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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
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:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
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Constituent
Test material form:
gas: vapour
Details on test material:
EPA described ‘‘baseline gasoline’’ (without oxygenate) (BGVC), identified and analysed as described in Henley, M., Daniel, J., Letinsky, D.J., Carr, J., Caro, M., Daughtrey, W., White, R., 2014. Health assessment of gasoline and fuel oxygenate vapors: generation and characterization of test materials, Regul. Toxicol. Pharm. 70 (2S), S13–S17.

Test animals

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 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.

Administration / exposure

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)
F1 premating phase 70 days: F1 males/females
F1 mating phase 14 days: F1 males/females
F1 gestation phase GD0 - 19: F1 males/females
F1 gestation/Lactation phase GD20 - LD4: F1 males
F1 lactation phase L5 - 28: F1 males/females F2 males/females indirectly via lactation
Weaning phase LD28: No exposure
Frequency of treatment:
6 hours per day, 7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
2 014 mg/m³ air (analytical)
Remarks:
Exposure chamber concentration ±96 mg/m³
Dose / conc.:
10 140 mg/m³ air (analytical)
Remarks:
Exposure chamber concentration ±369 mg/m³
Dose / conc.:
20 010 mg/m³ air (analytical)
Remarks:
Exposure chamber concentration ±599 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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reduction in weight gain during the premating period was observed in females exposed to 20,000 mg/m³ BGVC (P0) only. 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 / performance (P0)

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.

Details on results (P0)

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

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEC
Effect level:
10 000 mg/m³ air (nominal)
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other: Parental toxicity
Remarks:
The BGVC parental NOAEC was based on decreased body weight gains during the premating period in P0 females and F1 males and increased P0 female kidney weight which had no histopathologic correlate.
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

Target system / organ toxicity (P0)

Critical effects observed:
no
Lowest effective dose / conc.:
20 000 other: mg/m³
Organ:
kidney

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Description (incidence and severity):
See F1 generation
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
See F1 generation
Body weight and weight changes:
no effects observed
Description (incidence and severity):
See F1 generation
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
See F1 generation
Gross pathological findings:
no effects observed
Description (incidence and severity):
See F1 generation
Neuropathological findings:
no effects observed
Description (incidence and severity):
See F1 generation

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P1)

Dose descriptor:
NOAEC
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

Results: F1 generation

General toxicity (F1)

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):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
Expressions of sexual maturation (vaginal opening and preputial gland separation) were not altered by exposure to BGVC. Additionally there were no decrements in reproductive performance when the F1 animals (1/sex/litter) were bred to produce the second generation.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Lower spleen weights were seen in F1 offspring of BGVC exposed rats; however the effect was not expressed to the F2 offspring and was unlikely to be a toxicologically significant adverse finding. No other 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

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Neuropathology and GFAP assessments were performed on randomly selected F1 pups from BGVC exposed rats. No adverse neuropathology was observed and there were no differences between control and BGVC F1 offspring in brain length or width. Exposure to BGVC did not cause changes in GFAP levels in any brain region examined with the exception of a single decrease in the BGVC F1 male thalamus at 20,000 mg/m³ that was not considered biologically significant by the investigator. Overall, GFAP results indicated that none of these substances induced gliosis in the brain regions examined.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

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

Effect levels (F1)

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:
other: The NOAEL was the highest exposure tested for BGVC as no differences from controls were seen for fertility, days to mating, oestrous cycle length, sperm counts or morphology or developmental parameters in pups.

Results: F2 generation

General toxicity (F2)

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):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Dose descriptor:
NOAEC
Generation:
F2
Effect level:
20 000 mg/m³ air (nominal)
Based on:
test mat. (total fraction)
Sex:
male/female
Remarks on result:
other: The NOAEL was the highest exposure tested for BGVC as no differences from controls were seen for fertility, days to mating, oestrous cycle length, sperm counts or morphology or developmental parameters in pups.

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Effects on female rats (P0 and F1) from exposure to vapour condensates of baseline gasoline at 20,000 mg/m³.

Endpoints

Control range

BGVC

 

P0

F1

Premating body weight gain (g)

108 – 144 (P0)
175 – 200 (F1)

125 **

189

% of control

 

89.3%

94.5%

Gestation day 0–20 weight gain (g)

113 – 129 (P0) – 120, 121 (F1)

125

122

% of control

 

101.6%

100.8%

Lactation day 21–28 weight gain (g)

-3 to 13 (P0) 7 – 15 (F1)

-1

7

% of control

 

(control-3)

100.0%

Lung, discolored foci (macroscopic)

0/26–4/26

1/26

5/26

% of rats

0–15.4%

3.8%

19.2%

Relevant organ weight changes

 

kidney ↑

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 and F1) from exposure to vapour condensates of baseline gasoline at 20,000 mg/m³.

Endpoints

Control range

BGVC

 

P0

F1

Premating body weight gain (g)

225–313 (P0)
350–379 (F1)

250

364

% of control

95.8%

96.0%

 

Lung, discoloured foci (macroscopic)

0/26–4/26

2/26

1/26

% of rats

0–15.4%

7.7%

3.8%

Relevant organ weight changes

 

kidney ↑

kidney ↑

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

* p < 0.05, ** p < 0.01.

Effects on offspring (F1 and F2) from exposure of parents to vapour condensate of gasoline at 20,000 mg/m³.

Endpoints

Control range

BGVC

F1

F2

Litter size (Pups delivered)

12.1 – 14.5

13.7

13.2

Pup weights (g) sexes combined

LD 1

6.8 – 7.4

7

7

LD 4

9.7 – 11.4

10.1

10.2

LD 7

13.5 – 14.7

13.8

13.9

LD 14

23.7 – 26.0

23.3

24.7

LD 21

38.7 – 42.5

38.8

41.2

LD 28

69.5 – 78.8

66.1

75.4

Pup survival sexes combined

LD 0–4

93.1 – 99.6%

93.8%

95.4%

LD 5–21

98.7 – 100%

100.0%

99.6%

Other endpoints

Spleen weight (g)

0.263 – 0.327

0.232 *

0.306

GFAP assay

 

Negative

NE

Vaginal opening (day)

35.3 – 37.5

35

NE

Preputial separation (day)

46.2 – 46.4

48

NE

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

NE = not evaluated, * p < 0.05., ** p < 0.01.

Effects on reproductive parameters on parental animals (P0 and F1) from exposure to vapour condensates of gasoline or gasoline BGVC at 20,000 mg/m³.

Endpoint

Control range

BGVC

 

 

P0

F1

 

Male Fertility

20/25 – 25/26

25/26

24/25

 

 

80 – 96.2%

96.2%

96.0%

 

Female Fertility

21/24 – 25/25

25/25

25/25

 

 

87.5 – 100%

100.0%

100.0%

 

Number of Litters

20 – 25

25

25

 

Days to Mating

2.4 – 3.4

2.6

2.7

 

Estrus Cycle Length (days)

4.2 – 5.2, 5.7

4.5

4.1

 

Semen parameters

Sperm Count, testis (106/g)

81.2 – 124.1

115.4

100.6

 

Motility (%)

89 – 96%

94%

95%

 

Morphology (% abnormal)

0.6 – 1.7%a

1.20%

0.50%

 

Epididymal sperm count (106/g)

753.6 – 956.7

955.3

933.5

 

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

**p < 0.01, * p < 0.05,aDid not include control value of 7.4% from the G/DIPE study, which was influenced strongly by two males with abnormally high percentages (90.5% and 84%) of amorphous sperm heads.

Applicant's summary and conclusion

Conclusions:
In this two generation reproductive toxicity study with a gasoline blend, no developmental effects or decreases in fertility were observed and thus the NOAEC for reproductive toxicity in this study as 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.