Reaction mass of 2-methylpentane and Hexanol, branched and linear and diisopropyl ether

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The test item has the main constituent of isopropyl ether (approximately 50%). Minor constituents comprise propylene dimers (approximately 20% C6 hydrocarbons, mainly C6 alkenes), propylene trimers (approximately 10% C9 hydrocarbons), hexanols (approximately 10%) and C3 alcohols (approximately 10% consisting of both isopropanol and n-propanol). Effect on fertility has been assessed using data from well-conducted one-generation toxicity studies of the two read across substances diisopropyl ether (DIPE) and ethyl tert-butyl ether (ETBE). One-generation reproductive toxicity studies (Gray et al. 2014) have been conducted by inhalation exposure in rats to five blends of gasoline/oxygenated mixtures which included gasoline blended with the two read-across substances ethyl tert-butyl ether (G/ETBE) or diisopropyl ether (G/DIPE). The C6 and C9 hydrocarbons (alkanes and alkenes) present in the test item are also present in the gasoline composition used in these studies. The studies were of regulatory design (US EPA 1998a) and were well conducted with a negative control animal group receiving air only and a positive control group treated with the baseline gasoline vapour concentrate (BGVC) allowing assessment of the effects of the two read-across substances, DIPE and ETBE. The data generated for both gasoline blends (G/ETBE and G/DIPE) showed minimal toxicity up to and including the highest dose level of 20000 mg/m³ in both male and female parental animals. At these minimally toxic exposure levels in a one-generation reproductive toxicity setting, both read-across substances demonstrated no significant effects on reproduction capabilities of parent male and female rats or changes in the development of F1 offspring.

Link to relevant study records

Reference

Reference 1

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

US EPA, 1998 - Health Effects Guidelines Reproduction and Fertility Effects. OPPTS 870.3800 EPA—712-C-98-208.

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

CD (Sprague–Dawley derived) [Crl: CD@ IGS BR] albino rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rats (approximately 27–29 days of age) were received from Charles River Laboratories (Kingston, NY) for each study. Females were nulliparous and non-pregnant. Animals were acclimated for at least 13 days after receipt and examined to confirm suitability for study. After selection for study (P0 generation) each rat was identified with a metal ear-tag bearing its assigned animal number.
In the two-generation studies, selected F1 parental animals were ear-tagged with a unique number at the time of selection. Animals considered suitable for study on the basis of pre-test physical examinations and body weight data were randomly assigned, by sex, to control or treated groups in an attempt to equalize mean group body weights. Individual weights of animals placed on test were within ±20% of the mean weight for each sex for each study. Animals were approximately 40 – 42 days of age at initiation of exposure. Currently acceptable practices of good animal husbandry were followed (National Academy of Sciences, 1996). Certified Rodent Diet, No. 5002; (Meal) (PMI Nutrition International, St. Louis, Missouri) was available without restriction except during exposure. Water was available without restriction, except during exposures, via an automated watering system. Food and water were analysed for purity on a regular basis and there were no known contaminants which were expected to interfere with the results of this study.
Animals were individually housed in suspended stainless steel cages with wire mesh fronts and floors with the following exceptions: When mated, one male and one female were co-housed continuously (except during exposure) until mating occurred or for a maximum of 14 days; during lactation, dam and litter were housed together in a solid plastic ‘‘shoebox’’ cage with ground corn cob bedding, changed at least weekly until weaning.
A 12 h light/dark cycle controlled via an automatic timer was provided. Temperature and relative humidity were monitored in accordance with testing facility SOPs and maintained within the specified range (18 – 26 °C, and 30 – 70%, respectively) to the maximum extent possible. Air changes were maintained within a range of 10 – 15/h. Excursions outside the specified range were not considered to have affected the integrity of the study.

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

The test material was administered as a vapour in the breathing air of the animals (whole body exposure). Maximum exposure levels were 50% of the lower flammable limits of these test materials.
The exposure schedules are summarized on Table 3. P0 males and females received 70 consecutive days (10 weeks) of exposure prior to mating for 6 h/day, 7 days/week and continued to be exposed during the 14-day mating period. Mated females were exposed daily from Gestation day 0 (GD0) through GD19.
Females were not exposed after GD19 through lactation day 4 (LD4). Beginning on LD5, nursing P0 females were exposed daily until weaning on LD28. P0 females with no confirmed day of mating continued exposure for 25 days following completion of the mating period. P0 females with no confirmed day of mating but with evidence of pregnancy (weight gain) were exposed until presumed GD19 and females with a confirmed day of mating that did not deliver were sacrificed on presumed GD25. P0 males were exposed daily and sacrificed on the date proximate to the date of the first litter weaning or after the last day F1 pups were delivered (approximately 16 – 20 weeks of exposure).
Two generation studies: Selected F1 males and females (26 mating pairs/group) started exposure at weaning on LD28 and continued treatment for 10 weeks prior to pairing to produce the F2 generation. Exposure continued through the 14 day mating period.
Mated F1 females were exposed daily from GD0 through GD19. F1 females were not exposed after GD19 through lactation day 4 (LD4). Beginning on LD5, nursing F1 females were exposed daily until weaning of the F2 generation on LD28. F1 males were exposed daily and sacrificed on the date proximate to the date of the first F2 litter weaning.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A nominal exposure concentration was calculated. The flow of air through the chamber was monitored using appropriate calibrated equipment. The test substance consumed (weight difference of the 5 gallon cylinder) during the exposure (mg) was divided by the total volume of air (m³) passing through the chamber (volumetric combined flow rate for the 2 chambers times total exposure time) to calculate the nominal concentration mg/m³).
During each exposure, measurements of airborne concentrations were performed in the animals’ breathing zone at least 4 times using an appropriate sampling procedure and infrared (IR) spectrophotometric analytical procedure. Also, one charcoal tube sample was collected per chamber per week and analyzed by gas chromatography (GC) to characterize at least 18 major components (comprising at least 80% by weight of the test substance) to show test substance stability and comparison between the neat liquid test substance and the vaporized test atmospheres.
During each week of exposure, particle size determinations were performed using a TSI Aerodynamic Particle Sizer to characterize the aerodynamic particle size distribution of any aerosol present. The samples were drawn for 20 s at a flow rate of 5.00 L/min. The mass median aerodynamic diameter, geometric standard deviation and total mass concentration were calculated based on the amount of particles collected.

Duration of treatment / exposure:

Exposure schedule was as follows:
Premating phase 70 days: P0 males/females
Mating phase 14 days: P0 males/females
Gestation phase GD0 - 19: P0 males/females
Gestation/Lactation phase GD20 - LD4: P0 males
Lactation phase L5 - 28: P0 males/females F1 males/females indirectly via lactation
Weaning phase LD28: Female P0 dams and F1 pups not mating P0 males (sacrificed)

Frequency of treatment:

6 hours per day, 7 days per week

Dose / conc.:

2 034 mg/m³ air (analytical)

Remarks:

±128 mg/m³

Dose / conc.:

10 450 mg/m³ air (analytical)

Remarks:

±621 mg/m³

Dose / conc.:

20 230 mg/m³ air (analytical)

Remarks:

±775 mg/m³

No. of animals per sex per dose:

The test materials were administered via whole-body inhalation exposures to Sprague Dawley rats (26/sex/dose group) at target concentrations of 0, 2000, 10,000 and 20,000 mg/m³ for 6 h/day, 7 days/week

Control animals:

yes, concurrent vehicle

Positive control:

None

Parental animals: Observations and examinations:

See any other information on materials and methods

Litter observations:

See any other information on materials and methods

Postmortem examinations (parental animals):

See any other information on materials and methods

Postmortem examinations (offspring):

See any other information on materials and methods

Statistics:

For continuous data [body weights, body weight change, food consumption, organ weight data, gestation length, pup body weights, number of pups (live, dead, total), mean age-to-criteria for vaginal opening and preputial separation], mean values of all exposure groups were compared to the mean value for the concurrent control group at each time interval. The litter was considered the operative unit for offspring data (e.g., pups/litter). Evaluation of equality of group means was made with standard one-way analysis of variance (ANOVA) using the F ratio followed by Dunnett’s test (Dunnett, 1955, 1964; Dunlap and Duffy, 1975) if needed.
For sperm and ovary data the following parameters were analyzed statistically: mean sperm count (testicular sperm count and caudal epididymal sperm count), sperm morphology, and motility data and numbers of primordial and growing follicles by ovary and total. If a significant difference occurred (p < 0.05) between groups using the nonparametric Kruskal–Wallis test, the Wilcoxon (Mann–Whitney U) test was used for pair-wise comparisons of each treated group to the vehicle control group.
Incidence data [mortality, mating indices, pregnancy rates, male fertility indices, live birth indices, and pup viability indices (Days 0–4) and lactation indices (Days 4–28)] were analyzed using the Chi-square test (2 x n). If Chi-square analysis was not significant, no additional analyses were performed (Mantel, 1963; Dunlap et al., 1981). If Chi-square was significant, a Fisher Exact Test with Bonferroni correction was performed to identify differences between the groups.
Statistical methods for the GFAP assay employed separate one-way analysis of variance (ANOVA) for each of the brain areas from male and female rats (JMP, SAS Institute, 1995). The significance level was p < 0.05 and, to ensure detection of between group treatment effects, the Least Significance-Difference test (Keppel, 1973) was used for post hoc analyses.

Reproductive indices:

See any other information on materials and methods

Offspring viability indices:

See any other information on materials and methods

Clinical signs:

no effects observed

Description (incidence and severity):

There were no remarkable clinical observations reported

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Inhalation exposure, therefore, dermal effect not monitored

Mortality:

no mortality observed

Description (incidence):

There were no significant effects of treatment on survival in the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant effects obsered on body weight or weight gain. No effects on maternal body weight gains occurred during gestation (GD0–20) and lactation (LD1–28)

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was comparable to concurrent controls

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No remarkable histopathologic changes were reported in the study.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Light hydrocarbon nephropathy was strongly indicated in all studies by the presence of hyaline droplets in kidneys of 20,000 mg/m³ male rats, an effect already known for decade attributable to the hydrocarbon carrier.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrus cyclicity parameters were comparable between exposed and concurrent control groups.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Semen parameters were comparable between exposed and concurrent control groups.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences in male and female fertility or reproductive performance with exposure to any test material.

No treatment related macroscopic or microscopic changes were seen in male or female reproductive organs.

Dose descriptor:

NOAEC

Effect level:

2 000 mg/m³ air (nominal)

Based on:

test mat. (total fraction)

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: Parental effect

Dose descriptor:

NOAEC

Effect level:

20 000 mg/m³ air (nominal)

Based on:

test mat. (total fraction)

Sex:

male/female

Remarks on result:

other: Reproductive parameters

Remarks:

No effects

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No adverse effects were seen on offspring organ weights

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no findings of malformations

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

The NOAEL was the highest exposure tested for G/DIPE as no differences from controls were seen for fertility, days to mating, estrus cycle length, sperm counts or morphology or developmental parameters in pups.

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

20 000 mg/m³ air (nominal)

Based on:

test mat. (total fraction)

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

no

Effects on offspring (F1) from exposure of parents to G/DIPE at 20,000 mg/m³.

Endpoints	Control range	G/DIPE
		F1
Litter size (Pups delivered)	12.1 – 14.5	13.1
Pup weights (g) sexes combined
LD 1	6.8 – 7.4	7
LD 4	9.7 – 11.4	10.5*
LD 7	13.5 – 14.7	14.3
LD 14	23.7 – 26.0	24.3
LD 21	38.7 – 42.5	41.2
LD 28	69.5 – 78.8	76.9
Pup survival sexes combined
LD 0–4	93.1 – 99.6%	98.6%**^a
LD 5–21	98.7 – 100%	95.5%
Other endpoints
Spleen weight (g)	0.263 – 0.327	0.299
GFAP assay		NE
Vaginal opening (day)	35.3 – 37.5	NE
Preputial separation (day)	46.2 – 46.4	NE

Statistical significance based upon comparison to each study’s concurrent control.

NE = not evaluated, * p < 0.05., ** p < 0.01,^aSignificantly better survival than concurrent control (93.1%)

Effects on female rats (P0) from exposure to vapour condensates of G/DIPE at 20,000 mg/m³.

Endpoints	Control range	G/DIPE
		P0
Premating body weight gain (g)	108 – 144 (P0) 175 – 200 (F1)	126
% of control		97.7%
Gestation day 0–20 weight gain (g)	113 – 129 (P0) – 120, 121 (F1)	122
% of control		107.9%
Lactation day 21–28 weight gain (g)	-3 to 13 (P0) 7 – 15 (F1)	8
% of control		(control-1)
Lung, discolored foci (macroscopic)	0/26–4/26	10/26
% of rats	0–15.4%	38.5%
Relevant organ weight changes		NE

Statistical significance based upon comparison to each study’s concurrent control.

NE = no effect, * p < 0.05, ** p < 0.01.

 

Effects on male rats (P0) from exposure to vapour condensates of G/DIPE at 20,000 mg/m³.

Endpoints	Control range	G/DIPE
		P0
Premating body weight gain (g)	225–313 (P0) 350–379 (F1)	284
% of control	95.8%	97.3%
Lung, discoloured foci (macroscopic)	0/26–4/26	6/26
% of rats	0–15.4%	23.1%
Relevant organ weight changes		kidney, liver ↑

Statistical significance based upon comparison to each study’s concurrent control.

* p < 0.05, ** p < 0.01.

Effects on reproductive parameters on parental animals (P0) from exposure to vapour condensates of gasoline or gasoline/oxygenate G/DIPE at 20,000 mg/m³.

Endpoint	Control range	G/DIPE
		P0
Male Fertility	20/25 – 25/26	23/26
	80 – 96.2%	88.5%
Female Fertility	21/24 – 25/25	23/23
	87.5 – 100%	100.0%
Number of Litters	20 – 25	23
Days to Mating	2.4 – 3.4	3.1
Estrus Cycle Length (days)	4.2 – 5.2, 5.7	4.3
Semen parameters
Sperm Count, testis (10^6/g)	81.2 – 124.1	103.9
Motility (%)	89 – 96%	93%
Morphology (% abnormal)	0.6 – 1.7%^a	0.4% *^b
Epididymal sperm count (10^6/g)	753.6 – 956.7	927.9

Statistical significance based upon comparison to each study’s concurrent control.

**p < 0.01, * p < 0.05,^bSignificantly fewer abnormal sperm than concurrent control (7.4% abnormal)

Conclusions:

In this one generation reproductive toxicity study with a gasoline blend containing 17.8% di-isopropyl ether, no developmental effects or decreases in fertility were observed and thus the NOAEC in this study was set to the highest does tested being 20.000 mg/m³.

Executive summary:

Vapour condensates of baseline gasoline (BGVC), or gasoline-blended with methyl tertiary butyl ether (G/MTBE), ethyl t-butyl ether (G/ETBE), t-amyl methyl ether (G/TAME), diisopropyl ether (G/DIPE), ethanol (G/EtOH), or t-butyl alcohol (G/TBA) were evaluated for reproductive toxicity in rats at target concentrations of 2000, 10,000, or 20,000 mg/m³, 6 h/day, 7 days/week. BGVC and G/MTBE were assessed over two generations, the others for one generation. BGVC and G/MTBE F1 offspring were evaluated for neuropathology and changes in regional brain glial fibrillary acidic protein content. No neurotoxicity was observed. Male kidney weight was increased consistent with light hydrocarbon nephropathy. In adult rats, decreased body weight gain and increased liver weight were seen. Spleen weight decreased in adults and pups exposed to G/TBA. No pathological changes to reproductive organs occurred in any study.

Decreased food consumption was seen in G/TAME lactating females. Transient decreases in G/TAME off-spring weights were observed during lactation. Except for a minor increase in time to mating in G/TBA which did not affect other reproductive parameters, there were no adverse reproductive findings. The NOAEL for reproductive and offspring parameters was 20,000 mg/m³ for all vapour condensates except for lower offspring NOAELs of 10,000 mg/m³ for G/TBA and 2000 mg/m³ for G/TAME.

Thus, it can be concluded that di-isopropyl ether did not impact reproductive toxicity in this one-generation reproductive toxicity study.

Reference 2

Endpoint:

one-generation reproductive toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

The test item has the main constituent of isopropyl ether (approximately 50%). Minor constituents comprise propylene dimers (approximately 20% C6 hydrocarbons, mainly C6 alkenes), propylene trimers (approximately 10% C9 hydrocarbons), hexanols (approximately 10%) and C3 alcohols (approximately 10% consisting of both isopropanol and n-propanol). Effect on fertility has been assessed using data from well-conducted one-generation toxicity studies of the two read across substances diisopropyl ether (DIPE) and ethyl tert-butyl ether (ETBE).

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

US EPA, 1998 - Health Effects Guidelines Reproduction and Fertility Effects. OPPTS 870.3800 EPA—712-C-98-208.

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

CD (Sprague–Dawley derived) [Crl: CD@ IGS BR] albino rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rats (approximately 27–29 days of age) were received from Charles River Laboratories (Kingston, NY) for each study. Females were nulliparous and non-pregnant. Animals were acclimated for at least 13 days after receipt and examined to confirm suitability for study. After selection for study (P0 generation) each rat was identified with a metal ear-tag bearing its assigned animal number.
In the two-generation studies, selected F1 parental animals were ear-tagged with a unique number at the time of selection. Animals considered suitable for study on the basis of pre-test physical examinations and body weight data were randomly assigned, by sex, to control or treated groups in an attempt to equalize mean group body weights. Individual weights of animals placed on test were within ±20% of the mean weight for each sex for each study. Animals were approximately 40 – 42 days of age at initiation of exposure. Currently acceptable practices of good animal husbandry were followed (National Academy of Sciences, 1996). Certified Rodent Diet, No. 5002; (Meal) (PMI Nutrition International, St. Louis, Missouri) was available without restriction except during exposure. Water was available without restriction, except during exposures, via an automated watering system. Food and water were analysed for purity on a regular basis and there were no known contaminants which were expected to interfere with the results of this study.
Animals were individually housed in suspended stainless steel cages with wire mesh fronts and floors with the following exceptions: When mated, one male and one female were co-housed continuously (except during exposure) until mating occurred or for a maximum of 14 days; during lactation, dam and litter were housed together in a solid plastic ‘‘shoebox’’ cage with ground corn cob bedding, changed at least weekly until weaning.
A 12 h light/dark cycle controlled via an automatic timer was provided. Temperature and relative humidity were monitored in accordance with testing facility SOPs and maintained within the specified range (18 – 26 °C, and 30 – 70%, respectively) to the maximum extent possible. Air changes were maintained within a range of 10 – 15/h. Excursions outside the specified range were not considered to have affected the integrity of the study.

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

The test material was administered as a vapour in the breathing air of the animals (whole body exposure). Maximum exposure levels were 50% of the lower flammable limits of these test materials.
The exposure schedules are summarized on Table 3. P0 males and females received 70 consecutive days (10 weeks) of exposure prior to mating for 6 h/day, 7 days/week and continued to be exposed during the 14-day mating period. Mated females were exposed daily from Gestation day 0 (GD0) through GD19.
Females were not exposed after GD19 through lactation day 4 (LD4). Beginning on LD5, nursing P0 females were exposed daily until weaning on LD28. P0 females with no confirmed day of mating continued exposure for 25 days following completion of the mating period. P0 females with no confirmed day of mating but with evidence of pregnancy (weight gain) were exposed until presumed GD19 and females with a confirmed day of mating that did not deliver were sacrificed on presumed GD25. P0 males were exposed daily and sacrificed on the date proximate to the date of the first litter weaning or after the last day F1 pups were delivered (approximately 16 – 20 weeks of exposure).
Two generation studies: Selected F1 males and females (26 mating pairs/group) started exposure at weaning on LD28 and continued treatment for 10 weeks prior to pairing to produce the F2 generation. Exposure continued through the 14 day mating period.
Mated F1 females were exposed daily from GD0 through GD19. F1 females were not exposed after GD19 through lactation day 4 (LD4). Beginning on LD5, nursing F1 females were exposed daily until weaning of the F2 generation on LD28. F1 males were exposed daily and sacrificed on the date proximate to the date of the first F2 litter weaning.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A nominal exposure concentration was calculated. The flow of air through the chamber was monitored using appropriate calibrated equipment. The test substance consumed (weight difference of the 5 gallon cylinder) during the exposure (mg) was divided by the total volume of air (m³) passing through the chamber (volumetric combined flow rate for the 2 chambers times total exposure time) to calculate the nominal concentration mg/m³).
During each exposure, measurements of airborne concentrations were performed in the animals’ breathing zone at least 4 times using an appropriate sampling procedure and infrared (IR) spectrophotometric analytical procedure. Also, one charcoal tube sample was collected per chamber per week and analyzed by gas chromatography (GC) to characterize at least 18 major components (comprising at least 80% by weight of the test substance) to show test substance stability and comparison between the neat liquid test substance and the vaporized test atmospheres.
During each week of exposure, particle size determinations were performed using a TSI Aerodynamic Particle Sizer to characterize the aerodynamic particle size distribution of any aerosol present. The samples were drawn for 20 s at a flow rate of 5.00 L/min. The mass median aerodynamic diameter, geometric standard deviation and total mass concentration were calculated based on the amount of particles collected.

Duration of treatment / exposure:

Exposure schedule was as follows:
Premating phase 70 days: P0 males/females
Mating phase 14 days: P0 males/females
Gestation phase GD0 - 19: P0 males/females
Gestation/Lactation phase GD20 - LD4: P0 males
Lactation phase L5 - 28: P0 males/females F1 males/females indirectly via lactation
Weaning phase LD28: Female P0 dams and F1 pups not mating P0 males (sacrificed)

Frequency of treatment:

6 hours per day, 7 days per week

Dose / conc.:

2 034 mg/m³ air (analytical)

Remarks:

±128 mg/m³

Dose / conc.:

10 450 mg/m³ air (analytical)

Remarks:

±621 mg/m³

Dose / conc.:

20 230 mg/m³ air (analytical)

Remarks:

±775 mg/m³

No. of animals per sex per dose:

The test materials were administered via whole-body inhalation exposures to Sprague Dawley rats (26/sex/dose group) at target concentrations of 0, 2000, 10,000 and 20,000 mg/m³ for 6 h/day, 7 days/week

Control animals:

yes, concurrent vehicle

Positive control:

None

Parental animals: Observations and examinations:

See any other information on materials and methods

Litter observations:

See any other information on materials and methods

Postmortem examinations (parental animals):

See any other information on materials and methods

Postmortem examinations (offspring):

See any other information on materials and methods

Statistics:

For continuous data [body weights, body weight change, food consumption, organ weight data, gestation length, pup body weights, number of pups (live, dead, total), mean age-to-criteria for vaginal opening and preputial separation], mean values of all exposure groups were compared to the mean value for the concurrent control group at each time interval. The litter was considered the operative unit for offspring data (e.g., pups/litter). Evaluation of equality of group means was made with standard one-way analysis of variance (ANOVA) using the F ratio followed by Dunnett’s test (Dunnett, 1955, 1964; Dunlap and Duffy, 1975) if needed.
For sperm and ovary data the following parameters were analyzed statistically: mean sperm count (testicular sperm count and caudal epididymal sperm count), sperm morphology, and motility data and numbers of primordial and growing follicles by ovary and total. If a significant difference occurred (p < 0.05) between groups using the nonparametric Kruskal–Wallis test, the Wilcoxon (Mann–Whitney U) test was used for pair-wise comparisons of each treated group to the vehicle control group.
Incidence data [mortality, mating indices, pregnancy rates, male fertility indices, live birth indices, and pup viability indices (Days 0–4) and lactation indices (Days 4–28)] were analyzed using the Chi-square test (2 x n). If Chi-square analysis was not significant, no additional analyses were performed (Mantel, 1963; Dunlap et al., 1981). If Chi-square was significant, a Fisher Exact Test with Bonferroni correction was performed to identify differences between the groups.
Statistical methods for the GFAP assay employed separate one-way analysis of variance (ANOVA) for each of the brain areas from male and female rats (JMP, SAS Institute, 1995). The significance level was p < 0.05 and, to ensure detection of between group treatment effects, the Least Significance-Difference test (Keppel, 1973) was used for post hoc analyses.

Reproductive indices:

See any other information on materials and methods

Offspring viability indices:

See any other information on materials and methods

Clinical signs:

no effects observed

Description (incidence and severity):

There were no remarkable clinical observations reported

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Inhalation exposure, therefore, dermal effect not monitored

Mortality:

no mortality observed

Description (incidence):

There were no significant effects of treatment on survival in the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant effects obsered on body weight or weight gain. No effects on maternal body weight gains occurred during gestation (GD0–20) and lactation (LD1–28)

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was comparable to concurrent controls

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No remarkable histopathologic changes were reported in the study.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Light hydrocarbon nephropathy was strongly indicated in all studies by the presence of hyaline droplets in kidneys of 20,000 mg/m³ male rats, an effect already known for decade attributable to the hydrocarbon carrier.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrus cyclicity parameters were comparable between exposed and concurrent control groups.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Semen parameters were comparable between exposed and concurrent control groups.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences in male and female fertility or reproductive performance with exposure to any test material.

No treatment related macroscopic or microscopic changes were seen in male or female reproductive organs.

Dose descriptor:

NOAEC

Effect level:

2 000 mg/m³ air (nominal)

Based on:

test mat. (total fraction)

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: Parental effect

Dose descriptor:

NOAEC

Effect level:

20 000 mg/m³ air (nominal)

Based on:

test mat. (total fraction)

Sex:

male/female

Remarks on result:

other: Reproductive parameters

Remarks:

No effects

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No adverse effects were seen on offspring organ weights

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no findings of malformations

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

The NOAEL was the highest exposure tested for G/DIPE as no differences from controls were seen for fertility, days to mating, estrus cycle length, sperm counts or morphology or developmental parameters in pups.

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

20 000 mg/m³ air (nominal)

Based on:

test mat. (total fraction)

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

no

Effects on offspring (F1) from exposure of parents to G/DIPE at 20,000 mg/m³.

Endpoints	Control range	G/DIPE
		F1
Litter size (Pups delivered)	12.1 – 14.5	13.1
Pup weights (g) sexes combined
LD 1	6.8 – 7.4	7
LD 4	9.7 – 11.4	10.5*
LD 7	13.5 – 14.7	14.3
LD 14	23.7 – 26.0	24.3
LD 21	38.7 – 42.5	41.2
LD 28	69.5 – 78.8	76.9
Pup survival sexes combined
LD 0–4	93.1 – 99.6%	98.6%**^a
LD 5–21	98.7 – 100%	95.5%
Other endpoints
Spleen weight (g)	0.263 – 0.327	0.299
GFAP assay		NE
Vaginal opening (day)	35.3 – 37.5	NE
Preputial separation (day)	46.2 – 46.4	NE

Statistical significance based upon comparison to each study’s concurrent control.

NE = not evaluated, * p < 0.05., ** p < 0.01,^aSignificantly better survival than concurrent control (93.1%)

Effects on female rats (P0) from exposure to vapour condensates of G/DIPE at 20,000 mg/m³.

Endpoints	Control range	G/DIPE
		P0
Premating body weight gain (g)	108 – 144 (P0) 175 – 200 (F1)	126
% of control		97.7%
Gestation day 0–20 weight gain (g)	113 – 129 (P0) – 120, 121 (F1)	122
% of control		107.9%
Lactation day 21–28 weight gain (g)	-3 to 13 (P0) 7 – 15 (F1)	8
% of control		(control-1)
Lung, discolored foci (macroscopic)	0/26–4/26	10/26
% of rats	0–15.4%	38.5%
Relevant organ weight changes		NE

Statistical significance based upon comparison to each study’s concurrent control.

NE = no effect, * p < 0.05, ** p < 0.01.

 

Effects on male rats (P0) from exposure to vapour condensates of G/DIPE at 20,000 mg/m³.

Endpoints	Control range	G/DIPE
		P0
Premating body weight gain (g)	225–313 (P0) 350–379 (F1)	284
% of control	95.8%	97.3%
Lung, discoloured foci (macroscopic)	0/26–4/26	6/26
% of rats	0–15.4%	23.1%
Relevant organ weight changes		kidney, liver ↑

Statistical significance based upon comparison to each study’s concurrent control.

* p < 0.05, ** p < 0.01.

Effects on reproductive parameters on parental animals (P0) from exposure to vapour condensates of gasoline or gasoline/oxygenate G/DIPE at 20,000 mg/m³.

Endpoint	Control range	G/DIPE
		P0
Male Fertility	20/25 – 25/26	23/26
	80 – 96.2%	88.5%
Female Fertility	21/24 – 25/25	23/23
	87.5 – 100%	100.0%
Number of Litters	20 – 25	23
Days to Mating	2.4 – 3.4	3.1
Estrus Cycle Length (days)	4.2 – 5.2, 5.7	4.3
Semen parameters
Sperm Count, testis (10^6/g)	81.2 – 124.1	103.9
Motility (%)	89 – 96%	93%
Morphology (% abnormal)	0.6 – 1.7%^a	0.4% *^b
Epididymal sperm count (10^6/g)	753.6 – 956.7	927.9

Statistical significance based upon comparison to each study’s concurrent control.

**p < 0.01, * p < 0.05,^bSignificantly fewer abnormal sperm than concurrent control (7.4% abnormal)

Conclusions:

In this one generation reproductive toxicity study with a gasoline blend containing 17.8% di-isopropyl ether, no developmental effects or decreases in fertility were observed and thus the NOAEC in this study was set to the highest does tested being 20.000 mg/m³.

Executive summary:

Vapour condensates of baseline gasoline (BGVC), or gasoline-blended with methyl tertiary butyl ether (G/MTBE), ethyl t-butyl ether (G/ETBE), t-amyl methyl ether (G/TAME), diisopropyl ether (G/DIPE), ethanol (G/EtOH), or t-butyl alcohol (G/TBA) were evaluated for reproductive toxicity in rats at target concentrations of 2000, 10,000, or 20,000 mg/m³, 6 h/day, 7 days/week. BGVC and G/MTBE were assessed over two generations, the others for one generation. BGVC and G/MTBE F1 offspring were evaluated for neuropathology and changes in regional brain glial fibrillary acidic protein content. No neurotoxicity was observed. Male kidney weight was increased consistent with light hydrocarbon nephropathy. In adult rats, decreased body weight gain and increased liver weight were seen. Spleen weight decreased in adults and pups exposed to G/TBA. No pathological changes to reproductive organs occurred in any study.

Decreased food consumption was seen in G/TAME lactating females. Transient decreases in G/TAME off-spring weights were observed during lactation. Except for a minor increase in time to mating in G/TBA which did not affect other reproductive parameters, there were no adverse reproductive findings. The NOAEL for reproductive and offspring parameters was 20,000 mg/m³ for all vapour condensates except for lower offspring NOAELs of 10,000 mg/m³ for G/TBA and 2000 mg/m³ for G/TAME.

Thus, it can be concluded that di-isopropyl ether did not impact reproductive toxicity in this one-generation reproductive toxicity study.

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

Study duration:

subchronic

Experimental exposure time per week (hours/week):

42

Species:

rat

Quality of whole database:

The information for this endpoint was taken from a well-constructed toxicity study in rats incorporating two regulatory designed (OECD TG 416 and EPA OPPTS 870.3800) one-generation studies via whole-body inhalation exposure to three dose groups of gasoline vapour condensates mixed with either ETBE (G/ETBE) or DIPE (G/DIPE). The study was well-conducted, having a negative control group of rats receiving air only and also a positive control group receiving the baseline gasoline vapour condensate without oxygenate (BGVC). Both read-across substances, DIPE and ETBE, in the presence of hepatotoxicity and renal toxicity in males at the highest dose level of 20000 mg/m3, together with some small but statistically significant reduction in body weight gain in males and females at this dose level, did not demonstrate any reproductive toxicity effects in P0 or F1 generations. The NOAEC for reproductive and offspring effects was considered to be the highest dose level of 20000 mg/m3.

Effects on developmental toxicity

Description of key information

The test item has the main constituent of isopropyl ether (approximately 50%). Minor constituents comprise propylene dimers (approximately 20% C6 hydrocarbons, mainly C6 alkenes), propylene trimers (approximately 10% C9 hydrocarbons), hexanols (approximately 10%) and C3 alcohols (approximately 10% consisting of both isopropanol and n-propanol). Developmental toxicity has been assessed in three regulatory studies with the read-across substances, DIPE or ETBE. DIPE was investigated in rats (Dalbey and Feuston, 1996) via inhalation at concentrations of 0, 430, 3095 and 6745 ppm. ETBE was investigated in rabbits (Asano et al. 2011) and in rats (Aso et al. 2014) via oral gavage administration (0, 100, 300 and 1000 mg/kg/day). All three studies were of regulatory design with sufficient numbers of animals per group for the evaluation. All studies included appropriate vehicle control groups with pregnant rats being treated during gestation days 6-15 (Dalbey and Feuston, 1996) or 5 - 19 (Aso et al. 2014) and rabbits during gestation days 6-27 (Asano et al. 2011). In the rat study with DIPE, there was transient tolerated maternal toxicity at the high dose level and an increased incidence of rudimentary 14^th ribs in the foetuses from dams treated at 6745 and 3095 ppm. The toxicological significance of this finding was considered equivocal by the authors and not indicative of a teratogenic effect and also was not observed in the oral gavage study with ETBE up to and including the high dose level of 1000 mg/kg/day. In the rabbit study, transient minimal toxicity was reported in the dams at 1000 mg/kg/day but no effects on reproduction or foetal development parameters were observed.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY
- Age at study initiation: Rats were approximately 9 weeks old when received and approximately 11 weeks old when breeding began
- Weight at study initiation: Not reported
- Fasting period before study: None
- Housing: Individually housed in 1 cubic metre (H-1000) inhalation chambers.
- Diet (e.g. ad libitum): Certified Purina Rodent Chow 5002 ad libitum except during exposure period
- Water (e.g. ad libitum): Ad libitum except during exposure period
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22 ºC
- Humidity (%): 40 to 60%
- Air changes (per hr): At least 12/hour
- Photoperiod (hrs dark / hrs light): 12 hours: 12 hours

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: H-1000 1 cubic-metre chambers
- Source and rate of air: HEPA-filtered room air; rate of air flow = 290 lpm
- Temperature, humidity, pressure in air chamber: Mean temperature within the chambers was in the range of 23 to 24 ºC and relative humidity was 60 to 67%. Pressure was not reported.
- Air flow rate: 290 lpm
- Air change rate: 12 changes/hour
- Treatment of exhaust air: Air exiting the chambers was cleaned by passage through charcoal beds


TEST ATMOSPHERE
- Brief description of analytical method used: Samples of air (approximately 50 to 250 µL) from the chambers were drawn into a gas-tight syringe and the air was injected directly into a gas chromatograph with a flame ionization detector and a fused silica column. In addition, 500 µL samples of air from the chambers were periodically taken for analysis by gas chromatography/mass spectroscopy.

VEHICLE (if applicable)
- Justification for use and choice of vehicle: No vehicle

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of air (approximately 50 to 250 µL) from the chambers were drawn into a gas-tight syringe and the air was injected directly into a gas chromatograph with a flame ionization detector and a fused silica column. Analyzed concentrations included both DIPE and total hydrocarbons. In addition, 500 µL samples of air from the chambers were periodically taken for analysis by gas chromatography/mass spectroscopy. Time points of analysis were not reported.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: Not reported
- Proof of pregnancy: sperm plug/sperm in the vaginal lavage fluid referred to as day 0 of pregnancy

Duration of treatment / exposure:

6 hours/day on Days 6 to 15 of gestation

Frequency of treatment:

6 hours/day on Days 6 to 15 of gestation

Duration of test:

Approximately 5 weeks (acclimation = 2 weeks and study duration = approximately 3 weeks)

Dose / conc.:

430 ppm (analytical)

Remarks:

equivalent to 1800 mg/m³, basis analytical conc.

Dose / conc.:

3 095 ppm (analytical)

Remarks:

equivalent to 12940 mg/m³, basis analytical conc.

Dose / conc.:

6 745 ppm (analytical)

Remarks:

equivalent to 28200 mg/m³, basis analytical conc.

No. of animals per sex per dose:

22 females/group

Control animals:

yes, concurrent no treatment

yes, sham-exposed

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded on Days 0, 6, 13, 16, and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Publication states that "all organs were examined grossly;" however, specific organs were not reported.

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

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No data

Statistics:

ANOVA, Fisher's exact, or Dunnett's tests were used

Indices:

None reported

Historical control data:

None reported

Details on maternal toxic effects:

Maternal toxic effects: yes

Details on maternal toxic effects:
Lacrimation and salivation were noted in a few DIPE-exposed females at the highest concentration during, or immediately following, exposures. The animals returned to normal appearance shortly after cessation of each daily exposure.

In general, animals housed in chambers gained less weight and consumed less food during the exposure period than the untreated controls (statistically significant at 6745 ppm relative to both of the control groups).

No treatment-related effects were noted at the time of macroscopic examination.

Serum chemistry endpoints were not adversely affected by exposure to DIPE vapors.

Statistically significant decrease in body weight gains were seen on gestation days 6 to 16 at all dose levels (compared to untreated controls for the low- and mid-dose groups and compared to both controls at the high-dose group).

Statistically significant decrease was seen in food consumption on gestation days 6 to 16 at the mid- and high-dose groups (compared to untreated controls on gestation days 6 to 13 and compared to both control groups on gestation days 13 to 16).

Reproductive parameters (i.e., number of pregnant females, percent preimplantation loss, percent resorptions, and litter sizes) were not affected by exposure.

Dose descriptor:

NOEC

Effect level:

430 ppm (analytical)

Based on:

test mat. (dissolved fraction)

Basis for effect level:

other: maternal toxicity

Remarks on result:

not determinable

Remarks:

no NOAEC identified by the authors, as these effects were considered transient and non-adverse by the authors arguing that this "variation" is not conclusive evidence of developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Fetal development (i.e., fetal body weight) was not affected by exposure.

Evaluations of fetal skeletons revealed a significant increase in rudimentary (small, discrete ossification) or short (less than one-half the length of the preceding rib) 14th ribs in fetuses exposed to DIPE at concentrations of 3095 and 6745 ppm. All of the observed 14th ribs were rudimentary except for 2 fetuses from each of the mid- and high-dose groups that had either bilateral short 14th ribs or bilateral short and rudimentary 14th ribs. No other exposure-related findings were noted at the time of fetal evaluations. The study authors have stated that "the observed increase in the incidence of rudimentary 14th ribs does not appear to be indicative of an adverse effect on development" at the concentrations tested.

Dose descriptor:

NOEC

Effect level:

430 ppm (analytical)

Based on:

test mat. (dissolved fraction)

Sex:

male/female

Basis for effect level:

other: slight skeletal effects

Remarks on result:

other: no NOAEC identified by the authors, as these effects were considered transient and non-adverse by the authors arguing that this "variation" is not conclusive evidence of developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

No developmental effects in the absence of maternal toxicity were observed. The NOAEC in this study for maternal effects as well as developmental effects can be set to 430 ppm.

Executive summary:

A NOAEC was not reported by the study authors. Review of the study data suggests that a NOAEC of 430 ppm can be derived for maternal toxicity based on the decrease in body weight gain and food consumption at higher concentrations, and a NOAEC of 430 ppm can be derived for foetal toxicity based on increases in rudimentary/short 14th ribs at higher concentrations (although the authors argue that this "variation" is not conclusive evidence of developmental toxicity).

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Administration via oral gavage to pregnant female New Zealand rabbits at dose levels of 0, 100, 300, 1000 mg/kg

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Kbl:NZW
- Source: Kitayama Labs Co Ltd, Japan
- Age at study initiation: 17-18 wk
- Weight at study initiation: mean group weights approx. 3.25 kg
- Housing: single housed in aluminium cages
- Diet (ad libitum): RC4 pellets (Oriental Yeast Co. Ltd, Chiba, Japan)
- Water (ad libitum): domestic tap water
- Acclimation period: 27 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22 degrees
- Humidity (%): 50-72%
- Air changes (per hr): 10-15 per hr
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 11 June 2007 (initiation of treatment) - 3 July 2007 (start of cesarean section)

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

- Dosing solutions prepared at 60, 180 and 600 mg/l in olive oil.
- Frequency of preparation: at least weekly

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Target concentrations: test solutions were analyzed using GC-FID (HP6890N).
Results were within 5% of target

Details on mating procedure:

Females judged to be in oestrus (based on appearance of external genitalia) were housed together with untreated males on a 1:1 basis. Animals were considered pregnant when copulation was observed twice (designated GD 0).

Duration of treatment / exposure:

GD 6 - 27 (22 days)

Frequency of treatment:

daily, 7 d/wk

Duration of test:

GD 6 - 28

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
Treatment levels were based on a preliminary study in which ETBE was administered orally to pregnant rabbits (6 / group) at 0, 30, 100, 300 and 1000 mg/kg bw/d for 22 days. Reduced food intake was noted at 1000 mg/kg bw/d but embryo-foetal development was unaffected. No maternal or foetal effects recorded at 300 mg/kg bw/d or below. Therefore the highest dose level was set at 1000 mg/kg bw/d with lower doses of 300 and 100 mg/kg bw/d.

Rationale for animal assignment (if not random): stratified according to body weight

Dosing: the control and test solutions were administered at 1.67 ml/kg body weight

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS:
Dams were observed for clinical signs dosing, immediately after dosing and 1 hour post-dosing during the treatment period.

BODY WEIGHT:
Dams were weighed on GD 0 (at time of mating) and on GD 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28. Any dams sacrified early were weighed before necropsy.

FOOD CONSUMPTION:
Food intake was measured on GD 1, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28.

NECROPSY:
Animals were sacrificed by exsanguination (abdominal aorta) under pentobarbital sodium anesthesia, and the major organs in the thoracic and abdominal cavities examined macroscopically. Animals were examined to confirm pregnancy. No organ weights measurement or routine tissue sampling were performed.

Ovaries and uterine content:

The ovaries and uterus were removed from dams confirmed as pregnant, and the number of corpora lutea determined. The uterus was weighed, opened, and the number of live/dead foetuses, implantation sites, resorbed embryos, placental remnants and early/late macerated foetuses counted. The placenta was also examined. Uteri were immersed in 10 vol% ammonium sulphide solution if no signs of pregnancy were visible macroscopically.

Fetal examinations:

- Body Weight:
Recorded for all foetuses (including those with external malformations, but the data excluded from group calculations).

- Sex Determination:
Assessed from examination of the internal genital organs of all live foetuses (excluding any with external malformations).

- External examinations
Foetuses were examined for the presence or absence of external malformations, including those in the oral cavity. Any exhibiting external malformations were preserved (phosphate buffered 10 vol% formalin).

- Soft tissue examinations:
The contents of the thoracic and abdominal cavities were observed macroscopically for all live foetuses (excluding those with external malformations). The brain (Wilson technique) and heart (Nishimura microdissection technique) were excised and fixed (phosphate buffered 10% formalin).

- Skeletal examinations:
All live foetuses (excluding those with external malformations) were fixed (95% alcohol), stained with Alizarin red S (Dawson method), and examined for skeletal malformations or variations. The degree of ossification was determined by counting the number of ossified metacarpals, metatarsals and digital phalanges and sacral/caudal vertebrae together with the degree of ossification of the sternebrae.

Statistics:

Body weight, body weight gain, food consumption, uterine weight, number of corpora lutea, number of implantations, number of live foetuses, sex ratio, body weight and ossification data (phalanges and vertebrae) were analyzed by Bartletts test followed by Dunnetts test if the data were homogeneous, with a Dunnett-type mean rank test used if the results were heterogeneous. Implantation indices, embryo-fetal deaths, external malformations, visceral malformations, visceral variations, skeletal malformations and skeletal variations were calculated and the data were analyzed by Dunnett-type mean rank test.

Clinical signs:

no effects observed

Description (incidence and severity):

Apart from reduced production of faeces (recorded in 14, 10, 7 and 13 animals from the 0, 100, 300 and 1000 mg/kg bw/d groups) there were no findings of note.

Mortality:

no mortality observed

Description (incidence):

No deaths occurred in any group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight was statistically significantly decreased by 4 - 5% in the 1000 mg/kg bw/d group relative to controls on GD 12, 14 and 16 but was unremarkable; thereafter, body weight gain in this group on GD 6- 2 8 was approx. half that of the control, however the effect was not statistically significant. Body weight and weight gain in the 100 and 300 mg/kg bw/d groups was unremarkable.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was decreased significantly by 25 - 30% at 1000 mg/kg bw/d on GD 8, 10, 12 and 14, but was comparable to the control group from GD 20 onwards. Food intake for the 100 and 300 mg/kg bw/d groups was similar to that of the controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Dark red discoloration of the lung was observed in 1 animal from the 300 and 1000 mg/kg bw/d groups with mild to moderate haemorrhage and inflammatory cell infiltration noted microscopically; cause not known but judged of limited relevance. There were no other findings of note.

Number of abortions:

no effects observed

Description (incidence and severity):

Abortions occurred in the control (1 animal on GD 28), 300 mg/kg bw/d (2 animals on GD 19 and GD 26) and 1000 mg/kg bw/d groups (1 animal on GD 25), but no dose-dependence apparent.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were 1 or 2 non-pregnant animals in the control, 100 and 300 mg/kg bw/d groups (all animals pregnant at 1000 mg/kg bw/d).

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
GENERAL:
There were 1 or 2 non-pregnant animals in the control, 100 and 300 mg/kg bw/d groups (all animals pregnant at 1000 mg/kg bw/d). No deaths occurred in any group. Abortions occurred in the control (1 animal on GD 28), 300 mg/kg bw/d (2 animals on GD 19 and GD 26) and 1000 mg/kg bw/d groups (1 animal on GD 25), but no dose-dependence apparent. One control animal and one animal that aborted in the 300 mg/kg bw/d group exhibited reductions in body weight and food intake for at least 4 days before abortion whereas clinical signs, body weight and food consumption were unremarkable in the other animals.

CLINICAL SIGNS:
Apart from reduced production of faeces (recorded in 14, 10, 7 and 13 animals from the 0, 100, 300 and 1000 mg/kg bw/d groups) there were no findings of note.

BODY WEIGHT:
Body weight was statistically significantly decreased by 4 - 5% in the 1000 mg/kg bw/d group relative to controls on GD 12, 14 and 16 but was unremarkable thereafter; body weight gain in this group on GD 6 - 28 was approx. half that of the control, however the effect was not statistically significant. Body weight and weight gain in the 100 and 300 mg/kg bw/d groups was unremarkable.

FOOD CONSUMPTION:
Food consumption was decreased significantly by 25 - 30% at 1000 mg/kg bw/d on GD 8, 10, 12 and 14, but was comparable to the control group from GD 20 onwards. Food intake for the 100 and 300 mg/kg bw/d groups was similar to that of the controls.

NECROPSY:
Dark red discoloration of the lung was observed in 1 animal from the 300 and 1000 mg/kg bw/d groups with mild to moderate haemorrhage and inflammatory cell infiltration noted microscopically; cause not known but judged of limited relevance. There were no other findings of note.

CESAREAN SECTION DATA:
The number of corpora lutea, implantations and implantation index was similar in the control and treated groups. Uterus weight was comparable between the groups.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences in the index of embryo-foetal deaths, number of live foetuses, sex ratio, body weight of live foetuses or the type or incidence of external malformations.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no significant differences in the index of embryo-foetal deaths, number of live foetuses, sex ratio, body weight of live foetuses or the type or incidence of external malformations.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no significant differences in the index of embryo-foetal deaths, number of live foetuses, sex ratio, body weight of live foetuses or the type or incidence of external malformations.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no significant differences in the index of embryo-foetal deaths, number of live foetuses, sex ratio, body weight of live foetuses or the type or incidence of external malformations.

External malformations:

no effects observed

Description (incidence and severity):

There were no significant differences in the index of embryo-foetal deaths, number of live foetuses, sex ratio, body weight of live foetuses or the type or incidence of external malformations.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No significant differences in the incidence of skeletal malformations or variations and extent of ossification with any findings present occurring in control and treated groups with no obvious dose relationship present.

Visceral malformations:

no effects observed

Description (incidence and severity):

No significant differences in the incidence of visceral malformations or variations, with any findings present occurring in control and treated groups with no obvious dose relationship present.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
GENERAL:
There were no significant differences in the index of embryo-foetal deaths, number of live foetuses, sex ratio, body weight of live foetuses or the type or incidence of external malformations.

VISCERAL EXAMINATION:
No significant differences in the incidence of visceral malformations or variations, with any findings present occurring in control and treated groups with no obvious dose relationship present.

SKELETAL EXAMINATION:
No significant differences in the incidence of skeletal malformations or variations and extent of ossification with any findings present occurring in control and treated groups with no obvious dose relationship present.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

 Selected results:

Treatment (mg/kg bw/d)	0	100	300	1000
Mean no. corpora lutea	10.3	10.4	10.1	9.8
Mean no. implantations	8.4	8.7	9.0	7.7
Mean implantation index (%)	80.4	84.7	89.3	77.1
Mean no. resorbed foetuses	11.0	11.3	7.0	8.7
Total no. early resorptions	8	7	10	11
Total no. late resorptions	5	10	2	8
Mean no. live foetuses	7.8	7.9	8.4	6.9
Sex ratio	0.50	0.54	0.54	0.42
Male foetal bw (g)	33.5	33.4	33.9	32.3
Female foetal bw (g)	31.2	31.4	32.0	30.1
No. foetuses examined	171	173	167	158
No. visceral malformations	1	1	1	3
No. visceral variations	3	5	5	3
No. skeletal malformations	5	4	3	8
No. skeletal variations	9	11	6	15

 

Conclusions:

Female New Zealand Rabbits have been treated with ETBE (0 - 1000 mg/kg/day) during GD 6 - 27. It is concluded that the no observed adverse effect levels of ETBE were 300 mg/kg/day for dams and 1,000 mg/kg/day for fetuses in rabbits.

Executive summary:

Ethyl tertiary-butyl ether (ETBE) is commonly used as an oxygenated gasoline additive. In this study, the prenatal developmental toxicity of ETBE was determined in rabbits. New Zealand white rabbits were given ETBE by gavage at 100, 300, or 1,000 mg/kg/day on gestational days (GDs) 6 - 27, and the pregnancy outcome was determined on GD 28. Neither death nor abortion occurred in the pregnant rabbits at any dose. Slightly and significantly suppressed maternal body-weight gain and transiently decreased maternal food consumption were found at 1,000 mg/kg/day during the administration period. At this dose, no changes in clinical or macroscopic finding were noted in dams. No treatment-related changes were observed in any dam treated at 300 mg/kg/day or less. There was no significant effect of ETBE on the numbers of corpora lutea, implantations, live fetuses, resorptions and dead fetuses, incidences of pre- and postimplantation loss, viability of fetuses, fetal body weight, sex ratio of fetuses, or weights of gravid uteri. No significant difference was detected in the incidences of fetuses with malformations or variations between the ETBE-treated and control groups. Also, no adverse effects on the progress of ossification were noted in fetuses of dams given ETBE. Based on these findings, it is concluded that the no observed adverse effect levels of ETBE were 300 mg/kg/day for dams and 1,000 mg/kg/day for fetuses in rabbits.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:CD(SD)
- Source: Charles River Laboratories Japan
- Age at study initiation: 11 wks
- Weight at study initiation: mean group weights approx. 250 g
- Housing: stainless-steel cages
- Diet (ad libitum): MF pellets (Oriental Yeast Co. Ltd)
- Water (ad libitum): domestic tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25 degrees
- Humidity (%): 40-70%
- Air changes (per hr): 10-15 per hr
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 22 Jan 2007 (Start of administration) - 6 Feb 2007 (start of cesarean section)

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Dosing solutions prepared at 2, 6 and 20 w/v% in olive oil.
- Frequency of preparation: once in a week

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Target concentrations: test solutions were analyzed using GC-FID (Shimadzu GC-2010).
Results were within 10 % of target

Details on mating procedure:

Females were housed together with males on a 2:1 basis. Females showing a vaginal plug or presence of sperms in the vaginal smear were considere to be signs of succesful corpulation (designated GD 0).

Duration of treatment / exposure:

GD 5 - 19

Frequency of treatment:

Daily

Duration of test:

GD 5 - 20

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
Treatment levels were based on a preliminary study in which ETBE was administered at 5, 25, 100 and 400 mg/kg/d. No effects have been observed. Therefore, the highest dose level was set at 1000 mg/kg/d with lower doses of 300 and 100 mg/kg/d.

Rationale for animal assignment (if not random): stratified according to body weight

Dosing: the control and test solutions were administered at 5.0 ml/kg body weight

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS:
Dams were observed for clinical signs once or twice each day.

BODY WEIGHT:
Dams were weighed on GD 0 (at time of mating) and on GD 3, 5, 7, 10, 13, 15, 17 and 20 (day of cesarean section).

FOOD CONSUMPTION:
Food intake was measured on GD 0 (at time of mating) and on GD 3, 5, 7, 10, 13, 15, 17 and 20 (day of cesarean section).

NECROPSY:
Animals were sacrificed under ether anaesthesia. No organ weight measurements or routine tissue sampling were performed.

Ovaries and uterine content:

The ovaries and uterus were removed and the number of corpora lutea determined. The uterus was weighed, opened, and immersed in 10 vol% ammonium sulphide solution for visualisation of implantation sites. The ovary and uterus were preserved in 10% neutral buffered formalin. Placental weight was recorded.

Fetal examinations:

- Body Weight:
Recorded for all foetuses.

- Sex Determination:
Assessed from examination of the internal genital organs of all live foetuses.

- External examinations
Foetuses were examined for the external appearance, including the oral cavity.

- Soft tissue examinations:
The contents of the thoracic and abdominal cavities were observed macroscopically for all live foetuses. The brain (Wilson technique) and heart (Nishimura microdissection technique) were excised and fixed.

- Skeletal examinations:
Even numbered fetuses were fixed (70% alcohol), stained with Alizarin red S , and examined for skeletal malformations or variations. The degree of ossification was determined by counting the number of ossified metacarpals, metatarsals and digital phalanges and sacral/caudal vertebrae together with the degree of ossification of the sternebrae.

Statistics:

Maternal body weight, food consumption, number of corpora lutea, ovary weight, uterine weight, number of implantations, number of live foetuses, foetal live weight, placental weight, number of ossified cervical vertebrae and number of ossified lumbar vertebrae were analysed using Bartlett’s test for homogeneity of variance, with homogeneous data subject to one-way ANOVA while non-homogenous data were analyzed using the Kruskal-Wallis rank sum test. Differences from controls were analyzed using Dunnet’s multiple comparison method. The sex ratio analysed using the chi-square test. The ratio of non-implanted foetuses, embryo-foetal death index, index of early (late) resorptions, index of dead foetuses, ratios of malformations / variations and ossification progress were subject to the Kruskal- Wallis rank sum test followed by Dunnett’s multiple comparison test. The litter was the unit of analysis.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was noted immediately after dosing in 2, 7 and 22 animals from the 100, 300 and 1000 mg/kg bw/d groups, respectively.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Maternal body weights were unremarkable.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unremarkable.

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

not examined

Ophthalmological findings:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Discoloration of the liver was noted in 1 control and 1 dam from the 100 mg/kg bw/d group. The animal from the the 300 mg/kg bw/d group that died prematurely showed no abnormalities.

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions occurred in any group.

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were 3 or 4 non-pregnant animals in the control and test groups.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

One dam from the 300 mg/kg bw/d died on GD 7 (following 3 treatments) with no clinical or other toxicological signs.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
GENERAL:
There were 3 or 4 non-pregnant animals in the control and test groups. No abortions occurred in any group. One dam from the 300 mg/kg bw/d died on GD 7 (following 3 treatments) with no clinical or other toxicological signs.

CLINICAL SIGNS:
Salivation was noted immediately after dosing in 2, 7 and 22 animals from the 100- 300 and 1000 mg/kg bw/d groups, respectively.

BODY WEIGHT:
Maternal body weights were unremarkable.

FOOD CONSUMPTION:
Food consumption was unremarkable.

NECROPSY:
Discoloration of the liver was noted in 1 control and 1 dam from the 100 mg/kg bw/d group. The animal from the the 300 mg/kg bw/d group that died prematurely showed no abnormalities.

CESAREAN SECTION DATA:
The number of corpora lutea, implantations, ovary weights and uterus weights were comparable between the groups.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The number of corpora lutea, implantations, ovary weights and uterus weights were comparable between the groups.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No significant differences in the incidence of skeletal malformations or variations and extent of ossification with any findings present occurring in control and treated groups with no obvious dose relationship present.
The incidence of lumbar rib (a variation) was significantly increased at 1000 mg/kg bw/d (19% of foetuses affected versus 2.9% of controls) but was within the historical range (1.1 to 21.2%; CD(SD)IGS Study Group for this strain of rat, and is a transient finding that disappears after birth.

Visceral malformations:

no effects observed

Description (incidence and severity):

No significant differences in the incidence of visceral malformations or variations, with any findings present occurring in control and treated groups with no obvious dose relationship present.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
GENERAL:
There were no significant differences in the index of embryo-foetal deaths, number of live foetuses, sex ratio, body weight of live foetuses or the type or incidence of external malformations.

VISCERAL EXAMINATION:
No significant differences in the incidence of visceral malformations or variations, with any findings present occurring in control and treated groups with no obvious dose relationship present.

SKELETAL EXAMINATION:
No significant differences in the incidence of skeletal malformations or variations and extent of ossification with any findings present occurring in control and treated groups with no obvious dose relationship present.
The incidence of lumbar rib (a variation) was significantly increased at 1000 mg/kg bw/d (19% of foetuses affected versus 2.9% of controls) but was within the historical range (1.1 to 21.2%; CD(SD)IGS Study Group for this strain of rat, and is a transient finding that disappears after birth.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Abnormalities:

no effects observed

Developmental effects observed:

no

 Selected results:

Treatment (mg/kg bw/d)	0	100	300	1000
Mean no. corpora lutea	15.5	14.1	14.4	14.6
Mean no. implantations	14.4	13.0	13.2	12.9
Mean implantation index (%)
Mean no. resorbed foetuses	5.8	7.2	4.2	5.0
Total no. early resorptions	5.8	7.2	3.5	4.7
Total no. late resorptions	0	0	0.7	0.4
Mean no. live foetuses	13.6	12.0	12.6	12.3
Sex ratio	0.44	0.49	0.52	0.50
Male foetal bw (g)	4.10	4.14	4.23	4.14
Female foetal bw (g)	3.89	3.92	4.01	3.91
No. foetuses examined	146	137	132	139
No. visceral malformations	3	2	2	0
No. visceral variations	6	8	4	8
No. foetuses examined	139	126	119	131
No. skeletal malformations	0	0	0	0
No. skeletal variations	9	3	3	29
No. lumbar rib (rudimentary)	4	0	2	25

 

Conclusions:

Female SD rats have been treated with ETBE (0 - 1000 mg/kg/day) during GD 5 - 19.The no observed adverse effect level was 1000  mg/kg/day both for pregnant rats and their embryos and fetuses.

Executive summary:

Ethyl tertiary-butyl ether (ETBE) is commonly used as an oxygenated gasoline additive. In this study, we evaluated its developmental toxicity in rats. ETBE was administered by gavage to 21 or 22 pregnant female Sprague-Dawley rats per group at dose levels of 0, 100, 300 and 1000  mg/kg/day from days 5 through 19 postcoitum to assess its effects on pregnant animals and their embryos and fetuses applied to the OECD testing guideline (no. 414), correspondingly. There were no toxicological effects attributable to ETBE regarding clinical signs, body weight, food intake, necropsy or examination at caesarean section in pregnant animals. There were also no toxicological effects on external, visceral and skeletal examinations of embryos and fetuses. These results indicate that, under the conditions of this study, ETBE had no toxicological effects on pregnant rats or their embryos and fetuses and that the no observed adverse effect level was 1000  mg/kg/day both for pregnant rats and their embryos and fetuses.

Reference 4

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

The test item has the main constituent of isopropyl ether (approximately 50%). Minor constituents comprise propylene dimers (approximately 20% C6 hydrocarbons, mainly C6 alkenes), propylene trimers (approximately 10% C9 hydrocarbons), hexanols (approximately 10%) and C3 alcohols (approximately 10% consisting of both isopropanol and n-propanol). Read across from diisopropylether (DIPE) and ethyl tert-butyl ether (ETBE) is justified on the basis that NOAECs reported for the constituents of this UVCB range from 3500 mg/m3 for hexanol to 31680 mg/m3 for propylene dimers, which can be considered indicative of low toxicity for this UVCB substance. DIPE comprises approximately 50% of the UVCB substance and is, therefore, the main constituent.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY
- Age at study initiation: Rats were approximately 9 weeks old when received and approximately 11 weeks old when breeding began
- Weight at study initiation: Not reported
- Fasting period before study: None
- Housing: Individually housed in 1 cubic metre (H-1000) inhalation chambers.
- Diet (e.g. ad libitum): Certified Purina Rodent Chow 5002 ad libitum except during exposure period
- Water (e.g. ad libitum): Ad libitum except during exposure period
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22 ºC
- Humidity (%): 40 to 60%
- Air changes (per hr): At least 12/hour
- Photoperiod (hrs dark / hrs light): 12 hours: 12 hours

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: H-1000 1 cubic-metre chambers
- Source and rate of air: HEPA-filtered room air; rate of air flow = 290 lpm
- Temperature, humidity, pressure in air chamber: Mean temperature within the chambers was in the range of 23 to 24 ºC and relative humidity was 60 to 67%. Pressure was not reported.
- Air flow rate: 290 lpm
- Air change rate: 12 changes/hour
- Treatment of exhaust air: Air exiting the chambers was cleaned by passage through charcoal beds


TEST ATMOSPHERE
- Brief description of analytical method used: Samples of air (approximately 50 to 250 µL) from the chambers were drawn into a gas-tight syringe and the air was injected directly into a gas chromatograph with a flame ionization detector and a fused silica column. In addition, 500 µL samples of air from the chambers were periodically taken for analysis by gas chromatography/mass spectroscopy.

VEHICLE (if applicable)
- Justification for use and choice of vehicle: No vehicle

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of air (approximately 50 to 250 µL) from the chambers were drawn into a gas-tight syringe and the air was injected directly into a gas chromatograph with a flame ionization detector and a fused silica column. Analyzed concentrations included both DIPE and total hydrocarbons. In addition, 500 µL samples of air from the chambers were periodically taken for analysis by gas chromatography/mass spectroscopy. Time points of analysis were not reported.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: Not reported
- Proof of pregnancy: sperm plug/sperm in the vaginal lavage fluid referred to as day 0 of pregnancy

Duration of treatment / exposure:

6 hours/day on Days 6 to 15 of gestation

Frequency of treatment:

6 hours/day on Days 6 to 15 of gestation

Duration of test:

Approximately 5 weeks (acclimation = 2 weeks and study duration = approximately 3 weeks)

Dose / conc.:

430 ppm (analytical)

Remarks:

equivalent to 1800 mg/m³, basis analytical conc.

Dose / conc.:

3 095 ppm (analytical)

Remarks:

equivalent to 12940 mg/m³, basis analytical conc.

Dose / conc.:

6 745 ppm (analytical)

Remarks:

equivalent to 28200 mg/m³, basis analytical conc.

No. of animals per sex per dose:

22 females/group

Control animals:

yes, concurrent no treatment

yes, sham-exposed

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded on Days 0, 6, 13, 16, and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Publication states that "all organs were examined grossly;" however, specific organs were not reported.

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

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No data

Statistics:

ANOVA, Fisher's exact, or Dunnett's tests were used

Indices:

None reported

Historical control data:

None reported

Details on maternal toxic effects:

Maternal toxic effects: yes

Details on maternal toxic effects:
Lacrimation and salivation were noted in a few DIPE-exposed females at the highest concentration during, or immediately following, exposures. The animals returned to normal appearance shortly after cessation of each daily exposure.

In general, animals housed in chambers gained less weight and consumed less food during the exposure period than the untreated controls (statistically significant at 6745 ppm relative to both of the control groups).

No treatment-related effects were noted at the time of macroscopic examination.

Serum chemistry endpoints were not adversely affected by exposure to DIPE vapors.

Statistically significant decrease in body weight gains were seen on gestation days 6 to 16 at all dose levels (compared to untreated controls for the low- and mid-dose groups and compared to both controls at the high-dose group).

Statistically significant decrease was seen in food consumption on gestation days 6 to 16 at the mid- and high-dose groups (compared to untreated controls on gestation days 6 to 13 and compared to both control groups on gestation days 13 to 16).

Reproductive parameters (i.e., number of pregnant females, percent preimplantation loss, percent resorptions, and litter sizes) were not affected by exposure.

Dose descriptor:

NOEC

Effect level:

430 ppm (analytical)

Based on:

test mat. (dissolved fraction)

Basis for effect level:

other: maternal toxicity

Remarks on result:

not determinable

Remarks:

no NOAEC identified by the authors, as these effects were considered transient and non-adverse by the authors arguing that this "variation" is not conclusive evidence of developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Fetal development (i.e., fetal body weight) was not affected by exposure.

Evaluations of fetal skeletons revealed a significant increase in rudimentary (small, discrete ossification) or short (less than one-half the length of the preceding rib) 14th ribs in fetuses exposed to DIPE at concentrations of 3095 and 6745 ppm. All of the observed 14th ribs were rudimentary except for 2 fetuses from each of the mid- and high-dose groups that had either bilateral short 14th ribs or bilateral short and rudimentary 14th ribs. No other exposure-related findings were noted at the time of fetal evaluations. The study authors have stated that "the observed increase in the incidence of rudimentary 14th ribs does not appear to be indicative of an adverse effect on development" at the concentrations tested.

Dose descriptor:

NOEC

Effect level:

430 ppm (analytical)

Based on:

test mat. (dissolved fraction)

Sex:

male/female

Basis for effect level:

other: slight skeletal effects

Remarks on result:

other: no NOAEC identified by the authors, as these effects were considered transient and non-adverse by the authors arguing that this "variation" is not conclusive evidence of developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

No developmental effects in the absence of maternal toxicity were observed. The NOAEC in this study for maternal effects as well as developmental effects can be set to 430 ppm.

Executive summary:

A NOAEC was not reported by the study authors. Review of the study data suggests that a NOAEC of 430 ppm can be derived for maternal toxicity based on the decrease in body weight gain and food consumption at higher concentrations, and a NOAEC of 430 ppm can be derived for foetal toxicity based on increases in rudimentary/short 14th ribs at higher concentrations (although the authors argue that this "variation" is not conclusive evidence of developmental toxicity).

Reference 5

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

The test item has the main constituent of isopropyl ether (approximately 50%). Minor constituents comprise propylene dimers (approximately 20% C6 hydrocarbons, mainly C6 alkenes), propylene trimers (approximately 10% C9 hydrocarbons), hexanols (approximately 10%) and C3 alcohols (approximately 10% consisting of both isopropanol and n-propanol). Effect on fertility has been assessed using data from well-conducted one-generation toxicity studies of the two read across substances diisopropyl ether (DIPE) and ethyl tert-butyl ether (ETBE).

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Administration via oral gavage to pregnant female New Zealand rabbits at dose levels of 0, 100, 300, 1000 mg/kg

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Kbl:NZW
- Source: Kitayama Labs Co Ltd, Japan
- Age at study initiation: 17-18 wk
- Weight at study initiation: mean group weights approx. 3.25 kg
- Housing: single housed in aluminium cages
- Diet (ad libitum): RC4 pellets (Oriental Yeast Co. Ltd, Chiba, Japan)
- Water (ad libitum): domestic tap water
- Acclimation period: 27 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22 degrees
- Humidity (%): 50-72%
- Air changes (per hr): 10-15 per hr
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 11 June 2007 (initiation of treatment) - 3 July 2007 (start of cesarean section)

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

- Dosing solutions prepared at 60, 180 and 600 mg/l in olive oil.
- Frequency of preparation: at least weekly

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Target concentrations: test solutions were analyzed using GC-FID (HP6890N).
Results were within 5% of target

Details on mating procedure:

Females judged to be in oestrus (based on appearance of external genitalia) were housed together with untreated males on a 1:1 basis. Animals were considered pregnant when copulation was observed twice (designated GD 0).

Duration of treatment / exposure:

GD 6 - 27 (22 days)

Frequency of treatment:

daily, 7 d/wk

Duration of test:

GD 6 - 28

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
Treatment levels were based on a preliminary study in which ETBE was administered orally to pregnant rabbits (6 / group) at 0, 30, 100, 300 and 1000 mg/kg bw/d for 22 days. Reduced food intake was noted at 1000 mg/kg bw/d but embryo-foetal development was unaffected. No maternal or foetal effects recorded at 300 mg/kg bw/d or below. Therefore the highest dose level was set at 1000 mg/kg bw/d with lower doses of 300 and 100 mg/kg bw/d.

Rationale for animal assignment (if not random): stratified according to body weight

Dosing: the control and test solutions were administered at 1.67 ml/kg body weight

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS:
Dams were observed for clinical signs dosing, immediately after dosing and 1 hour post-dosing during the treatment period.

BODY WEIGHT:
Dams were weighed on GD 0 (at time of mating) and on GD 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28. Any dams sacrified early were weighed before necropsy.

FOOD CONSUMPTION:
Food intake was measured on GD 1, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28.

NECROPSY:
Animals were sacrificed by exsanguination (abdominal aorta) under pentobarbital sodium anesthesia, and the major organs in the thoracic and abdominal cavities examined macroscopically. Animals were examined to confirm pregnancy. No organ weights measurement or routine tissue sampling were performed.

Ovaries and uterine content:

The ovaries and uterus were removed from dams confirmed as pregnant, and the number of corpora lutea determined. The uterus was weighed, opened, and the number of live/dead foetuses, implantation sites, resorbed embryos, placental remnants and early/late macerated foetuses counted. The placenta was also examined. Uteri were immersed in 10 vol% ammonium sulphide solution if no signs of pregnancy were visible macroscopically.

Fetal examinations:

- Body Weight:
Recorded for all foetuses (including those with external malformations, but the data excluded from group calculations).

- Sex Determination:
Assessed from examination of the internal genital organs of all live foetuses (excluding any with external malformations).

- External examinations
Foetuses were examined for the presence or absence of external malformations, including those in the oral cavity. Any exhibiting external malformations were preserved (phosphate buffered 10 vol% formalin).

- Soft tissue examinations:
The contents of the thoracic and abdominal cavities were observed macroscopically for all live foetuses (excluding those with external malformations). The brain (Wilson technique) and heart (Nishimura microdissection technique) were excised and fixed (phosphate buffered 10% formalin).

- Skeletal examinations:
All live foetuses (excluding those with external malformations) were fixed (95% alcohol), stained with Alizarin red S (Dawson method), and examined for skeletal malformations or variations. The degree of ossification was determined by counting the number of ossified metacarpals, metatarsals and digital phalanges and sacral/caudal vertebrae together with the degree of ossification of the sternebrae.

Statistics:

Body weight, body weight gain, food consumption, uterine weight, number of corpora lutea, number of implantations, number of live foetuses, sex ratio, body weight and ossification data (phalanges and vertebrae) were analyzed by Bartletts test followed by Dunnetts test if the data were homogeneous, with a Dunnett-type mean rank test used if the results were heterogeneous. Implantation indices, embryo-fetal deaths, external malformations, visceral malformations, visceral variations, skeletal malformations and skeletal variations were calculated and the data were analyzed by Dunnett-type mean rank test.

Clinical signs:

no effects observed

Description (incidence and severity):

Apart from reduced production of faeces (recorded in 14, 10, 7 and 13 animals from the 0, 100, 300 and 1000 mg/kg bw/d groups) there were no findings of note.

Mortality:

no mortality observed

Description (incidence):

No deaths occurred in any group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight was statistically significantly decreased by 4 - 5% in the 1000 mg/kg bw/d group relative to controls on GD 12, 14 and 16 but was unremarkable; thereafter, body weight gain in this group on GD 6- 2 8 was approx. half that of the control, however the effect was not statistically significant. Body weight and weight gain in the 100 and 300 mg/kg bw/d groups was unremarkable.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was decreased significantly by 25 - 30% at 1000 mg/kg bw/d on GD 8, 10, 12 and 14, but was comparable to the control group from GD 20 onwards. Food intake for the 100 and 300 mg/kg bw/d groups was similar to that of the controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Dark red discoloration of the lung was observed in 1 animal from the 300 and 1000 mg/kg bw/d groups with mild to moderate haemorrhage and inflammatory cell infiltration noted microscopically; cause not known but judged of limited relevance. There were no other findings of note.

Number of abortions:

no effects observed

Description (incidence and severity):

Abortions occurred in the control (1 animal on GD 28), 300 mg/kg bw/d (2 animals on GD 19 and GD 26) and 1000 mg/kg bw/d groups (1 animal on GD 25), but no dose-dependence apparent.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were 1 or 2 non-pregnant animals in the control, 100 and 300 mg/kg bw/d groups (all animals pregnant at 1000 mg/kg bw/d).

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
GENERAL:
There were 1 or 2 non-pregnant animals in the control, 100 and 300 mg/kg bw/d groups (all animals pregnant at 1000 mg/kg bw/d). No deaths occurred in any group. Abortions occurred in the control (1 animal on GD 28), 300 mg/kg bw/d (2 animals on GD 19 and GD 26) and 1000 mg/kg bw/d groups (1 animal on GD 25), but no dose-dependence apparent. One control animal and one animal that aborted in the 300 mg/kg bw/d group exhibited reductions in body weight and food intake for at least 4 days before abortion whereas clinical signs, body weight and food consumption were unremarkable in the other animals.

CLINICAL SIGNS:
Apart from reduced production of faeces (recorded in 14, 10, 7 and 13 animals from the 0, 100, 300 and 1000 mg/kg bw/d groups) there were no findings of note.

BODY WEIGHT:
Body weight was statistically significantly decreased by 4 - 5% in the 1000 mg/kg bw/d group relative to controls on GD 12, 14 and 16 but was unremarkable thereafter; body weight gain in this group on GD 6 - 28 was approx. half that of the control, however the effect was not statistically significant. Body weight and weight gain in the 100 and 300 mg/kg bw/d groups was unremarkable.

FOOD CONSUMPTION:
Food consumption was decreased significantly by 25 - 30% at 1000 mg/kg bw/d on GD 8, 10, 12 and 14, but was comparable to the control group from GD 20 onwards. Food intake for the 100 and 300 mg/kg bw/d groups was similar to that of the controls.

NECROPSY:
Dark red discoloration of the lung was observed in 1 animal from the 300 and 1000 mg/kg bw/d groups with mild to moderate haemorrhage and inflammatory cell infiltration noted microscopically; cause not known but judged of limited relevance. There were no other findings of note.

CESAREAN SECTION DATA:
The number of corpora lutea, implantations and implantation index was similar in the control and treated groups. Uterus weight was comparable between the groups.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences in the index of embryo-foetal deaths, number of live foetuses, sex ratio, body weight of live foetuses or the type or incidence of external malformations.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no significant differences in the index of embryo-foetal deaths, number of live foetuses, sex ratio, body weight of live foetuses or the type or incidence of external malformations.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no significant differences in the index of embryo-foetal deaths, number of live foetuses, sex ratio, body weight of live foetuses or the type or incidence of external malformations.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no significant differences in the index of embryo-foetal deaths, number of live foetuses, sex ratio, body weight of live foetuses or the type or incidence of external malformations.

External malformations:

no effects observed

Description (incidence and severity):

There were no significant differences in the index of embryo-foetal deaths, number of live foetuses, sex ratio, body weight of live foetuses or the type or incidence of external malformations.

Skeletal malformations:

no effects observed

Description (incidence and severity):

No significant differences in the incidence of skeletal malformations or variations and extent of ossification with any findings present occurring in control and treated groups with no obvious dose relationship present.

Visceral malformations:

no effects observed

Description (incidence and severity):

No significant differences in the incidence of visceral malformations or variations, with any findings present occurring in control and treated groups with no obvious dose relationship present.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
GENERAL:
There were no significant differences in the index of embryo-foetal deaths, number of live foetuses, sex ratio, body weight of live foetuses or the type or incidence of external malformations.

VISCERAL EXAMINATION:
No significant differences in the incidence of visceral malformations or variations, with any findings present occurring in control and treated groups with no obvious dose relationship present.

SKELETAL EXAMINATION:
No significant differences in the incidence of skeletal malformations or variations and extent of ossification with any findings present occurring in control and treated groups with no obvious dose relationship present.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

 Selected results:

Treatment (mg/kg bw/d)	0	100	300	1000
Mean no. corpora lutea	10.3	10.4	10.1	9.8
Mean no. implantations	8.4	8.7	9.0	7.7
Mean implantation index (%)	80.4	84.7	89.3	77.1
Mean no. resorbed foetuses	11.0	11.3	7.0	8.7
Total no. early resorptions	8	7	10	11
Total no. late resorptions	5	10	2	8
Mean no. live foetuses	7.8	7.9	8.4	6.9
Sex ratio	0.50	0.54	0.54	0.42
Male foetal bw (g)	33.5	33.4	33.9	32.3
Female foetal bw (g)	31.2	31.4	32.0	30.1
No. foetuses examined	171	173	167	158
No. visceral malformations	1	1	1	3
No. visceral variations	3	5	5	3
No. skeletal malformations	5	4	3	8
No. skeletal variations	9	11	6	15

 

Conclusions:

Female New Zealand Rabbits have been treated with ETBE (0 - 1000 mg/kg/day) during GD 6 - 27. It is concluded that the no observed adverse effect levels of ETBE were 300 mg/kg/day for dams and 1,000 mg/kg/day for fetuses in rabbits.

Executive summary:

Ethyl tertiary-butyl ether (ETBE) is commonly used as an oxygenated gasoline additive. In this study, the prenatal developmental toxicity of ETBE was determined in rabbits. New Zealand white rabbits were given ETBE by gavage at 100, 300, or 1,000 mg/kg/day on gestational days (GDs) 6 - 27, and the pregnancy outcome was determined on GD 28. Neither death nor abortion occurred in the pregnant rabbits at any dose. Slightly and significantly suppressed maternal body-weight gain and transiently decreased maternal food consumption were found at 1,000 mg/kg/day during the administration period. At this dose, no changes in clinical or macroscopic finding were noted in dams. No treatment-related changes were observed in any dam treated at 300 mg/kg/day or less. There was no significant effect of ETBE on the numbers of corpora lutea, implantations, live fetuses, resorptions and dead fetuses, incidences of pre- and postimplantation loss, viability of fetuses, fetal body weight, sex ratio of fetuses, or weights of gravid uteri. No significant difference was detected in the incidences of fetuses with malformations or variations between the ETBE-treated and control groups. Also, no adverse effects on the progress of ossification were noted in fetuses of dams given ETBE. Based on these findings, it is concluded that the no observed adverse effect levels of ETBE were 300 mg/kg/day for dams and 1,000 mg/kg/day for fetuses in rabbits.

Reference 6

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

supporting study

Study period:

2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

The test item has the main constituent of isopropyl ether (approximately 50%). Minor constituents comprise propylene dimers (approximately 20% C6 hydrocarbons, mainly C6 alkenes), propylene trimers (approximately 10% C9 hydrocarbons), hexanols (approximately 10%) and C3 alcohols (approximately 10% consisting of both isopropanol and n-propanol). Effect on fertility has been assessed using data from well-conducted one-generation toxicity studies of the two read across substances diisopropyl ether (DIPE) and ethyl tert-butyl ether (ETBE).

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:CD(SD)
- Source: Charles River Laboratories Japan
- Age at study initiation: 11 wks
- Weight at study initiation: mean group weights approx. 250 g
- Housing: stainless-steel cages
- Diet (ad libitum): MF pellets (Oriental Yeast Co. Ltd)
- Water (ad libitum): domestic tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25 degrees
- Humidity (%): 40-70%
- Air changes (per hr): 10-15 per hr
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 22 Jan 2007 (Start of administration) - 6 Feb 2007 (start of cesarean section)

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Dosing solutions prepared at 2, 6 and 20 w/v% in olive oil.
- Frequency of preparation: once in a week

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Target concentrations: test solutions were analyzed using GC-FID (Shimadzu GC-2010).
Results were within 10 % of target

Details on mating procedure:

Females were housed together with males on a 2:1 basis. Females showing a vaginal plug or presence of sperms in the vaginal smear were considere to be signs of succesful corpulation (designated GD 0).

Duration of treatment / exposure:

GD 5 - 19

Frequency of treatment:

Daily

Duration of test:

GD 5 - 20

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
Treatment levels were based on a preliminary study in which ETBE was administered at 5, 25, 100 and 400 mg/kg/d. No effects have been observed. Therefore, the highest dose level was set at 1000 mg/kg/d with lower doses of 300 and 100 mg/kg/d.

Rationale for animal assignment (if not random): stratified according to body weight

Dosing: the control and test solutions were administered at 5.0 ml/kg body weight

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS:
Dams were observed for clinical signs once or twice each day.

BODY WEIGHT:
Dams were weighed on GD 0 (at time of mating) and on GD 3, 5, 7, 10, 13, 15, 17 and 20 (day of cesarean section).

FOOD CONSUMPTION:
Food intake was measured on GD 0 (at time of mating) and on GD 3, 5, 7, 10, 13, 15, 17 and 20 (day of cesarean section).

NECROPSY:
Animals were sacrificed under ether anaesthesia. No organ weight measurements or routine tissue sampling were performed.

Ovaries and uterine content:

The ovaries and uterus were removed and the number of corpora lutea determined. The uterus was weighed, opened, and immersed in 10 vol% ammonium sulphide solution for visualisation of implantation sites. The ovary and uterus were preserved in 10% neutral buffered formalin. Placental weight was recorded.

Fetal examinations:

- Body Weight:
Recorded for all foetuses.

- Sex Determination:
Assessed from examination of the internal genital organs of all live foetuses.

- External examinations
Foetuses were examined for the external appearance, including the oral cavity.

- Soft tissue examinations:
The contents of the thoracic and abdominal cavities were observed macroscopically for all live foetuses. The brain (Wilson technique) and heart (Nishimura microdissection technique) were excised and fixed.

- Skeletal examinations:
Even numbered fetuses were fixed (70% alcohol), stained with Alizarin red S , and examined for skeletal malformations or variations. The degree of ossification was determined by counting the number of ossified metacarpals, metatarsals and digital phalanges and sacral/caudal vertebrae together with the degree of ossification of the sternebrae.

Statistics:

Maternal body weight, food consumption, number of corpora lutea, ovary weight, uterine weight, number of implantations, number of live foetuses, foetal live weight, placental weight, number of ossified cervical vertebrae and number of ossified lumbar vertebrae were analysed using Bartlett’s test for homogeneity of variance, with homogeneous data subject to one-way ANOVA while non-homogenous data were analyzed using the Kruskal-Wallis rank sum test. Differences from controls were analyzed using Dunnet’s multiple comparison method. The sex ratio analysed using the chi-square test. The ratio of non-implanted foetuses, embryo-foetal death index, index of early (late) resorptions, index of dead foetuses, ratios of malformations / variations and ossification progress were subject to the Kruskal- Wallis rank sum test followed by Dunnett’s multiple comparison test. The litter was the unit of analysis.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was noted immediately after dosing in 2, 7 and 22 animals from the 100, 300 and 1000 mg/kg bw/d groups, respectively.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Maternal body weights were unremarkable.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unremarkable.

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

not examined

Ophthalmological findings:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Discoloration of the liver was noted in 1 control and 1 dam from the 100 mg/kg bw/d group. The animal from the the 300 mg/kg bw/d group that died prematurely showed no abnormalities.

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions occurred in any group.

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were 3 or 4 non-pregnant animals in the control and test groups.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

One dam from the 300 mg/kg bw/d died on GD 7 (following 3 treatments) with no clinical or other toxicological signs.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
GENERAL:
There were 3 or 4 non-pregnant animals in the control and test groups. No abortions occurred in any group. One dam from the 300 mg/kg bw/d died on GD 7 (following 3 treatments) with no clinical or other toxicological signs.

CLINICAL SIGNS:
Salivation was noted immediately after dosing in 2, 7 and 22 animals from the 100- 300 and 1000 mg/kg bw/d groups, respectively.

BODY WEIGHT:
Maternal body weights were unremarkable.

FOOD CONSUMPTION:
Food consumption was unremarkable.

NECROPSY:
Discoloration of the liver was noted in 1 control and 1 dam from the 100 mg/kg bw/d group. The animal from the the 300 mg/kg bw/d group that died prematurely showed no abnormalities.

CESAREAN SECTION DATA:
The number of corpora lutea, implantations, ovary weights and uterus weights were comparable between the groups.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The number of corpora lutea, implantations, ovary weights and uterus weights were comparable between the groups.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No significant differences in the incidence of skeletal malformations or variations and extent of ossification with any findings present occurring in control and treated groups with no obvious dose relationship present.
The incidence of lumbar rib (a variation) was significantly increased at 1000 mg/kg bw/d (19% of foetuses affected versus 2.9% of controls) but was within the historical range (1.1 to 21.2%; CD(SD)IGS Study Group for this strain of rat, and is a transient finding that disappears after birth.

Visceral malformations:

no effects observed

Description (incidence and severity):

No significant differences in the incidence of visceral malformations or variations, with any findings present occurring in control and treated groups with no obvious dose relationship present.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
GENERAL:
There were no significant differences in the index of embryo-foetal deaths, number of live foetuses, sex ratio, body weight of live foetuses or the type or incidence of external malformations.

VISCERAL EXAMINATION:
No significant differences in the incidence of visceral malformations or variations, with any findings present occurring in control and treated groups with no obvious dose relationship present.

SKELETAL EXAMINATION:
No significant differences in the incidence of skeletal malformations or variations and extent of ossification with any findings present occurring in control and treated groups with no obvious dose relationship present.
The incidence of lumbar rib (a variation) was significantly increased at 1000 mg/kg bw/d (19% of foetuses affected versus 2.9% of controls) but was within the historical range (1.1 to 21.2%; CD(SD)IGS Study Group for this strain of rat, and is a transient finding that disappears after birth.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Abnormalities:

no effects observed

Developmental effects observed:

no

 Selected results:

Treatment (mg/kg bw/d)	0	100	300	1000
Mean no. corpora lutea	15.5	14.1	14.4	14.6
Mean no. implantations	14.4	13.0	13.2	12.9
Mean implantation index (%)
Mean no. resorbed foetuses	5.8	7.2	4.2	5.0
Total no. early resorptions	5.8	7.2	3.5	4.7
Total no. late resorptions	0	0	0.7	0.4
Mean no. live foetuses	13.6	12.0	12.6	12.3
Sex ratio	0.44	0.49	0.52	0.50
Male foetal bw (g)	4.10	4.14	4.23	4.14
Female foetal bw (g)	3.89	3.92	4.01	3.91
No. foetuses examined	146	137	132	139
No. visceral malformations	3	2	2	0
No. visceral variations	6	8	4	8
No. foetuses examined	139	126	119	131
No. skeletal malformations	0	0	0	0
No. skeletal variations	9	3	3	29
No. lumbar rib (rudimentary)	4	0	2	25

 

Conclusions:

Female SD rats have been treated with ETBE (0 - 1000 mg/kg/day) during GD 5 - 19.The no observed adverse effect level was 1000  mg/kg/day both for pregnant rats and their embryos and fetuses.

Executive summary:

Ethyl tertiary-butyl ether (ETBE) is commonly used as an oxygenated gasoline additive. In this study, we evaluated its developmental toxicity in rats. ETBE was administered by gavage to 21 or 22 pregnant female Sprague-Dawley rats per group at dose levels of 0, 100, 300 and 1000  mg/kg/day from days 5 through 19 postcoitum to assess its effects on pregnant animals and their embryos and fetuses applied to the OECD testing guideline (no. 414), correspondingly. There were no toxicological effects attributable to ETBE regarding clinical signs, body weight, food intake, necropsy or examination at caesarean section in pregnant animals. There were also no toxicological effects on external, visceral and skeletal examinations of embryos and fetuses. These results indicate that, under the conditions of this study, ETBE had no toxicological effects on pregnant rats or their embryos and fetuses and that the no observed adverse effect level was 1000  mg/kg/day both for pregnant rats and their embryos and fetuses.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

Study duration:

subacute

Species:

rabbit

Quality of whole database:

The study was of regulatory design with sufficient numbers of animals to provide robust statistical comparisons. The GLP status of the conducting laboratory is not reported, however, all data represented would be indicative of Klimisch 1 rating for reliability.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

Study duration:

subacute

Experimental exposure time per week (hours/week):

42

Species:

rat

Quality of whole database:

The study was of regulatory design with sufficient numbers of animals to provide robust statistical comparisons. The GLP status of the conducting laboratory is not reported, however, all data represented would be indicative of Klimisch 1 rating for reliability.

Justification for classification or non-classification

It is concluded that the conduct of an extended one generation reproductive toxicity study for this UVCB substance can be waived on the basis that sufficient data for this endpoint exists for the two read-across substances, DIPE and ETBE. In a rat inhalation one generation reproductive toxicity study with both read-across substances, in a rat inhalation developmental toxicity study with DIPE and in both a rat and rabbit oral developmental toxicity study with ETBE, there were no indications of effects on reproduction and fertility or on foetal growth and development parameters at dose levels causing transient parental toxicity.