethylene glycol hexyl ether). Based on the data from this study, 41 ppm (245 mg/m3) is considered to be NOAEC. At this concentration no biologically significant toxic effects were observed. Decreases in body weight and increases in male kidney and female liver weights occurring at this concentration were considered to be adaptive (and not adverse) since there were no correlative changes in histopathology or serum chemistry. The changes in liver enzymes in animals exposed to 71 ppm (425 mg/m3) are difficult to interpret since levels of 3 out of 4 enzymes were decreased and only 1 out of 4 was increased. Whereas the effects on the kidney were not dose-dependent, liver weights increased in a dose-dependent manner and were not reversed after 4 weeks of recovery in animals exposed to 71 ppm. No effects on reproductive organs, red blood cells or histologic changes in the liver or kidney were noted at concentrations up to and including the highest concentration tested (71 ppm or 425 mg/m3).

Additional information for EGHE:

EGHE has also been evaluated in the EPA Endocrine Disruption Screening Program for the 21^stCentury (EDSP21) for potential endocrine-related activity. It was evaluate in 8 androgen, 15 estrogen and 3 thyroidin vitroassays. EGHE was not called active in any of these endocrine-related assays.

Data available for EGBE:

A continuous breeding study in CD1-mice is available for ethylene glycol butyl ether (EGBE). The main study comprised 3 stages. In the first stage 40 male/female pairs of mice (control group) and 20 male/female pairs of mice (treated groups) were house together for a period of approximately 90 days. The dose levels were 0.5, 1 and 2% in the drinking water corresponding to approximately 0.72, 1.35, and 2.0 g/kg/d respectively. During the cohabitation period the animals mated several times (5-6 litters) and the fecundity/fertility of these animals was assessed. At the end of the cohabitation period the animals were separated and housed individually and the final litter allowed to be weaned. The final litter from the mid dose group was used in a cross breeding phase (treated males and females mated with control males and females – three groups, treated male + control female; treated female + control male; control male and control female). The F1 generation from the low dose group was used to generate the F2 generation due to the reduced fertility of the mid dose group and the excessive toxicity in the high dose group.

Excessive mortality (65%) was seen in the high-dose females during cohabitation; and moderate morality was seen in the mid dose group with 6/20 females dying in the 1% group, and 1/20 in the control and 0.5% groups. Male mice in the middle and high dose groups showed less weight gain. Water consumption was reduced in all treatment groups in both sexes (22 %, 18 % and 36 % reduction relative to controls at 0.5 %, 1.0 % and 2.0 %, respectively after 14 weeks of dosing). The number of live pups/litter was reduced by 52% and 71% in the middle and high dose groups, respectively, and the proportion of pups born alive was reduced in these groups by nearly equal to 40% and 45%. As EGBE concentration increased, adjusted pup weight decreased by 5%, 11%, and 16%.

At the crossover mating trial, using the controls and the 1.0% EGBE group, EGBE-treated females delivered 34% fewer pups which weighed 12% less than control pups.

At necropsy after crossbreeding, F₀females from the 1.0% EGBE group weighed 10% less than controls, while the kidneys and liver (adjusted for body weight) weighed 17% and 21% more than controls. EGBE-treated F₀males weighed 9% less than controls, with kidneys that weighed 13% more than controls, and right testis that weighed 8% less (but not statistically significant). There were no changes in sperm indices.

Due to the reduced fertility in the 1% EGBE group, insufficient animals were available to test the second generation at this dose. Thus, the second generation was evaluated using the 0.5% group and the controls. There was no indication of reduced body weight gain during maturation to mating at 74 ± 10 days of age. While there was no difference between the two groups in the proportion of pairs mating or becoming pregnant, there was a 5% reduction in the weight of the F₂pups.

At F₁necropsy, body weights were not different between the groups, although in females, liver weight and kidney weight were increased by 7% and 22%, respectively. In males, liver weight was increased by 9%.

Based on the findings of this study, a NOAEL for reproductive toxicity was determined to be the low dose of 720 mg/kg bw. Although reproductive effects were observed in the mid and high doses in this study, it is clear that these doses were in excess of the maximum tolerated dose given the mortality observed in these groups and that the fertility effects are likely to be a direct consequence of the parental toxicity rather than a direct effect on fertility or fecundity. In addition, both these dose groups are greater than the limit dose of 1000 mg/kg bw/day currently employed in reproductive toxicity testing protocols. 

Although there are some methodological differences between this study design and a conventional 2 generation or the more recent extended one generation protocol, the study along with the other supporting information summarized in the dossier is considered sufficient to evaluate the potential reproductive toxicity of EGBE and consequently, form the basis of the prediction of the reproductive toxicity of EGHE.

The absence of direct effects on reproductive organs is confirmed by the 90-day repeated dose toxicity studies in rats and mice with EGBE at doses of 750, 1,500, 3,000, 4,500 or 6,000 ppm (actual ingested rats: ~ 75, 140, 290, 365 or 460 mg/kg bw/day; mice: ~ 150, 300, 610, 770 or 1000 mg/kg bw/day), effects were not seen macroscopic/microscopic on reproductive organs or on sperm morphology or estrous cycle length in the absence of effects on general toxicity.

Data available for DEGBE:

In a well conducted single generation fertility study which conformed to the basic requirements of an OECD guideline study, DEGBE produced no signs of toxicity to reproduction in either male or female rats when tested at oral doses up to 1000mg/kg. The only finding that was attributed to treatment was a small but statistically significant reduction in pup weight gain seen at the highest dose tested and at a single time point during gestation. The minor and transient nature of this finding is not deemed biologically significant. A similar study by the dermal route of exposure showed no evidence of reproductive toxicity in either male or female rats when tested with a dermally dose of 2000mg/kg. The only finding that was dermal irritation resulting from repeated application of the test substance to the same application site.

Another single generation reproductive toxicity study was undertaken in drinking water following the OECD 415 guideline in rats with additional satellite groups for the analysis of clinical chemistry, haematology and oxidative stress markers. There was no effect seen on any reproductive parameters up to the maximum tested dose of 1000mg/kg. Parameters used to assess the general toxicity indicated that males receiving DEGBE were more sensitive than females: effects included significantly greater, dose-dependent relative spleen weight, significant decrease in hematological parameters from 8% to 15% depending on the dose, were observed. Clinical chemistry parameters (HDL-cholesterol, BUN) and some markers of oxidative stress differ between the exposed groups and the control one, but without adverse health effect manifesting. No adverse histopathology was observed. The same authors also conducted a fertility study alongside the main study where the substance was dosed by oral gavage to assess the impact on the estrous cycle in the rat. There was no effect seen on any reproductive parameters 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).

In a 90-day repeated dose toxicity study in rats with DEGBE with doses up to 1,000 mg/kg bw/day, evaluation of sperm and testes histopathology showed no adverse effects at the maximum dose.

Reproductive toxicity data on DEGHE:

In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422), groups of 12 male and 12 female CD rats were administered DEGHE via the diet at concentrations supplying 0, 100, 300 and 1000 mg/kg/day. Females were dosed daily for two weeks prior to breeding, through breeding (up to two weeks), gestation (three weeks), and through post-partum day 4. Females were necropsied on post-partum day 5. The males were dosed for two weeks prior to breeding and continuing through breeding (two weeks) up until necropsy (test day 34). Effects on reproductive and neurological function as well as general toxicity were evaluated. In addition, post-mortem examinations included a gross necropsy of the adults with collection of organ weights and extensive histopathologic examination of tissues. Litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were also assessed.

Dietary administration of DEGHE to CD rats resulted in slight treatment-related decreases in body weights of males and females given 1000 mg/kg/day. Decreased maternal body weights persisted throughout the gestation and lactation phases of the study. Females given 1000 mg/kg/day had treatment-related increases in serum ALT and ALP activities, along with increases in relative liver weight. These effects corresponded with treatment-related histopathologic changes in the liver that consisted of very slight panlobular hepatocyte hypertrophy. Males given 1000 mg/kg/day had treatment-related increases in relative liver weight, which corresponded with the histological alteration of very slight periportal hepatocyte hypertrophy. At 1000 mg/kg/day, other findings of lesser significance included slightly increased blood urea nitrogen and decreased absolute thymus weights in females, decreased urine pH and increased relative kidney weights in males. Additional histopathologic findings of minor toxicological significance at 1000 mg/kg/day included very slight diffuse acinar hypertrophy of the submandibular salivary gland in males, and very slight atrophy of the mesenteric adipose tissue in females. No adverse effects of DEGHE on neurological function were found.

There were no treatment-related effects at any dose level on mating, conception, fertility and gestation indices, time to mating, gestation length, post-implantation loss, pup survival or pup sex ratio. In addition, there were no adverse gross or histopathological findings in the reproductive organs of males or females at the highest exposure level, despite the presence of treatment-related maternal toxicity at this dose.

Based on the results of this test, the no-observed-effect level (NOEL) for general toxicity was 300 mg/kg/day. The NOEL for reproductive and neurological effects was 1000 mg/kg/day, the highest dose level tested.

Summary:

Although there are no reproductive toxicity data available for EGHE, there are sufficient data available for the structurally related analogues to make a conclusion about the reproductive toxicity of EGHE to support classification and risk assessment.

Repeated dose toxicity data on EGHE do not highlight the reproductive organs as a target organ following inhalation exposure. A continuous breeding study using EGBE, one generation studies using DEGBE and an OECD 422 reproductive screening study using DEGHE all support the conclusion that these substance are not reproductive toxicants up to the limit dose of 1000 mg/kg bw and in the absence of frank maternal toxicity. We note that although some of the studies reported here may have been performed using different guidelines than those currently required under REACH, based on the parameters covered in these studies the overall assessment indicates there would be no reproductive effects expected, in the absence of general toxicity, for EGHE. This is in agreement with the relationship between increasing length of the alkyl chain (see Analogue document) and decrease/absence of reproductive effects for the E-series glycol ethers.

Effects on developmental toxicity

Description of key information

GLP studies in rats and rabbits, conducted similar to OECD guideline 414 are available for ethylene glycol hexyl ether.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

The study methodology followed was equivalent or similar to OECD guideline 414 and was conducted in accordance with the Principles of GLP

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc.
- Age at study receipt: males (69 days upon arrival) and females (62 days upon arrival)
- Weight at study initiation: not specified in the report
- Fasting period before study: none
- Housing: mating - (1:1, male:female), post-mating - females housed individually
- Diet (e.g. ad libitum): Certified Ground Rodent Chow provided ad libitum, except during exposure
- Water (e.g. ad libitum): Municipal water provided ad libitum, except during exposure
- Acclimation period: Animals were quarantined for two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-28 °C
- Humidity (%): 50-61 %
- Air changes (per hr): standard conditions
- Photoperiod (hrs dark / hrs light): 12 hours (light:dark cycle)

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4 chambers made of glass and stainless steel
- Method of holding animals in test chamber: in cages
- Source and rate of air: ambient
- Method of conditioning air: not specified in the report
- Temperature, humidity, pressure in air chamber: mean temperatures - 25.1-26.5 °C, mean RH - 52.9-56.5%
- Air flow rate: 1000 liters/minute
- Air change rate: 14 air changes/hour
- Treatment of exhaust air: Chamber atmospheres containing hexyl CELLOSOLVE were filtered before leaving an exhaust stack

TEST ATMOSPHERE
- Brief description of analytical method used: A Perkin-Elmer Model 3920B gas chromatograph (GC) equipped with a flame ionization detector was used to monitor the hexyl CELLOSOLVE vapor concentrations in the chambers. The GC column was a 10-feet x 1/8 inch (i.d.) stainless steel column packed with 20% SP-2100 on 80/100 mesh Supelcoport (Supelco, Inc., Bellefonte, PA). Calibration of the gas chromatograph was done with dynamically generated gas standards of hexyl CELL0S0LVE prepared by syringe injection of test material into Tedlar (DuPont) gas bags. The series of standards encompassed the entire range of vapor concentrations generated in the exposure chambers. A linear calibration curve was obtained when areas (integration counts) were plotted versus the gas equivalent concentrations of the standards. Each chamber atmosphere was analyzed for hexyl CELLOSOLVE approximately once every 30 minutes during each 6-hour exposure. Daily nominal concentrations (an estimated concentration calculated from the amount of test material delivered and the chamber airflow during the exposure period) were also calculated for each chamber.
- Samples taken from breathing zone: not specified in the report
- Liquid hexyl CELLOSOLVE® was metered from a piston pump into a heated glass evaporator. The temperature in the evaporator was maintained at the lowest level sufficient to vaporize the liquid (33-52°C). The resultant vapor was carried into the chamber by passage of conditioned air through the evaporator. Chamber atmospheres containing hexyl CELLOSOLVE were filtered before leaving an exhaust stack.
The four chambers employed in this study were rectangular in shape, constructed of glass and stainless steel (Wahmann Manufacturing Company, Timonium, MD), and measured approximately 2.1 m x 1 m x 2.1 m (height). Total volume in each chamber was approximately 4320 L. An orifice plate was positioned in the exhaust duct of the chamber and was connected to a Dwyer Magnehelic Pressure Gauge.
Airflow in each chamber was approximately 1000 L/minute (14 air changes per hour) with a t99 (theoretically-derived time required for the chamber to reach 99% of the equilibrium concentration) of approximately 20 minutes.
The chambers were illuminated with artificial room light. Chamber temperature, relative humidity and air flow rate were recorded at least five (5) times during each exposure. Within each chamber, the animal cages were rotated daily to compensate for any possible, but undetected, variation in chamber exposure conditions.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

target concentrations - 0, 20, 40 and 85 ppm
analytical concentrations - 0, 20.8 ± 0.90, 41.1 ± 1.77 and 79.2 ± 10.80 ppm
nominal concentrations - 0, 26.1 ± 1.84, 39.2 ± 1.80 and 91.0 ± 6.28 ppm

Details on mating procedure:

- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1:1 (male:female)
- Length of cohabitation: 5 days
- Further matings after two unsuccessful attempts: [no]
- Verification of same strain and source of both sexes: [no]
- Proof of pregnancy: [vaginal plug] referred to as [day 0] of pregnancy

Duration of treatment / exposure:

6 hours/day, gd 6 to gd 15

Frequency of treatment:

daily from gd 6 to gd 15

Duration of test:

9 days

Dose / conc.:

20.8 ppm (analytical)

Remarks:

± 0.90 ppm

Dose / conc.:

41.1 ppm (analytical)

Remarks:

± 1.77 ppm

Dose / conc.:

79.2 ppm (analytical)

Remarks:

± 10.80 ppm

No. of animals per sex per dose:

25 plug positive females/group

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: based on range-fiding study
- Rationale for animal assignment: random assignment

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily from gd 0 to gd 21

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: gd 0, 6, 9, 12, 15 and 21

FOOD CONSUMPTION AND WATER CONSUMPTION: Yes
- Time schedule for examinations: at intervals gd 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: all organs

OTHER: Hematological examination

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: percent live and dead fetuses

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter] - fetuses examined for soft-tissue examinations

Statistics:

The unit of comparison was the pregnant female or the litter. Results of the quantitative continuous variables (e.g.., maternal body weights, liver weights, etc.) were intercompared for the three exposure groups and a control group for each species by use of Levene's test for equal variances, analysis of variance (ANOVA), and t-tests with Bonferroni probabilities. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated homogeneous variances, and the ANOVA was significant, the pooled t-test was used. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by the separate variance t-test.
Nonparametric data obtained following laparohysterectomy were statistically treated using the Kruskal-Wallis test followed by the Mann-Whitney U test when appropriate. Incidence data were compared using Fisher's Exact Test. For all statistical tests, the fiducial limit of 0.05 (two-tailed) was used as the criterion for significance.

Indices:

Pre-and post-implantation loss, percent live fetuses, sex ratio

Historical control data:

not applicable

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment related clinical signs of toxicity which included urine stains on fur, lacrimation, ocular wetness and encrustation were observed only at 85 ppm were observed during the exposure period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were significant decreases in maternal body weight and weight gain at both the 40 and 85 ppm dose group

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There were significant decreases in food consumption at 85 ppm dose group during the exposure period.

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Water consumption was increased at the 85 ppm dose group during the exposure period.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no changes in the hematological examination.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no changes on maternal organ weights or absolute/relative liver weight

Gross pathological findings:

no effects observed

Description (incidence and severity):

At scheduled necropsy, there were no treatment related findings at maternal gross examination.

Pre- and post-implantation loss:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no changes in gravid uterine weight or gestational body weight

Dose descriptor:

NOEL

Effect level:

20.8 ppm (analytical)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

not specified

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

There were no effects of exposure on gestational parameters including number of corpora lutea per dam, number of total, non-viable or live implantations per litter, pre- and post-implantation loss, sex ratio and fetal body weight. The incidence of fetal malformations and variations were comparable between the treatment groups

Dose descriptor:

NOEL

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

None

Conclusions:

Exposure to ethylene glycol hexyl ether vapor during organogenesis in Fischer 344 rats resulted in transient maternal toxicity at 85 and 40 ppm, and no embryofetal toxicity (including teratogenicity) at any exposure concentrations employed. The "no observable effect level" (NOEL) for maternal toxicity was 20 ppm for rats and the developmental toxicity NOEL was at the ambient temperature generated vapor concentration of about 85 ppm.

Executive summary:

Timed-pregnant Fischer 344 rats, 25 per group, were exposed to ethylene glycol hexyl ether vapor for six hours/day on gestational days (gd) 6 through 15 for rats at target concentrations of 0, 20, 40 or 85 ppm (analytical concentrations of 20.8 ± 0.90, 41.1 ± 1.77 and 79.2 ± 10.80 ppm for the 20, 40 and 85 ppm groups, respectively). Eighty-five ppm was close to the saturated vapor concentration of the material at 20°C. Maternal body weights were measured on gd 0, 6, 9, 12, 15 and 21 (rats). Food and water consumption was measured, for rats only, for three-day intervals throughout gestation. At scheduled sacrifice on gd 21 (rats), maternal body weight, gravid uterine weight, and liver weight were taken. Maternal blood samples were taken examined for hematologic changes including differential leukocyte counts. Ovarian corpora lutea of pregnancy were counted and all uterine implantation sites were identified and recorded: resorptions (early or late), dead fetuses and live fetuses.All live fetuses were examined for external malformations, including cleft palate, and variations. About 50% of the rat fetuses in each litter were examined for visceral malformations and variations, 50% of the fetuses in each litter were examined for craniofacial defects and the other 50% (intact fetuses) were examined for skeletal malformations and variations.

In rats, maternal toxicity was observed at 40 and 85 ppm, including transient reductions in maternal body weight and weight gain during the exposure period and clinical signs of toxicity at 85 ppm, and reduced weight gain during the exposure period at 40 ppm. Maternal food consumption was reduced at 85 ppm during the exposure period (and increased in the postexposure period). Water consumption was increased at 85 ppm during and after the exposure period. There were no treatment-related clinical signs, gross necropsy observations or hematologic changes. Gestational parameters, including corpora lutea, total, nonviable or live implantations per litter, pre- or postimplantation loss, sex ratio or fetal body weights (males, females or total) per litter, were unaffected by exposures. There were no effects of treatment on the incidence of malformations or variations when examined by individual finding, findings by category or total findings.

Exposure to ethylene glycol hexyl ether vapor during organogenesis in Fischer 344 rats resulted in transient maternal toxicity at 85 and 40 ppm, and no embryofetal toxicity (including teratogenicity) at any exposure concentrations employed. The "no observable effect level" (NOEL) for maternal toxicity was 20 ppm for rats and the NOEL for developmental toxicity was 85 ppm.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

The study methodology followed was equivalent or similar to OECD guideline 414 and was conducted in accordance with the Principles of GLP

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Dutchland Laboratories Inc.
- Age at study receipt: approximately 5-5 1/2 months old
- Weight at study initiation: at least 2.5 kg
- Fasting period before study: none
- Housing: mating - (1:1, male:female), post-mating - females housed individually
- Diet (e.g. ad libitum): Prolab Certified Rabbit Food provided ad libitum, except during exposure
- Water (e.g. ad libitum): Municipal water provided ad libitum, except during exposure
- Acclimation period: Animals were quarantined for two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-28 °C
- Humidity (%): 50-61 %
- Air changes (per hr): standard conditions
- Photoperiod (hrs dark / hrs light): 12 hours (light:dark cycle)

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4 chambers made of glass and stainless steel
- Method of holding animals in test chamber: in cages
- Source and rate of air: ambient
- Method of conditioning air: not specified in the report
- Temperature, humidity, pressure in air chamber: mean temperatures - 25.1-26.5 °C, mean RH - 52.9-56.5%
- Air flow rate: 1000 liters/minute
- Air change rate: 14 air changes/hour
- Treatment of exhaust air: Chamber atmospheres containing hexyl CELLOSOLVE were filtered before leaving an exhaust stack

TEST ATMOSPHERE
- Brief description of analytical method used: A Perkin-Elmer Model 3920B gas chromatograph (GC) equipped with a flame ionization detector was used to monitor the hexyl CELLOSOLVE vapor concentrations in the chambers. The GC column was a 10-feet x 1/8 inch (i.d.) stainless steel column packed with 20% SP-2100 on 80/100 mesh Supelcoport (Supelco, Inc., Bellefonte, PA). Calibration of the gas chromatograph was done with dynamically generated gas standards of hexyl CELL0S0LVE prepared by syringe injection of test material into Tedlar (DuPont) gas bags. The series of standards encompassed the entire range of vapor concentrations generated in the exposure chambers. A linear calibration curve was obtained when areas (integration counts) were plotted versus the gas equivalent concentrations of the standards. Each chamber atmosphere was analyzed for hexyl CELLOSOLVE approximately once every 30 minutes during each 6-hour exposure. Daily nominal concentrations (an estimated concentration calculated from the amount of test material delivered and the chamber airflow during the exposure period) were also calculated for each chamber.
- Samples taken from breathing zone: not specified in the report
- Liquid hexyl CELLOSOLVE® was metered from a piston pump into a heated glass evaporator. The temperature in the evaporator was maintained at the lowest level sufficient to vaporize the liquid (33-52°C). The resultant vapor was carried into the chamber by passage of conditioned air through the evaporator. Chamber atmospheres containing hexyl CELLOSOLVE were filtered before leaving an exhaust stack.
The four chambers employed in this study were rectangular in shape, constructed of glass and stainless steel (Wahmann Manufacturing Company, Timonium, MD), and measured approximately 2.1 m x 1 m x 2.1 m (height). Total volume in each chamber was approximately 4320 L. An orifice plate was positioned in the exhaust duct of the chamber and was connected to a Dwyer Magnehelic Pressure Gauge.
Airflow in each chamber was approximately 1000 L/minute (14 air changes per hour) with a t99 (theoretically-derived time required for the chamber to reach 99% of the equilibrium concentration) of approximately 20 minutes.
The chambers were illuminated with artificial room light. Chamber temperature, relative humidity and air flow rate were recorded at least five (5) times during each exposure. Within each chamber, the animal cages were rotated daily to compensate for any possible, but undetected, variation in chamber exposure conditions.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

target concentrations - 0, 20, 40 and 85 ppm
analytical concentrations - 0, 20.8 ± 0.90, 41.1 ± 1.77 and 79.2 ± 10.80 ppm
nominal concentrations - 0, 26.1 ± 1.84, 39.2 ± 1.80 and 91.0 ± 6.28 ppm

Details on mating procedure:

- Impregnation procedure: [copulation]
- If cohoused:
- M/F ratio per cage: not specified in the report
- Further matings after two unsuccessful attempts: [no]
- Verification of same strain and source of both sexes: [no]
- Proof of pregnancy: [copulation] referred to as [day 0] of pregnancy

Duration of treatment / exposure:

6 hours/day, gd 6 to gd 18

Frequency of treatment:

daily from gd 6 to gd 18

Duration of test:

13 days

Dose / conc.:

20.8 ppm (analytical)

Remarks:

± 0.90 ppm

Dose / conc.:

41.1 ppm (analytical)

Remarks:

± 1.77 ppm

Dose / conc.:

79.2 ppm (analytical)

Remarks:

± 10.80 ppm

No. of animals per sex per dose:

22 mated rabbits/group

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: based on range-fiding study
- Rationale for animal assignment: random assignment

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily from gd 0 to gd 29

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: gd 0, 6, 9, 12, 18 and 29

FOOD CONSUMPTION AND WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: all organs

OTHER: Hematological examination

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: percent live and dead fetuses

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]

Statistics:

The unit of comparison was the pregnant female or the litter. Results of the quantitative continuous variables (e.g.., maternal body weights, liver weights, etc.) were intercompared for the three exposure groups and a control group for each species by use of Levene's test for equal variances, analysis of variance (ANOVA), and t-tests with Bonferroni probabilities. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated homogeneous variances, and the ANOVA was significant, the pooled t-test was used. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by the separate variance t-test.
Nonparametric data obtained following laparohysterectomy were statistically treated using the Kruskal-Wallis test followed by the Mann-Whitney U test when appropriate. Incidence data were compared using Fisher's Exact Test. For all statistical tests, the fiducial limit of 0.05 (two-tailed) was used as the criterion for significance.

Indices:

Pre-and post-implantation loss, percent live fetuses, sex ratio

Historical control data:

not applicable

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Maternal body weights were comparable between groups, however, there were significant reductions in body weight gains in the 85 ppm group, during the exposure period.

Pre- and post-implantation loss:

no effects observed

Details on maternal toxic effects:

There were no treatment related clinical signs of toxicity during the gestation period.

Dose descriptor:

NOEL

Effect level:

41.1 ppm (analytical)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

There were no effects of treatment on any parameters examined, including number of corpora lutea, number of total, non-viable and viable implantations, pre- and post-implantation loss, sex ratio and fetal body weights. The incidence and frequency of rabbit fetal malformations and variations observed were comparable between the groups.

Dose descriptor:

NOEL

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

none

Conclusions:

Exposure to ethylene glycol hexyl ether vapor during organogenesis in New Zealand White rabbits resulted in transient maternal toxicity at 85 ppm, and no embryofetal toxicity (including teratogenicity) at any exposure concentrations employed. The "no observable effect level" (NOEL) for maternal toxicity was 40 ppm for rabbits and the developmental toxicity NOEL was at the ambient temperature generated vapor concentration of about 85 ppm.

Executive summary:

Timed-pregnant Fischer 344 rats, 25 per group, were exposed to ethylene glycol hexyl ether vapor for six hours/day on gestational days (gd) 6 through 15 for rats at target concentrations of 0, 20, 40 or 85 ppm (analytical concentrations of 20.8 ± 0.90, 41.1 ± 1.77 and 79.2 ± 10.80 ppm for the 20, 40 and 85 ppm groups, respectively). Eighty-five ppm was close to the saturated vapor concentration of the material at 20°C. Maternal body weights were measured on gd 0, 6, 9, 12, 15 and 21 (rats). Food and water consumption was measured, for rats only, for three-day intervals throughout gestation. At scheduled sacrifice on gd 21 (rats), maternal body weight, gravid uterine weight, and liver weight were taken. Maternal blood samples were taken examined for hematologic changes including differential leukocyte counts. Ovarian corpora lutea of pregnancy were counted and all uterine implantation sites were identified and recorded: resorptions (early or late), dead fetuses and live fetuses.All live fetuses were examined for external malformations, including cleft palate, and variations. About 50% of the rat fetuses in each litter were examined for visceral malformations and variations, 50% of the fetuses in each litter were examined for craniofacial defects and the other 50% (intact fetuses) were examined for skeletal malformations and variations.

For rabbits, maternal toxicity was observed at 85 ppm in the form of transient weight gain depression during the exposure period. There were no treatment-related clinical signs, or effects on hematologic parameters or gross necropsy observations. Gestational parameters were unaffected by treatment, including number of corpora lutea, total, nonviable or live implantations per litter, sex ratio, pre- and postimplantation loss, and fetal body weights (total, males or females) per litter. There were no significant changes in the incidence of any individual malformations, malformations by category (external, visceral or skeletal) or of total malformations. There were also no treatment-related changes in the incidence of individual variations, variations by category or of total variations.

Exposure to ethylene glycol hexyl ether vapor during organogenesis in New Zealand White rabbits resulted in transient maternal toxicity at 85 ppm, and no embryofetal toxicity (including teratogenicity) at any exposure concentrations employed. The "no observable effect level" (NOEL) for maternal toxicity was 40 ppm for rabbits and the NOEL for developmental toxicity was 85 ppm.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Additional information

Exposure to ethylene glycol hexyl ether vapor during organogenesis in Fischer 344 rats resulted in transient maternal toxicity at 85 and 40 ppm, and no embryofetal toxicity (including teratogenicity) at any exposure concentrations employed. The "no observable effect level" (NOEL) for maternal toxicity was 20 ppm for rats: the NOEL for developmental toxicity was 85 ppm.

Exposure to ethylene glycol hexyl ether vapor during organogenesis in New Zealand White rabbits resulted in transient maternal toxicity at 85 ppm, and no embryofetal toxicity (including teratogenicity) at any exposure concentrations employed. The "no observable effect level" (NOEL) for maternal toxicity was 40 ppm for rabbits; the NOEL for developmental toxicity was 85 ppm.

Justification for classification or non-classification

No developmental effects were observed in rats or rabbits. Therefore, no classification for reproductive toxicity is required.