Naphtha (Fischer-Tropsch), light, C4-10-branched and linear

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: peer reviewed international scientific journal

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

not specified

Principles of method if other than guideline:

The study was conducted in accordance with the United States Environmental Protection Agency’s (EPA) Good Laboratory Practice Standards (US EPA, 1994a), and complied with all appropriate parts of the Animal Welfare Act Regulations (USDA, 1989, 1991).The study also met the requirements of US EPA OPPTS 870.3800 guidelines for one and two-generation reproductive toxicity studies (US EPA, 1998a).

The test item was evaluated in a two-generation studies which included neuropathology assessments and quantitative changes in regional brain glial fibrillary acidic protein (GFAP) content, a measurement of reactive gliosis and an index of underlying neurotoxicity, in F1 offspring.
At weaning one pup/sex/group was selected for mating to produce the F2 generation. F1 pups [5/sex/group/assessment] not selected for F1 mating were evaluated for standard Tier 2 neuropathology (40 CFR79.66; US EPA, 1998b) or for GFAP assessments (40 CFR79.67; US EPA, 1994b) on postpartum day 28. Methods employed for both procedures are provided in O’Callaghan et al. (2014). The standard Tier 2 neuropathologic evaluation was
performed at Huntingdon Life Sciences. For GFAP analyses, brains were removed, weighed and processed, then shipped on dry ice to the US Centers for Disease Control and Prevention, Health Effects Laboratory Division, Morgantown, WV for analysis by Dr. James O’Callaghan. The remaining pups were examined for external abnormalities and sacrificed. Pups with abnormalities were preserved intact in 10% neutral buffered formalin. Three pups/sex/litter in each group (F1 and F2) were selected for macroscopic examination and selected organs [brain, spleen, thymus] were weighed from one pup /sex/litter.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Gasoline, vapor-recovery

EC Number:

271-025-4

EC Name:

Gasoline, vapor-recovery

Cas Number:

68514-15-8

IUPAC Name:

Gasoline, vapor-recovery

Test material form:

gas: vapour

Details on test material:

- Name of test material (as cited in study report): baseline gasoline vapour condensate (BGVC)
- Test materials included vapor condensates prepared from an EPA described ‘‘baseline gasoline’’ (BGVC), identified as API Lot 99-01. Analytical characterisation of the cross-reference Henley et al. (2014) is attached in the background material.
- Other: Test substance is closely related to 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear'

Specific details on test material used for the study:

Information on the composition of the test item is given in table 1 and 2, see any other information on material and methods.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal selection, assignment and care:
CD (Sprague–Dawley derived) [Crl: CD@ IGS BR] albino 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. 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 pretest 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). Huntingdon Life Sciences, East Millstone, New Jersey is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). 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 analyzed for purity on a regular basis and there were no known contaminants which were expected to interfere with the results of this study.

Housing and environmental conditions:
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, (Bed-O-Cobs 1/4 inch irradiated, The Andersons Inc. Maumee, OH), 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:

Exposure conditions:
Details of exposure procedures and chamber operation and diagram of the exposure chambers are described in the cross-reference Clark et al. (2014) (see below). The flow of air through the chamber was monitored using appropriate calibrated equipment. Exposure levels were analyzed using an infra-red spectrophotometer 4 times per chamber per day. The test material’s major components were assayed once per chamber per week. Particle size distribution measurements were also made once per chamber per week using a TSI aerodynamic particle sizer.

Exposure chambers according to Clark et al. (2014):
Unexposed control group:
Houseline nitrogen was delivered from a regulator with a backpressure gauge via 1/400 tubing to a flow meter regulated by a metering valve. This nitrogen flow was then split via a 1/400 stainless steel ‘‘T’’ to both chamber turrets of the 1 cubic meter glass and stainless steel exposure chamber where it was mixed with room air as it was drawn into the chamber.
Low, middle and high exposure groups:
Houseline nitrogen was delivered from a regulator with a backpressure gauge through a stainless steel fitting to create three flow systems: the test substance pressurization flow, the purge flow and the volatilization flow.
The nitrogen for the test substance pressurization flow was directed through a metering valve, attached to a back pressure gauge, into the vapor inlet valve of the test substance cylinder. The metering valve was used to adjust and maintain the pressure within the cylinder. From the pressurized cylinder, the test substance flowed from the liquid outlet valve through a disconnect fitting (equipped with a toggle valve) and through a filter to prevent equipment contamination. From the filter, the test substance flowed to a liquid flow meter via 1/800 tubing. The outlet of the flow meter was regulated by a metering valve. From this metering valve, the test substance flowed via 1/800 tubing onto the glass helix of a counter current volatilization chamber. This glass helix was heated by a nichrome wire which was controlled by a variable autotransformer and inserted in the center of the glass tube that supported the helix external to the volatilization chamber.
The nitrogen for the purge flow system was directed, via 1/400 tubing to a flow meter regulated by a metering valve. The purge nitrogen was delivered via 1/800 tubing to the bottom of the tube containing the nichrome wire. This nitrogen flow continuously purged the area surrounding the nichrome wire within the tube, thereby, protecting the wire from oxidation. The nitrogen for the volatilization system was directed via 1/400 tubing to a flow meter regulated by a metering valve. From the flow meter, the volatilization nitrogen flowed via 1/400 tubing to a ball and socket joint at the bottom of the volatilization chamber. This nitrogen flowed up through the volatilization chamber passing over the coil and volatilizing the test substance. The pressure within the counter-current volatilization chamber was maintained slightly negative to the room and was monitored with a pressure gauge.
This test substance laden nitrogen exited the top of the volatilization chamber through a stainless steel ‘‘T’’ which divided the flow, via 1/200 tubing, to the turrets of two 1 cubic meter glass and stainless steel exposure chambers. As the test substance laden nitrogen was drawn into each of the chambers, it was mixed with room air. The whole-body exposure chambers each had a volume of approximately 1000 L (l m3). Each chamber was operated at a minimum flow rate of 200 L/min. The final airflow was set to provide at least one air change (calculated by dividing the chamber volume by the airflow rate) in 5.0 min (12 air changes/h) and a T99 equilibrium time (calculated by multiplying the air change by the exponential factor 4.6) of at most 23 min.
This chamber size and airflow rate was considered adequate to maintain the oxygen level at least 19% and the animal loading factor below 5%. At the end of each exposure, all animals remained in the chamber for a minimum of 30 min. During this time, each chamber was operated at approximately the same flow rate using clean air only. The chambers were exhausted through the in house filtering system, which consisted of a coarse filter, a HEPA filter, activated charcoal and then through a fume incinerator.

Details on mating procedure:

Vaginal smears were taken daily for each female beginning 3 weeks prior to cohabitation for P0 and F1 rats and continuing until there was evidence of mating or until the 14-day mating period was ended. Following 10 weeks premating exposure, one male and one female from the same group were mated overnight until evidence of mating was observed or 14 days had elapsed. The day evidence of mating was observed (a copulation plug in the vagina and/or microscopic observation of sperm in a vaginal smear) was defined as Day 0 of gestation (GD0). Animals were not paired during the daily exposure period. During mating of F1 generation, male and female littermates were never paired together. At weaning of each F1 litter on Lactation day (LD) 28, one pup/sex/litter was chosen at random to continue with exposure to the test item as the F1 parental generation.
When less than 26 litters were available in a group, additional pups from other litters within the group were selected at random to make up 26 mating pairs/group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test material was administered as a vapor in the breathing air of the animals as described in the cross reference Clark et al. (2014):
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:

6 h / day

Frequency of treatment:

7 days / week

Details on study schedule:

Exposure schedule is attached as illustration ( see overall remarks, attachments).
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).
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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/m³ air

Doses / concentrations 2

Dose / conc.:

10 000 mg/m³ air

Doses / concentrations 3

Dose / conc.:

20 000 mg/m³ air

No. of animals per sex per dose:

26/sex/dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Animals considered suitable for study on the basis of pretest 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.

Examinations

Parental animals: Observations and examinations:

Viability checks were performed twice daily for mortality and signs of severe health effects. Physical observations and body weights were recorded twice pretest (P0 generation) and at least weekly during the study. Food consumption was measured beginning the week prior to treatment initiation (P0 generation) and at least weekly during the study. For P0 and F1 dams, body weight and food consumption were measured on Gestation Days [GD] 0, 7, 14, 20 and on Lactation Days [LD] 1, 4, 7, 14, 21 and 28.

Oestrous cyclicity (parental animals):

Reproductive organs from all bred female rats in control and high dose groups were evaluated. Examination of all parental females included a vaginal smear at time of necropsy to determine stage of estrus and a count of uterine implantation scars if present. Ovary histopathology included evaluation of the primordial follicle population, number of growing follicles and corpora lutea.

Sperm parameters (parental animals):

Reproductive organs from all male rats in control and high dose groups were evaluated. Right testes and right epididymis from each animal were removed intact, weighed (testes weighed together and separately) and fixed in modified Davidson’s solution for 48 h prior to permanent storage in 10% neutral buffered formalin for possible histopathology at Huntingdon Life Sciences. Sperm evaluations included motility, testicular homogenization-resistant sperm and cauda epididymal sperm count and sperm morphology. Sperm evaluations were performed on specimens shipped frozen on dry ice to Pathology Associates International, Frederick MD (PAI). Analyses were performed on the left epididymis, vas deferens and left testis.

Litter observations:

On GD18, exposure was ended and each female was transferred to a plastic shoebox with bedding material and observed for evidence of parturition twice daily. The day on which parturition was observed was LD0. These females were not exposed from GD19 [P0 and F1 dams] until exposure was resumed on LD5 to weaning at LD28.
Pups of the F1 and F2 generations were observed as soon as possible after delivery for sex, number of live and dead pups and pup abnormalities. All pups were uniquely identified within the litter by toe tattoo. Pups dead at delivery were identified as stillborn or liveborn found dead based on lung floatation (air in the lungs) evaluation. Thereafter litters were observed twice daily and litter size was recorded daily from LD1 to LD28. On LD4, F1 litters with more than 10 pups were randomly culled to 10 pups with sex distribution equalized if possible. Pups were examined and weighed on LD1 (delivery day), 4 (preculled), 7, 14, 21 and 28.

Postmortem examinations (parental animals):

All parental male animals were sacrificed during the lactation period for a total of 16–20 weeks of exposure and all parental females (P0 and F1) were sacrificed on their respective LD28. Females that failed to mate were sacrificed 25 days after the end of the mating period and females with confirmed mating but without delivery were sacrificed on presumed GD25. Non-pregnant status was confirmed by staining with ammonium sulfide for implantation sites. Selected organs [adrenals, brain, heart, liver, lungs, kidneys, spleen, thymus, ovaries, uterus, testes, seminal vesicles, prostate, epididymides] were weighed and organ/body weight and organ/brain weight ratios calculated. Macroscopic examinations were performed on all parental rats and histological evaluations of the tissue samples from the weighed organs of 10 randomly selected rats in the control and 20,000 mg/m3 groups were performed. Details of general histopathology procedures are found in Clark et al. (2014).

Postmortem examinations (offspring):

F1 pups [5/sex/group/assessment] not selected for F1 mating were evaluated for standard Tier 2 neuropathology (40 CFR79.66; US EPA, 1998b) or for GFAP assessments (40 CFR79.67; US EPA, 1994b) on postpartum day 28. Methods employed for both procedures are provided in O’Callaghan et al. (2014). The standard Tier 2 neuropathologic evaluation was performed at Huntingdon Life Sciences. For GFAP analyses, brains were removed, weighed and processed, then shipped on dry ice to the US Centers for Disease Control and Prevention, Health Effects Laboratory Division, Morgantown, WV for analysis by Dr. James O’Callaghan. The remaining pups were examined for external abnormalities and sacrificed. Pups with abnormalities were preserved intact in 10% neutral buffered formalin. Three pups/sex/litter in each group (F1 and F2) were selected for macroscopic examination and selected organs [brain, spleen, thymus] were weighed from one pup /sex/litter.

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 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 + 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 (Games and Howell, 1976; Kruskal and Wallis, 1952, 1953; Siegel, 1956). 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 oneway 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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

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 baseline gasoline vapour condensate, however 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

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

Mortality:
There were no significant effects of treatment on survival.

Systemic parental effects:
Parental animal data are summarized in Table 3a (Females) and Table 3b (Males). To facilitate comparisons across test materials the data are presented for the 20,000 mg/m3 groups only. Statistical significance was determined by comparison with concurrent air control values.

Clinical observations:
There were no remarkable clinical observations. Statistically significant reduction in weight gain during the premating period was observed in females exposed to 20,000 mg/m3 BGVC (P0). However, no effects on maternal body weight gains occurred during gestation (GD0–20) and lactation (LD1–28). Additionally, food consumption (data not shown) was comparable to concurrent controls for BGVC.

Organ weights:
All test material exposure caused a statistically significant increase in male kidney weights in the 10,000 and 20,000 mg/m3 groups consistent with light hydrocarbon nephropathy (data not shown). Slight increases in female kidney weights were observed at 20,000 mg/m3 in BGVC P0.

Histopathological observations:
No remarkable histopathologic changes were reported in the study. Light hydrocarbon nephropathy was strongly indicated by the presence of hyaline droplets in kidneys of 20,000 mg/m3 male rats (Alden, 1986). No treatment related macroscopic or microscopic changes were seen in male or female reproductive organs.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

10 000 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Remarks on result:

other: NOAEL determination excluded male rat kidney hydrocarbon nephropathy as not relevant to human risk assessment.

2

Dose descriptor:

NOAEL

Effect level:

20 000 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive: fertility, days to mating, estrus cycle length, sperm counts or morphology or developmental parameters in pups.

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

NOAEL determination excluded male rat kidney hydrocarbon nephropathy as not relevant to human risk assessment

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Exposure to baseline gasoline vapour condensate caused a statistically significant increase in male kidney weights in the 10,000 and 20,000 mg/m3 groups consistent with light hydrocarbon nephropathy (data not shown).

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Description (incidence and severity):

Exposure to BGVC did not cause changes in GFAP levels in any brain region examined with the exception of a single decrease in the 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.

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P1)

Mortality:
There were no significant effects of treatment on survival.

Systemic parental effects:
Parental animal data are summarized in Table 3a (Females) and Table 3b (Males). To facilitate comparisons across test materials the data are presented for the 20,000 mg/m3 groups only. Statistical significance was determined by comparison with concurrent air control values.

Clinical observations:
There were no remarkable clinical observations.

Organ weights:
All test material exposure caused a statistically significant increase in male kidney weights in the 10,000 and 20,000 mg/m3 groups consistent with light hydrocarbon nephropathy (data not shown).

Histopathological observations:
No remarkable histopathologic changes were reported in the study. Light hydrocarbon nephropathy was strongly indicated by the presence of hyaline droplets in kidneys of 20,000 mg/m3 male rats (Alden, 1986). No treatment related macroscopic or microscopic changes were seen in male or female reproductive organs.

Effect levels (P1)

Dose descriptor:

NOAEL

Effect level:

10 000 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

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

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.

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Offspring observations:
Offspring observations are summarized in Table 4 (see any information on results). There were no effects attributable to test material exposure on litter size (pups/litter, pups born dead/litter), number of implantation sites/litter, pup birth weight, offspring survival, or sex ratio. Additionally, no adverse effects were seen on offspring organ weights.
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. 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.
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. GFAP results are presented in Tables 5, see any information on results. Exposure to either test material 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.

Reproductive paramters:
Reproductive parameters are summarized in Table 6, see any information on results. There were no differences in male and female fertility or reproductive performance with exposure to the test material. Estrus cyclicity and semen parameters were comparable between exposed and concurrent control group.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

20 000 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observable

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

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not specified

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

Offspring observations:
Offspring observations are summarized in Table 4 (see any information on results). There were no effects attributable to test material exposure on litter size (pups/litter, pups born dead/litter), number of implantation sites/litter, pup birth weight, offspring survival, or sex ratio. Additionally, no adverse effects were seen on offspring organ weights.
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.

Reproductive paramters:
Reproductive parameters are summarized in Table 6, see any information on results. There were no differences in male and female fertility or reproductive performance with exposure to the test material. Estrus cyclicity and semen parameters were comparable between exposed and concurrent control group.

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

20 000 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observable

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 3a Effects on female rats (P0 and F1) from exposure to vapor condensates of baseline gasoline vapour condensate at 20,000 mg/m3.

Endpoints	Control range	BGVC
FEMALES		P0	F1
Premating body weight gain (g) % of control	108-144 (P0); 175-200 (F1)	125**	189
		89.3%	94.5%
Gestation day 0-20 weight gain (g) % of control	113-129 (P0); 120, 121(F1)	125	122
		101.6%	100.8%
Lactation day 21-28 weight gain (g) % of control	-3 to13 (P0); 7-15 (F1)	-1	7
		(control-3)	100.0%
Lung, discolored foci (macroscopic) % of rats	0/26-4/26	1/26	5/26
	0-15.4%	3.8%	19.2%
Relevant organ weight changes		increase: kidney	NE

Statistical significance based upon comparison to each study’s concurrent control. NE = no effect. * p<0.05; ** p<0.01

Table 3b Effects on female rats (P0 and F1) from exposure to vapor condensates of baseline gasoline vapour condensate at 20,000 mg/m3.

Endpoints	Control range	BGVC
		P0	F1
MALES
Prematingbody weight gain (g) % of control	225-313 (P0); 350-379 (F1)	250	364
		95.8%	96.0%
Lung, discolored foci (macroscopic) % of rats	0/26-4/26	2/26	1/26
	0-15.4%	7.7%	3.8%
Relevant organ weight changes		Increase: kidney	Increase: kidney

Statistical significance based upon comparison to each study’s concurrent control. * p<0.05; ** p<0.01

Table 4 Effects on offspring (F1 and F2) from exposure of parents to baseline gasoline vapour condensate 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
LD1	6.8-7.4	7	7
LD4	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^b		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 = no effect. * p<0.05; ** p<0.01

Table 5 Mean GFAP levels on specific regions of rat brains of F1 generation offspring following whole body inhalation exposure of maternal rats to gasoline (BGVC) vapor condensate.

Brain Area	Control	2000 mg/m^3	10,000 mg/m^3	20,0000 mg/m^3
Males (N=5)
Striatum	0.33 ±0.01^a	0.38 ± 0.02	0.40 ± 0.04	0.33 ± 0.05
Hippocampus	2.01 ± 0.14	2.36 ± 0.22	2.48 ± 0.20	1.75 ±0.20
Cortex	0.61 ± 0.05	0.72 ± 0.08	0.76 ± 0.07	0.54 ± 0.07
Olfactory Bulb	1.34 ±0.08	1.19 ±0.07	1.28 ±0.08	1.13 ±0.09
Thalamus	0.86 ± 0.07	0.88 ± 0.07	0.94 ± 0.07	0.65 ± 0.05^b
Hypothalamus	1.81 ±0.18	1.99 ±0.20	1.73 ±0.14	1.36 ±0.16
Cerebellum	3.04 ± 0.07	3.34 ± 0.26	3.13 ±0.18	2.47 ± 0.27
Rest of Brain	2.56 ± 0.25	3.00 ± 0.22	3.07 ± 0.27	2.41 ± 0.39
Females (N=5)
Striatum	0.34 ± 0.04	0.42 ± 0.02	0.37 ± 0.04	0.35 ± 0.03
Hippocampus	2.04 ± 0.09	2.18 ±0.09	2.26 ± 0.14	2.06 ± 0.09
Cortex	0.60 ± 0.02	0.67 ± 0.04	0.69 ± 0.04	0.63 ± 0.07
Olfactory Bulb	1.29 ±0.11	1.37 ±0.11	1.22 ±0.09	1.12 ±0.07
Thalamus	0.77 ± 0.03	0.86 ± 0.06	0.93 ± 0.06	0.73 ± 0.07
Hypothalamus	1.89 ±0.12	1.68 ±0.09	1.75 ±0.18	1.68 ±0.20
Cerebellum	2.84 ± 0.18	3.42 ± 0.30	2.94 ± 0.23	2.44 ± 0.27
Rest of Brain	2.96 ± 0.52	2.94 ± 0.20	3.32 ± 0.42	2.64 ± 0.22

a Each value represents the mean ± SEM for the concentration of GFAP (lg/mg total protein). b Statistically different from control (p<0.05).

Table 6 Effects on reproductive parameters on parental animals (P0 and F1) from exposure to vapor condensates of gasoline or gasoline/oxygenate blends 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 paramters
Sperm Count, testis (x10^6/g)	81.2-124.1	115.4	100.6
Motility(%)	89-96%	94%	95%
Morphology(%abnormal)	0.6-1.7%	1.20%	0.50%
Epididymalsperm Count (x 10^6/g)	753.6-956.7	955.3	933.5

Applicant's summary and conclusion

Conclusions:

The BGVC parental NOAEL of 10,000 mg/m³ 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. The NOAEL for reproductive and offspring parameters was 20,000 mg/m3 for Baseline gasoline vapor condensate (BGVC) based on no differences from controls were seen for fertility, days to mating, estrus cycle length, sperm counts or morphology or developmental parameters in pups, whereby the result should be also applicable for the closely related substance 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear'.
Additionally, no neuropathology or neurotoxicity expressed as GFAP changes were observed in F1 offspring in BGVC. The single decrease in the BGVC F1 male thalamus at 20,000 mg/m³ was not considered biologically significant by the investigator.

Executive summary:

Vapor condensates of baseline gasoline (BGVC) was evaluated for reproductive toxicity in Sprague-Dawley rats at target concentrations of 2000, 10,000, or 20,000 mg/m3, 6 h/day, 7 days/week. BGVC was assessed over two generations. The F1 offspring was evaluated for neuropathology and changes in regional brain glial fibrillary acidic protein content. No neurotoxicity was observed. Male (P0 and F1) kidney weight was increased consistent with light hydrocarbon nephropathy. In adult female P0 rats, decreased body weight gain and increased kidney weight were seen. No pathological changes to reproductive organs occurred in the study. The NOAEL for reproductive and offspring parameters was 20,000 mg/m3 for BGVC.