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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No pathological changes to reproductive organs occurred in the study. The NOAEL for reproductive and offspring parameters was >= 20000 mg/m3 for the read-across substance Baseline Gasoline Vapour Condensate (BGVC) (CAS 68514-15-8; EC 271-025-4), whereby the result should also be applicable for the closely related substance 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear'.

Link to relevant study records

Reference

Reference 1

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

Reason / purpose for cross-reference:

reference to other study

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

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.

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.

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.

Dose / conc.:

2 000 mg/m³ air

Dose / conc.:

10 000 mg/m³ air

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.

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.

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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.

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.

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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.

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

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

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not examined

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.

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

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

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not examined

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.

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

Reproductive effects observed:

not specified

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

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.

Reference 2

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.35 (Two-Generation Reproduction Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4700 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

Animals were exposed 6 h/day, 7 days/week in 1.5-m³ chambers. A schematic of the test atmosphere generation and exposure system is presented in Fig. 1 (attached in the background material). The temperature was maintained between 20 and 24°C; the humidity ranged between 40 and 60%, and there were 12–15 air changes/h. The target vapor concentrations were 5000, 10 000, and 20 000 mg/m3. The chamber concentrations were monitored hourly via on-line gas chromatography.
The study was carried out with groups of 30 randomly selected male and female Sprague–Dawley rats, approximately 7 weeks old at the start of exposure. Singly housed animals were exposed for 10 weeks prior to mating. The animals were exposed throughout the 3-week mating period. Exposure of the males continued until the end of the mating period, when they were sacrificed. Exposure of the females continued until gestation day (GD) 20. Exposure was suspended until postpartum day (PPD) 5 to avoid unduly stressing the dams during birth, and then resumed. Exposure of the parental females continued until sacrifice after weaning. The pups were culled on a random basis to approximately 5/sex/litter. At weaning on postnatal day (PND) 28, the F1 pups selected for the second generation were sorted by exposure group. The selected pups were exposed for a 13-week premating period and then for a 3-week mating period as described above. The males were sacrificed at that point, and the females continued to be exposed until GD 20. Exposure was then resumed on PPD 5 and continued to weaning (PND 21), when all remaining animals were sacrificed. Other than the period between GD20 and PPD 5, as described above, all F1 offspring were exposed from conception to sacrifice.

Details on mating procedure:

During a 3-week mating period, mating pairs were cohoused. After 2 weeks, females that had not mated were cohoused for a third week with males of the same exposure group that were known to be fertile. Exposure of the males continued until the end of the mating period, when they were sacrificed. Females confirmed to have mated by observation of a vaginal plug or the presence of sperm in a vaginal rinse were single housed for the gestational period.
At weaning on postnatal day (PND) 28, the F1 pups selected for the second generation were sorted by exposure group. The selected pups were exposed for a 13-week premating period and then for a 3-week mating period as described above.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The overall mean chamber concentrations of VRU gasoline during the exposure periods throughout the study were 0 +/- 0, 5076 +/- 146, 10247 +/- 249, and 20241 +/- 373 mg/m³. The concentrations of the test atmosphere were determined approximately hourly during each exposure by on-line gas chromatography.

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

7 days/ week

Details on study schedule:

The study was carried out with groups of 30 randomly selected male and female Sprague–Dawley rats, approximately 7 weeks old at the start of exposure.
Singly housed animals were exposed for 10 weeks prior to a 3-week mating period.
The pups were culled on a random basis to approximately 5/sex/litter. At weaning on postnatal day (PND) 28, the F1 pups selected for the second generation were sorted by exposure group. The selected pups were exposed for a 13-week premating period and then for a 3-week mating period as described above.

Dose / conc.:

5 000 mg/m³ air

Remarks:

Chamber concentration, 12–15 air changes/h.

Dose / conc.:

10 000 mg/m³ air

Remarks:

Chamber concentration, 12–15 air changes/h.

Dose / conc.:

20 000 mg/m³ air

Remarks:

Chamber concentration, 12–15 air changes/h.

No. of animals per sex per dose:

The study was carried out with groups of 30 randomly selected male and female Sprague–Dawley rats

Control animals:

yes

Details on study design:

The study was carried out with groups of 30 randomly selected male and female Sprague–Dawley rats. Also the pups were culled on a random basis to approximately 5/sex/litter.
The highest concentration, approximately half of the lower explosive limit, was the highest level that was considered safe to test. The suitability of these levels was assessed in preliminary range-finding studies [EBSI Report No. 115233A and Report No. 115233B (both 1997)] and, as no effects were observed, they were also used for the definitive study.

Positive control:

None tested.

Parental animals: Observations and examinations:

All test animals were checked twice daily for viability, and clinical observations were carried out on a daily basis. Body weights and food consumption were measured weekly until confirmation of mating and then on GD 0, 7, 14, and 21 and PPD 0, 4, 7, 14, and 21 for P0 and P1, and on PPD 28 for P0 only.

Oestrous cyclicity (parental animals):

There was also an ovarian examination that included confirmation of growing follicles and corpora lutea and quantification of primordial oocytes. Five sections per ovary for all ovaries from the high-dose and control group animals were evaluated under light microscopy. As there were no differences between the high dose (20000 mg/m³) and control, tissues from animals in the intermediate groups were not examined.
Reproductive parameters evaluated included: female fertility indices, female fecundity and gestational indices, mean litter size, mean days of gestation, female estrous cycle length, and number of females cycling normally. Live birth index, survival indices (PPD 1, 4, 7, 14, 21), viability index at weaning, mean live and dead offspring on Day 0, sex ratio at Day 0, offspring inlife observations, offspring body weight, and offspring gross postmortem findings were also assessed.

Sperm parameters (parental animals):

Reproductive parameters evaluated included: male fertility indices, male mating index. The sperm analysis was carried out on both P0 and on P1(F1) males.

Samples of sperm from the left distal cauda epididymis were collected from all males at terminal sacrifice for evaluation of sperm parameters. These included assessments of total caudal epididymal sperm number, percent progressively motile sperm, and homogenization resistant spermatid count, percent morphologically normal sperm, and percent of sperm with an identified abnormality. To assess progressively motile sperm, the left cauda was sliced and suspended in a petri dish in 10 ml Dulbecco’s phosphate- buffered saline (PBS) with 1.0% bovine serum albumen at 37°C for 15 min. The resulting suspension was swirled gently, and a 1.0-ml sample was added to 9 ml warmed media. A sample was taken up in a cannula by capillary action and assessed by the Hamilton Thorne Research IVOS (Integrated Visual Optical System) from the suspension in the petri dish. Total cauda epididymal sperm counts were also assessed by the IVOS. For evaluation of homogenization-resistant spermatid counts, the testes were placed in 20-30 ml SMT solution (100 mg merthiolate and 0.5 ml Triton X-100 in 1000 ml 0.9% saline), homogenized for 2 min, and allowed to settle. The volume was brought up to 50 ml SMT solution, homogenized again, and allowed to settle for 1 min. An aliquot of the homogenate was then stained for DNA, and quantified using the IVOS system.

Sperm morphology was determined manually under phase contrast microscopy. Ten males were randomly selected from each treatment group. Samples were collected from the left cauda of each animal, and four slides were prepared from each sample. Two of the slides were stained with 1.0% Eosin Y and two with Papanicolaou stain. The Eosin Y slides were evaluated, and the Papanicolaou stained slides were retained for future evaluation. Five hundred sperm from each animal were evaluated.

Litter observations:

All pups were counted and examined externally on a daily basis until PND 21, and weighed on PND 0, 4, 7, 14, and 21. F1 pups were also examined daily from PND 21 to 28 and weighed on PND 28 and 35. All surviving F1 and F2 pups were evaluated for developmental landmarks, including pinna detachment, hair growth, incisor eruption, eye opening, and the development of the surface righting reflex. All surviving F1 female offspring were monitored for vaginal opening beginning on PND 29, and F1 male offspring were monitored for preputial separation beginning on PND 35.

Postmortem examinations (parental animals):

Parental animals were approximately 7 weeks old at the start of exposure. Animals were exposed for 10 weeks prior to the 3-week mating period. Exposure of the males continued until the end of the mating period, when they were sacrificed. Exposure of the females continued until gestation day (GD) 20. Exposure was suspended until postpartum day (PPD) 5 to avoid unduly stressing the dams during birth, and then resumed. Exposure of the parental females continued until sacrifice after weaning.
All animals dying spontaneously or sacrificed in a moribund condition were necropsied.
Organs weighed included liver, adrenals, brain, uterus, testes, right epididymis, and left caudal epididymis, seminal vesicles (with coagulating glands and fluid), kidneys, spleen, thymus, ovaries, prostate, and lungs. The list of tissues taken for microscopic examination included vagina, uterus, ovaries, right epididymis, seminal vesicles, prostate, oviducts, thymus, trachea, nasal turbinates, spleen, coagulating gland, pituitary, kidneys, liver, mammary gland (females only), testes, brain, tissue masses/gross lesions, larynx, lungs, and adrenals. The tissues from the high-dose (20000 mg/m³) and control animals were evaluated. There were histologic findings in the kidneys, and, as a consequence, kidney sections from the intermediate exposure groups were examined. However, as there was no other evidence of treatment related effects, the other tissues from the other treatment groups were not evaluated.

Postmortem examinations (offspring):

All animals dying spontaneously or sacrificed in a moribund condition were necropsied.
The pups were culled on a random basis to approximately 5/sex/litter. Culled pups were examined and sacrificed.
Culled pups were examined externally but were not necropsied unless there was external evidence of abnormalities. Randomly selected pups were necropsied, and the following organs were weighed: ovaries, liver, adrenals, thymus, testes, kidneys, spleen, and brain. Additionally, the following tissues were taken for microscopic examination: vagina, ovaries, epididymides, prostate, pituitary, spleen, kidneys, thymus, uterus (with cervix), testes, seminal vesicles, coagulating gland, adrenals, liver, brain, and any gross lesions. The majority of the tissues were fixed in 10% neutral buffered formalin, routinely processed, embedded in paraffin, and stained with hematoxylin and eosin (H&E). The right testes were fixed in Bouin’s fixative and stained with the periodic acid-Schiff reaction (PAS). Sections of the kidneys of male rats from all groups of the P0 and P1 generations were stained by Mallory’s Heidenhain technique for the identification of hyaline droplet accumulation.

Statistics:

Statistics were describes in "any other information on materials and methods"

Reproductive indices:

Reproductive parameters evaluated included: male and female fertility indices, male mating index, female fecundity and gestational indices, mean litter size, mean days of gestation, female estrous cycle length, and number of females cycling normally. Live birth index, survival indices (PPD 1, 4, 7, 14, 21), viability index at weaning, mean live and dead offspring on Day 0, sex ratio at Day 0, offspring inlife observations, offspring body weight, and offspring gross postmortem findings were also assessed.

Offspring viability indices:

All pups were counted and examined externally on a daily basis until PND 21, and weighed on PND 0, 4, 7, 14, and 21. F1 pups were also examined daily from PND 21 to 28 and weighed on PND 28 and 35. All surviving F1 and F2 pups were evaluated for developmental landmarks, including pinna detachment, hair growth, incisor eruption, eye opening, and the development of the surface righting reflex. All surviving F1 female offspring were monitored for vaginal opening beginning on PND 29, and F1 male offspring were monitored for preputial separation beginning on PND 35.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no treatment-related clinical signs of toxicity or mortality. In the first parental generation, there were two deaths during the exposure period, one control male and one female in the 5000 mg/m3 group. However, all animals in the highest exposure group survived in both generations. Analysis of the data revealed that there were no significant effects on weight gain (Figs. 2 and 3, see background material) or food consumption between treated and control animals of either generation. Similarly, there were no differences in gestational or postpartum body weights (Figs. 4 and 5, see background material) or food consumption in either generation. Finally, there were no postmortem findings that were unusual or appeared to have been treatment-related (data not shown).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

see Figure 2 and 4, background material.

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:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Mortality, weight gain, and clinical signs:
There were no treatment-related clinical signs of toxicity or mortality. In the P0 generation, there were two deaths during the exposure period, one control male and one female in the 5000 mg/m3 group. In the second parental generation, one male in the control group and one in the 10 000 mg/m3 group died during exposure. The deaths were not considered treatment related. All animals in the highest exposure group survived in both generations. Analysis of the data revealed that there were no significant effects on weight gain or food consumption between treated and control animals of either generation. Similarly, there were no differences in gestational or postpartum body weights or food consumption in either generation. Finally, there were no postmortem findings that were unusual or appeared to have been treatment-related.

Organ weights:
There were no significant differences in absolute organ weights in either males or females from the P0 generation.

Pathologic investigation:
There were no compound-related microscopic changes in any of the reproductive tissues or in the tissues of the upper or lower respiratory tract from any of the P0 or P1 generation rats exposed to 20000 mg/m3 VRU gasoline. The only treatment-related histologic changes were observed in the kidneys of male rats of both generations and consisted of exposure-related increases in the amount and size of hyaline droplets. The hyaline droplets in several of the exposed rats were larger and stained more densely with the Heidenhain stain. The enlargement of these droplets was due to coalescing of clusters of hyaline droplets and the accumulation of irregular to somewhat angular-shaped hyaline droplets. The only other treatment related effect in the kidney was seen in three male rats of the high-dosage groups from both the P0 and P1 generations and consisted of granular casts in medullary tubules. These granular casts often accompany increased hyaline droplet accumulations and are consistent with the “hydrocarbon/hyaline droplet nephropathy,” which is unique to male rats, reflecting the exacerbated accumulation of alpha-2-u-globulin in the kidney. This finding is not relevant to human risk assessment.

Reproductive parameters:
In the first parental generation, there were no differences in mating index, fecundity, pregnancy, or length of gestation.

Sperm parameters and Estrous cycle:
The sperm analysis was carried out on both P0 and on P1(F1) males. There were no significant effects on sperm count, progressive motility, or gross appearance in either group. There were no statistically significant differences in mean estrous cycle length, quantification of primordial oocytes, or percent females with abnormal cycles between treated and control females in the P0 or P1 generations.

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 20 000 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: no adverse effects

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the second parental generation, one male in the control group and one in the 10 000 mg/m3 group died during exposure. However, all animals in the highest exposure group survived in both generations. Finally, there were no postmortem findings that were unusual or appeared to have been treatment-related (data not shown). Analysis of the data revealed that there were no significant effects on weight gain (Figs. 2 and 3, see background material) or food consumption between treated and control animals of either generation. Similarly, there were no differences in gestational or postpartum body weights (Figs. 4 and 5, see background material) or food consumption in either generation. Finally, there were no postmortem findings that were unusual or appeared to have been treatment-related (data not shown).

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, non-treatment-related

Description (incidence and severity):

Among the second parental (P1) generation animals, there were some statistically significant increases in absolute organ weights, including liver, kidney, and
testis in the males and lungs in the females (Table 3, see any further information on results), but none of the organ weight differences between the high exposure group and control animals were significantly different. In the absence of clear dose-response relationships, the toxicologic significance of these data is unclear. When the data were expressed on an organ to body weight basis, the only significant differences were an elevation of relative kidney weights in the males from the low exposure group of the first parental generation (in the absence of a dose-response this observation was assumed not to be treatment-related) and an elevation of relative kidney weights from the high-exposure group males from the second parental generation (data not shown). The latter observation may have been treatment-related, but, as described below, was not clinically important.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The only treatment-related histologic changes were observed in the kidneys of male rats of both generations and consisted of exposure-related increases in the amount and size of hyaline droplets. The hyaline droplets in several of the exposed rats were larger and stained more densely with the Heidenhain stain. The enlargement of these droplets was due to coalescing of clusters of hyaline droplets and the accumulation of irregular to somewhat angular-shaped hyaline droplets. The only other treatment related effect in the kidney was seen in three male rats of the high-dosage groups from both the P0 and P1 generations and consisted of granular casts in medullary tubules. These granular casts often accompany increased hyaline droplet accumulations and are consistent with the “hydrocarbon/hyaline droplet nephropathy,” which is unique to male rats, reflecting the exacerbated accumulation of a-2 u-globulin in the kidney.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Mortality, weight gain, and clinical signs:
There were no treatment-related clinical signs of toxicity or mortality. In the P0 generation, there were two deaths during the exposure period, one control male and one female in the 5000 mg/m3 group. In the second parental generation, one male in the control group and one in the 10 000 mg/m3 group died during exposure. The deaths were not considered treatment related. All animals in the highest exposure group survived in both generations. Analysis of the data revealed that there were no significant effects on weight gain (Figs. 2 and 3, see background material) or food consumption between treated and control animals of either generation. Similarly, there were no differences in gesta-tional or postpartum body weights (Figs. 4 and 5, see background material) or food consumption in either generation. Finally, there were no postmortem findings that were unusual or appeared to have been treatment-related (data not shown).

Organ weights:
Among the second parental (P1) generation animals, there were some statistically significant increases in absolute organ weights, including liver, kidney, and testis in the males and lungs in the females (Table 3, see any further information on results), but none of the organ weight differences between the high exposure group and control animals were significantly different. In the absence of clear dose-response relationships, the toxicologic significance of these data is unclear. When the data were expressed on an organ to body weight basis, the only significant differences were an elevation of relative kidney weights in the males from the low exposure group of the first parental generation (in the absence of a dose-response this observation was assumed not to be treatment-related) and an elevation of relative kidney weights from the high-exposure group males from the second parental generation (data not shown). The latter observation may have been treatment-related, but, as described below, was not clinically important.

Pathologic investigation:
There were no compound-related microscopic changes in any of the reproductive tissues or in the tissues of the upper or lower respiratory tract from any of the P0 or P1 generation rats exposed to 20000 mg/m3 VRU gasoline. The only treatment-related histologic changes were observed in the kidneys of male rats of both generations and consisted of exposure-related increases in the amount and size of hyaline droplets. The hyaline droplets in several of the exposed rats were larger and stained more densely with the Heidenhain stain. The enlargement of these droplets was due to coalescing of clusters of hyaline droplets and the accumulation of irregular to somewhat angular-shaped hyaline droplets. The only other treatment related effect in the kidney was seen in three male rats of the high-dosage groups from both the P0 and P1 generations and consisted of granular casts in medullary tubules. These granular casts often accompany increased hyaline droplet accumulations and are consistent with the “hydrocarbon/hyaline droplet nephropathy,” which is unique to male rats, reflecting the exacerbated accumulation of alpha-2-u-globulin in the kidney. This finding is not relevant to human risk assessment.

Reproductive parameters:
There were no differences in mating index, fecundity, pregnancy, or length of gestation.

Sperm parameters and Estrous cycle:
The sperm analysis was carried out on both P0 and on P1(F1) males. There were no significant effects on sperm count, progressive motility, or gross appearance in either group. There were no statistically significant differences in mean estrous cycle length, quantification of primordial oocytes, or percent females with abnormal cycles between treated and control females in the P0 or P1 generations.

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 20 000 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: no adverse 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):

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:

no effects observed

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Pathological Examinations
As noted for the parental animals, the only treatment-related histologic changes were observed in the kidneys of F1 male rats consisted of exposure-related increases in the amount and size of hyaline droplets, granular casts in medullary tubules of the kidney. These findings are consistent with “hydrocarbon/hyaline droplet nephropathy,” which is unique to male rats, reflecting the exacerbated accumulation of alpha-2-u-globulin in the kidney. This finding is not relevant to human risk assessment.

Reproductive parameters
Among the offspring of the first parental generations, there were no differences in mean litter size, fraction of live births, or sex ratio. Among the offspring, there were no differences in survival of offspring through weaning in the first generation. There were no differences in the weight of the offspring through weaning. Finally, there were no unusual postmortem observations that were considered to be treatment related.

Developmental landmarks
There were no significant differences in incisor eruption, pinna detachment, or surface righting reflex in the F1 or F2 offspring, or vaginal patency or preputial separation in the F1 offspring. There was a significant delay in hair growth in the males but not the females of the F1 pups.

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Generation:

F1

Effect level:

>= 20 000 mg/m³ air (nominal)

Sex:

male/female

Basis for effect level:

other: No adverse effects on reproductive parameters (sperm parameters)

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):

no effects observed

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:

no effects observed

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Developmental landmarks:
There were no significant differences in incisor eruption, pinna detachment, or surface righting reflex in the F1 or F2 offspring. Eye opening was advanced by approximately one-half a day for the high-dose males, and hair growth was delayed in the low-dose males and females of the F2 offspring.

Reproductive parameters:
Among the offspring, there were no differences in survival of offspring through weaning in the second generation, early survival was slightly higher among the offspring from exposed dams than those from controls (Table 6, see any further information on results). There were no differences in the weight of the offspring through weaning in either generation (Table 7, see any further information on results). Finally, there were no unusual postmortem observations that were considered to be treatment related (data not shown).

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

Reproductive effects observed:

not specified

Table 2: Organ weight (mean g +/- SD) of male and female rats of the first parental (P0) generation from the two-generation reproductive toxicity study of Gasoline, vapor-recovery

Group (mg/m^3)

Terminal body weight

Liver

Kidneys

Adrenals

Brain

Thymus

Spleen

Lungs

MALES

0

496.8 ± 43.2

15.50 ± 1.66

3.45 ± 0.29

0.060 ± 0.010

2.20 ± 0.10

0.292 ± 0.083

0.76 ± 0.11

1.85 ± 0.19

5000

502.6 ± 44.6

15.95 ± 2.04

3.71 ± 0.40

0.063 ± 0.013

2.15 ± 0.13

0.271 ± 0.077

0.77 ± 0.11

1.88 ± 0.22

10000

501.1 ± 46.1

15.67 ± 1.58

3.61 ± 0.37

0.056 ± 0.010

2.18 ± 0.12

0.307 ± 0.097

0.79 ± 0.10

1.82 ± 0.18

20000

491.6 ± 47.2

15.29 ± 2.02

3.60 ± 0.43

0.057 ± 0.010

2.16 ± 0.14

0.275 ± 0.086

0.74 ± 0.11

1.86 ± 0.18

FEMALES

0

313.9 ± 22.5

12.81 ± 1.22

2.40 ± 0.16

0.079 ± 0.011

1.99 ± 0.07

0.212 ± 0.053

0.60 ± 0.08

1.51 ± 0.10

5000

325.7 ± 20.4

13.68 ± 1.52

2.48 ± 0.18

0.077 ± 0.011

1.97 ± 0.08

0.267 ±0.086

0.61 ± 0.06

1.54 ± 0.12

10000

323.6 ± 23.9

12.55 ± 1.39

2.50 ± 0.16

0.076 ± 0.010

1.98 ± 0.09

0.229 ± 0.064

0.62 ± 0.08

1.56 ± 0.13

20000

317.2 ± 21.7

12.48 ± 1.41

2.48 ± 0.20

0.075 ± 0.011

1.94 ± 0.09

0.245 ± 0.077

0.59 ± 0.06

1.52 ± 0.12

Group (mg/m^3)

Left testes

Right testes

Seminal vesicles Prostate

Left cauda

Right epididymis

Uterus

Right ovary

Left ovary

MALES

FEMALES

0

1.67 ± 0.17

1.67 ± 0.12

2.56 ± 0.36

0.685 ± 0.161

0.180 ± 0.031

0.764 ± 0.081

0.58 ± 0.19

0.054 ±0.012

0.053 ± 0.009

5000

1.68 ± 0.12

1.68 ± 0.11

2.52 ± 0.40

0.682 ± 0.188

0.189 ± 0.029

0.763 ± 0.089

0.63 ± 0.17

0.059 ± 0.009

0.057 ± 0.010

10000

1.67 ± 0.14

1.68 ± 0.14

2.44 ± 0.43

0.717 ± 0.176

0.187 ± 0.035

0.720 ± 0.074

0.63 ± 0.22

0.055 ± 0.009

0.051 ± 0.009

20000

1.65 ± 0.09

1.66 ± 0.09

2.49 ± 0.39

0.702 ± 0.218

0.180 ± 0.027

0.732 ± 0.079

0.67 ± 0.22

0.053 ± 0.011

0.054 ± 0.009

No statistically significant differences between control and treated groups at P < 0.05 or P < 0.01

Table 3: Organ weight (mean g +/- SD) of male and female rats of the second parental (P1) generation from the two-generation reproductive toxicity study of Gasoline, vapor-recovery

Group (mg/m^3)

Terminal body weight

Liver

Kidneys

Adrenals

Brain

Thymus

Spleen

Lungs

MALES

0

539.9 ± 54.5

18.18 ± 3.09

3.67 ± 0.37

0.056 ± 0.010

2.17 ± 0.11

0.281 ± 0.069

0.76 ± 0.13

1.86 ± 0.16

5000

569.4 ± 67.2

20.46 ± 3.94*

3.95 ± 0.50*

0.058 ± 0.009

2.16 ± 0.13

0.273 ± 0.78

0.80 ± 0.14

1.92 ± 0.20

10000

566.2 ± 58.3

19.25 ± 3.32

3.95 ± 0.38*

0.055 ± 0.011

2.14 ± 0.13

0.300 ± 0.097

0.80 ± 0.12

1.97 ± 0.17

20000

540.3 ± 43.0

17.93 ± 2.13

3.89 ± 0.38

0.054 ± 0.009

2.13 ± 0.10

0.254 ± 0.084

0.80 ± 0.11

1.90 ± 0.16

FEMALES

0

321.2 ± 32.1

16.65 ± 2.13

2.55 ± 0.23

0.087 ± 0.014

1.91 ± 0.09

0.180± 0.044

0.61 ± 0.10

1.47 ± 0.14

5000

325.0 ± 26.5

16.18 ± 2.15

2.58 ± 0.21

0.087 ± 0.013

1.92 ± 0.12

0.182 ± 0.052

0.59 ± 0.08

1.57 ± 0.10*

10000

324.6 ± 33.4

16.01 ± 2.43

2.59 ± 0.25

0.086 ± 0.016

1.95 ± 0.11

0.169 ± 0.051

0.62 ± 0.08

1.57 ± 0.17*

20000

327.6 ± 30.0

16.15 ± 2.24

2.56 ± 0.22

0.082 ± 0.012

1.93 ± 0.09

0.166 ± 0.033

0.59 ± 0.09

1.52 ± 0.14

Group (mg/m^3)

Left testes

Right testes

Seminal vesicles

Prostate

Left cauda

Right epididymis

Uterus

Right ovary

Left ovary

MALES

FEMALES

0

1.61 ± 0.27

1.65 ± 0.23

2.47 ± 0.35

0.658 ± 0.127

0.182 ± 0.039

0.745 ± 0.080

0.62 ± 0.18

0.052 ± 0.016

0.053 ± 0.015

5000

1.76 ± 0.14*

1.77 ± 0.14*

2.47 ± 0.40

0.643 ± 0.188

0.191 ± 0.046

0.753 ± 0.095

0.62 ± 0.16

0.057 ± 0.014

0.053 ±0.012

10000

179 ± 0.16**

1.79 ± 0.15*

2.50 ± 0.39

0.677 ± 0.201

0.199 ± 0.039

0.755 ± 0.092

0.59 ± 0.17

0.053 ± 0.012

0.052 ± 0.011

20000

1.70 ± 0.24

1.75 ± 0.15

2.48 ± 0.32

0.730 ± 0.209

0.196 ± 0.045

0.774 ± 0.071

0.60 ± 0.16

0.047 ± 0.013

0.050 ±0.013

Significant different from the control mean; * P < 0.05; ** P < 0.01.

Table 4: Results of reproductive and live birth indices of the first parental (P0) generation from the two-generation reproductive toxicity study of Gasoline, vapor-recovery

Dose (mg/m^3)	Male		Female		Number of litters	Gestation index (%)	Mean gestation days
	Mating index (%)	Fertility index (%)	Fertility index (%)	Fecundity index (%)
0	89.7	86.2	93.3	85.7	26	100.0	22.2
5000	93.3	76.7	96.7	82.8	23	100.0	22.3
10000	96.7	90.0	100.0	90.0	27	100.0	22.1
20000	100.0	96.7	100.0	96.7	29	100.0	22.0
Dose (mg/m^3)	Meanlitter size	Mean live	Mean dead	Percent		Sex ratio
				Live (%)             Dead(%)		Male (%)        Female (%)
0	14.6	14.4	0.2	98.4	1.6	48.7	51.3
5000	13.5	13.3	0.3	98.1	1.9	50.3	49.7
10000	14.1	13.8	0.3	98.2	1.8	50.9	49.1
20000	14.6	14.2	0.4	97.4	2.6	47.9	52.1

Male mating index, male fertility index, female fertility index, female fecundity index, percent live and dead and sex ratio were evaluated by chi square analysis. Gestational index, mean days of gestation, mean litter size, and mean live and dead were not evaluated statistically. There were no statistically significant differences between control and treated groups at P < 0.05 or P < 0.01 for any of the parameters evaluated statistically.

Table 5: Results of reproductive and live birth indices of the second parental (P1) generation from the two-generation reproductive toxicity study of Gasoline, vapor-recovery

Dose (mg/m^3)	Male		Female		Number of litters	Gestation index (%)	Mean gestation days
	Mating index (%)	Fertility index (%)	Fertility index (%)	Fecundity index (%)
0	93.3	80.0	93.3	82.1	24	100.0	21.9
5000	100.0	90.0	100.0	90.0	27	100.0	22.1
10000	96.6	89.7	96.7	93.1	27	100.0	22.0
20000	93.3	83.3	93.3	89.3	25	100.0	21.9
Dose (mg/m^3)	Mean litter size	Mean live	Mean dead	Percent		Sex ratio
				Live (%)	Dead (%)	Male (%)	Female (%)
0	14.8	14.5	0.29	98.0	2.0	48.3	51.7
5000	13.8	13.3	0.44	96.8	3.2	43.9	56.1
10000	14.8	14.5	0.30	98.0	2.0	52.4	47.6
20000	15.2	15.1	0.04	99.7*	0.3*	53.7	46.3

* Significantly different from the control mean (P # 0.05) Male mating index, male fertility index, female fertility index, female fecundity index, percent live and dead and sex ratio were evaluated by chi square analysis. Gestational index, mean days of gestation, mean litter size and mean live and dead were not evaluated statistically.

Table 6: Results of postnatal survival indices of the first (F1)- and second (F2)-generation offspring from the two-generation reproductive study of Gasoline, vapor-recovery

Dose (mg/m^3)	Live birthindex(%)	Day1survival index (%)	Day 4 survival index (%)	Day 7 survival index (%)	Day 14 survival index (%)	Day 21 survival index (%)	Viability at weaning index (%)
0	98.4	98.4	97.9	100.0	100.0	100.0	100.0
5000	98.1	98.0	97.7	99.5	100.0	100.0	99.5
10000	98.2	99.5	98.7	100.0	100.0	99.2	99.2
20000	97.4	99.0	98.3	100.0	100.0	99.3	99.3
Dose (mg m^3)	Live birth index (%)	Day 1 survival index (%)	Day 4 survival index (%)	Day 7 survival index (%)	Day 14 survival index (%)	Day 21 survival index (%)	Viability at weaning index (%)
0	98.0	95.4	94.3	99.6	98.7	100.0	98.3
5000	96.8	96.7	95.6	100.0	98.8	99.2	98.0
10000	98.0	99.2**	98.0*	100.0	99.6	99.3	98.9
20000	99.7*	98.4*	97.6*	99.6	99.6	99.6	98.8

Significantly different from the control mean; * P </= 0.05; ** P </= 0.01.

Table 7: Offspring body weight (mean g 6 SD) during the postnatal phase of the first (F1)- and second (F2)-generation offspring from the two-generation reproductive toxicity study of Gasoline, vapor-recovery

Dose (mg m^3)	Day 0	Day 4		Day 7	Day 14	Day 21	Day 28		Day 35
F1: MALES
0	6.50±0.66	9.90 ± 1.17		13.22 ± 1.86	23.57 ± 2.55	36.59 ± 4.81	75.5 ± 8.4		124.0 ± 13.5
5000	6.61±0.76	10.20±1.51		14.45±1.89*	25.06 ± 2.56	39.95 ± 6.22	79.6 ± 9.3		129.9 ± 12.0
10000	6.66 ± 0.60	10.31±1.37		14.29±1.90*	24.22 ± 2.77	38.45 ± 5.64	78.5 ± 10.1		129.6 ±15.0
20000	6.58 ± 0.53	9.89 ± 1.07		13.71 ± 1.36	24.68 ± 2.24	38.76 ± 4.51	77.5 ± 7.7		127.0 ± 11.7
F1: FEMALES
0	6.22 ± 0.67	9.58 ± 1.16		12.95±1.83	22.92 ± 2.41	35.91 ± 4.83	69.5 ± 7.4		109.9 ± 10.9
5000	6.27 ± 0.70	9.59 ± 1.41		13.66 ±1.84	24.11 ± 2.34	38.76 ± 5.34	74.1 ± 9.4		115.6±12.2
10000	6.29 ± 0.65	9.74 ± 1.38		13.49 ± 2.05	23.10±2.94	36.50 ± 5.92	71.8 ± 10.6		113.4±14.7
20000	6.30 ± 0.58	9.54 ± 1.10		13.30±1.38	23.89 ± 2.44	37.48 ± 4.61	73.1 ± 6.8		113.5 ± 9.1
Dose (mg m^3)	Day 0		Day 4		Day 7	Day 14		Day 21
F2: MALES
0	6.27 ± 0.70		9.17±1.32		12.99±1.77	22.91 ± 2.74		34.30 ± 4.09
5000	6.43 ± 0.82		9.30 ± 1.59		12.52±2.32	23.10±3.25		35.44 ± 5.80
10000	6.44 ± 0.77		9.61 ± 1.47		13.13 ± 2.30	23.54 ± 3.82		36.14±5.71
20000	6.28 ± 0.59		9.37±1.32		13.20±2.12	224.30 ± 3.32		37.13 ± 5.31
F2: FEMALES
0	5.91 ± 0.65		8.93 ± 1.21		12.61 ± 1.56	22.38 ± 2.53		33.84 ± 3.92
5000	6.08 ± 0.75		8.88 ± 1.52		12.06±2.40	22.19±3.48		34.34 ± 5.92
10000	6.22 ± 0.64		9.50 ± 1.29		12.84±2.16	23.16±3.67		35.39 ± 6.12
20000	5.94 ± 0.67		8.95 ± 1.40		12.42±2.15	23.38 ± 3.32		35.69 ± 5.24

* Significantly different from the control mean (P < 0.05).

Table 8 Sperm quality indices of the first generation offspring (F1) of the two-generation toxicity study of Gasoline, vapor-recovery (means +/- SD)

Dose (mg/m^3)	Homogenization-resistant spermatid count (x 10^6per g testis)	Total cauda sperm count (x 10^6)	Percent progressively motile	Percent normal	Daily sperm count (x 10^6per g testis per d)
0	124.6 ± 37.2	717.6 ± 182.9	55 ± 10.4	95.6 ± 1.8	20.4
5000	132.1 ± 25.0	685.1 ± 145.8	51 ± 13.0	95.9 ± 1.9	21.7
10000	130.4 ± 20.7	707.4 ± 127.8	55 ± 10.8	95.0 ±1.6	21.4
20000	119.4 ± 32.5	696.9 ± 164.6	56 ± 11.3	96.4 ± 1.2	19.6

No statistically significant differences between control and treated groups at P < 0.05 or P < 0.01.

Table 9 Mean estrous cycle length and oocyte count of the first (P1)- and second (P2)-parental females from the two-generation reproductive toxicity study of Gasoline, vapor-recovery

Dose (mg/m^3)	P1 estrous cycle length (d)	P1 normal cycles (%)	P1 oocyte counts	P2 estrous cycle length (d)	P2 normal cycles (%)	P2 oocyte counts
0	4.11	80.0	122.2	4.33	80.0	119.3
5000	4.27	80.0	NE	4.29	80.0	NE
10000	4.06	90.0	NE	4.31	83.0	NE
20000	4.21	97.0	143.7	4.54	80.0	122.9

No statistically significant differences between control and treated groups at P < 0.05 or P < 0.01 for estrous cycle length or oocyte count. NE, Not Examined

Table 10 Offspring developmental landmarks during the postnatal phase of the first and second generation from the two generation reproductive toxicity study of Gasoline, vapor-recovery

Dose (mg/m^3)	Eye opening	Hair growth	Incisor eruption	Pinna detachment	Righting reflex		Vaginalpatency	Preputial separation
F1: MALES
0	14.73 ± 0.89	6.20 ± 0.89	11.13 ± 0.98	3.18 ± 0.64	5.15 ± 1.70			46.07 ± 1.94
5000	14.39 ± 0.84	6.21 ± 0.94	10.76 ± 1.22	3.02 ± 0.70	4.51 ± 1.29			45.56 ± 2.40
10000	14.78 ± 0.89	7.12 ± 0.74**	10.93 ± 0.91	3.14 ± 0.58	4.83 ± 1.83			45.00 ± 1.66
20000	14.61 ± 0.79	7.10 ± 0.61**	11.12 ± 0.82	3.25 ± 0.53	5.61 ± 1.24			46.40 ± 2.44
F1: FEMALES
0	14.52 ± 0.97	6.22 ± 0.94	11.02 ± 0.92	3.10 ± 0.63	5.68 ± 1.73		33.86 ± 1.41
5000	14.23 ± 0.86	6.16 ± 1.02	10.78 ±1.21	3.09 ± 0.88	5.37 ± 1.84		34.34 ± 2.09
10000	14.59 ± 0.91	6.16 ± 0.74	10.88 ± 0.92	3.16 ± 0.59	5.56 ± 1.68		34.90 ± 1.97
20000	14.32 ± 0.78	6.22 ± 0.65	10.95 ± 0.90	3.18 ± 0.61	5.69 ± 1.70		34.23 ± 1.76
Dose (mg/m^3)	Eye opening		Hair growth	Incisor eruption		Pinna detachment		Righting reflex
F2: MALES
0	15.12 ± 0.98		6.87 ± 1.06	11.53 ± 1.04		3.27 ± 0.60		3.81 ± 1.17
5000	15.09 ± 0.95		7.29 ± 1.02	11.21 ± 1.12		3.18 ± 0.75		3.96 ± 1.33
10000	15.05 ± 0.91		6.74 ± 0.90	11.15 ± 1.05		3.16 ± 0.57		4.17 ± 1.80
20000	14.70 ± 0.74*		6.94 ± 0.78	11.17 ± 0.93		3.07 ± 0.47		4.27 ± 1.29
F2: FEMALES
0	14.87 ± 0.90		6.68 ± 1.05	11.32 ± 1.14		3.18 ± 0.64		4.82 ± 2.04
5000	14.81 ± 0.95		7.32 ± 0.99**	11.23 ± 1.15		3.14 ± 0.71		4.75 ± 1.53
10000	14.83 ±1.06		6.76 ± 0.82	10.97 ± 1.08		3.12 ± 0.57		4.76 ± 2.18
20000	14.52 ± 0.70		6.90 ± 0.87	11.06 ± 0.83		3.20 ± 0.58		5.24 ± 1.74

* Significantly different from the control mean; * P </= 0.05, ** P </= 0.01.

Conclusions:

It was apparent that the test item did not produce any pathologic changes in reproductive organs. Additionally, there were no differences in mating, fertility, live births, birth weights, and survival or weight gain through weaning. Finally, there were no differences in sperm count, sperm quality, estrous cycling, quantification of primordial oocytes, or developmental landmarks, other than a delay in hair growth in some treated offspring. Thus, the reproductive NOAEL as defined by this study is >= 20000 mg/m3, the highest dose tested.

Executive summary:

The generational reproductive study on the closely related substance 'Gasoline, vapor-recovery' was conducted at levels up to 20000 mg/m3, approximately half of the lower explosive limit, and the highest level considered safe for use in the laboratory. VRU gasoline did not produce any pathologic changes in reproductive organs. Additionally, there were no differences in mating, fertility, live births, birth weights, and survival or weight gain through weaning. Finally, there were no differences in sperm count, sperm quality, estrous cycling, quantification of primordial oocytes, or developmental landmarks, other than a delay in hair growth in some treated offspring.

 

There were weight and histopathological changes noted in the kidneys of the high-dose (20000 mg/m3) exposed males from the second parental generation, as well as microscopic evidence of hyaline droplets in the male rat kidneys from both generations. However, as the weight difference was slight (<6%), found only in one generation, and seen only in the males, it was not considered to be adverse. The microscopic changes were consistent with an alpha-2u globulin-mediated process that is unique to male rats and not toxicologically relevant to humans.

 

Based on the data reported, the reproductive NOAEL as defined by this study is >20000 mg/m3. The NOAEL should be also applicable for the closely related substance 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear'.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

20 000 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Study conducted in compliance with agreed protocols and has a klimisch score of 1, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done according to a valid guideline and the study was conducted under GLP conditions.

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No specific reproductive toxicity studies are currently available for 'Naphtha (Fischer-Tropsch), light, C4 -C10 - branched and linear'. However, evidence from two-generation reproductive toxicity studies (OECD 416) conducted with 'baseline gasoline vapour condensate (BGVC)' in rats indicate that the substances are not reproductive toxins at dose levels ≥ 20000 mg/m3. Based on the similarity of constituents and carbon-range coverage, read-across is justified.

Relevant information is available from two-generation reproductive toxicity studies with 'Baseline gasoline vapour condensate (BGVC, C4-C11)':

In a two-generation reproductive toxicity study (OECD 416) with BGVC (Gray et al., 2014), 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 F1 offspring in brain length or width. Exposure to the test material 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 20000 mg/m3 that was not considered biologically significant by the investigator. Overall, GFAP results indicated that BGVC did not induce gliosis in the brain regions examined. In addition, 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.

In another two-generation study conducted with BGVC (McKee et al., 2000) it could also be shown that the test item did not produce any pathologic changes in reproductive organs. Additionally, there were no differences in mating, fertility, live births, birth weights, and survival or weight gain through weaning. Finally, there were no differences in sperm count, sperm quality, estrous cycling, quantification of primordial oocytes, or developmental landmarks, other than a delay in hair growth in some treated offspring.

Thus, the reproductive NOAEL as defined by this study is >= 20000 mg/m3, the highest dose tested.

Short description of key information: Reproductive toxicity: Subchronic, two-generation study inhalation (whole body), rat (Sprague-Dawley) m/f (OECD guideline 416, GLP): NOAEL: 20000 mg/m3 (male/female, F1/F2 litters, highest dose tested).

Effects on developmental toxicity

Description of key information

Pre-natal developmental toxicity studies (OECD 414) were conducted with 'Unleaded gasoline vapour condensate' or 'Baseline gasoline vapour condensate' (BGVC). In the BGVC inhalation study (whole body) in mice (Roberts et al., 2014), the NOAEL was determined to be 2000 mg/m3 (based on reduced fetal body weight), whereby the result should also be applicable for the closely related substance 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear'.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4350 (Inhalation Developmental Toxicity Screen)

Deviations:

yes

Remarks:

exposure period was extended to encompass the phase of fetal development

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: 71 days
- Weight at study initiation: 261-264 g
- Housing: Individually housed in suspended stainless-steel cages with wire mesh floors and fronts
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 38-74
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: nitrogen (purity 99.98%)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- System of generating particulates/aerosols: Charging a glass-lined Pfaulder kettle (closed system) with 6746 lbs of unleaded gasoline. The sample was slowly heated and stirred as the liquid temperature was raised to 150 F, resulting in a vapor temperature of 130 F. The vapor was condensed by passing through a series of two receiving vessels chilled with cold water and dry ice, then through additional vapor traps chilled in dry ice/ isopropyl alcohol. The vapor condensate collected by this method represented 10.4% of the initial sample weight. The chilled condensate was uniformly mixed, transferred to 5-gallon containers, and shipped to the testing laboratory where it was stored at ambient temperature in a solvent storage building until use.
- Exposure apparatus: 1000-L glass and stainless steel exposure chamber (Wahlmann Mfg. Co., Timonium, MD)
- Source and rate of air: Test material was pumped directly from the storage container, refrigerated to minimize volatilization during transfer, into a countercurrent volatilization chamber
- Method of conditioning air: Gasoline vapor laden nitrogen flowed through the top of the volatilization chamber into the turret of the 1000 L exposure chamber, where it mixed with room air to the appropriate exposure concentration as it was drawn into the chamber.
- Temperature and humidity in air chamber: 20-25 °C, 34-70 %; monitored every 1/2 hour
- Air flow rate: 200 L/min
- Air change rate: 5 min
- Method of particle size determination: TSI Aerodynamic Particle Sizer at least once during every exposure
- Treatment of exhaust air: Chambers were exhausted through a system of a coarse filter, a HEPA filter, and a charcoal filter

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

One sample per chamber per week was collected by syringe grab sampling and analyzed by gas chromatography (Hewlett Packard 5890II with flame ionization detector) to characterize 12 major components of the test atmosphere to demostrate stability of test vapor over the course of the study. Composition and stability of the test material were evaluated by characterizing the liquid unleaded gasoline vapor condensate and comparing the major components with the generated atmospheres throughout the study.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: nightly
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

GD6-GD19

Frequency of treatment:

once daily, 6 h

Duration of test:

through day 20 of gestation

Remarks:

Doses / Concentrations:
2653 mg/m3
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
7960 mg/m3
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
23900 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

24 females/dose

Control animals:

yes, sham-exposed

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily; during days 6 through 19, animals were evaluated pre- and post- exposure when animals were removed from the inhalation chambers.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Days 0 and 6 through 20.

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 3, 6, 9, 12, 15, 18, and 20 of gestation.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus and ovaries.

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: Yes.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Where no uterine implants were grossly apparent, the uterus was stained with ammonium sulfide to visualize any uterine foci.

Fetal examinations:

- External examinations, weighed and sexed: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]

Statistics:

conducted where appropriate

Indices:

no data

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
All mated females survived to scheduled sacrifice. Physical examinations performed pre- and post-exposure did not indicate any adverse effect from exposure to unleaded gasoline vapor condensate. Mean maternal body weight and weight gain during gestation were not adversely affected by treatment.

Dose descriptor:

NOAEL

Effect level:

23 900 mg/m³ air (analytical)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Pregnancy rates in treated groups were statistically indistinguishable from the sham treated control group. No adverse effects of treatment were evident from uterine implantation data. There were no aborted pregnancies or premature deliveries in any group. No macroscopic abnormalities related to test material exposre were observed in postmortem examination of maternal animals. No external malformations or variations were recorded among fetuses from control or treated groups.

Dose descriptor:

NOAEL

Effect level:

23 900 mg/m³ air (analytical)

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Table: Summary of reproduction and mean fetal weight data

	Exposure Levels (ppm)
		0	1000	3000	9000
	Number of Females Mated	24	24	24	24
	No. Pregnant (%)	24 (100.0)	22 (91.7)	22 (91.7)	21 (87.5)
	No. Pregnancies Aborted	0	0	0	0
	No. Premature Births	0	0	0	0
	No. Litters with Viable Fetuses	24	22	22	21
	Female Mortality No.	0	0	0	0
	Corpora Lutea
	Mean ± SD	16.7 ± 3.9	18.1 ± 2.1	17.3 ± 2.9	17.3 ± 2.0
	Implanation Sites
	Mean ± SD	14.8 ± 4.5	16.1 ± 2.7	16.1 ± 3.1	16.1 ± 2.4
	Preimplantation Loss Index
	Mean ± SD	0.121 ± 0.193	0.104 ± 0.131	0.071 ± 0.118	0.063 ± 0.107
	Number of Viable Fetuses	327	334	336	325
	Number of Dead Fetuses	0	0	0	0
	Mean Litter Size ± SD	13.6 ± 5.0	15.2 ± 2.6	15.3 ± 3.4	15.5 ± 2.3
	Mean No. Males ± SD	7.0 ± 3.2	7.5 ± 2.6	8.0 ± 2.7	7.6 ± 2.7
	Mean No. Female ± SD	6.6 ± 3.0	7.7 ± 2.3	7.3 ± 3.3	7.9 ± 2.0
	Resorptions
	Mean ± SD	1.2 ± 2.7	1.0 ± 0.8	0.8 ± 1.0	0.7 ± 0.7
	Resorptions/Implants Ratio
	Mean ± SD	0.075 ± 0.017	0.058 ± 0.054	0.055 ± 0.063	0.041 ± 0.044
	No. of Litters with Resorptions (%)	10(41.7)	14 (63.6)	11 (50.0)	11 (52.4)
	Mean Body Weight (g) of Viable	3.66 ± 0.33	3.71 ± 0.30	3.68 ± 0.17	3.66 ± 0.32
	Fetuses ± SD
	Male Fetuses	3.75 ± 0.36	3.80 ± 0.28	3.78 ± 0.20	3.82 ± 0.34
	Female Fetuses	3.61 ± 0.24	3.64 ± 0.36	3.54 ± 0.20	3.54 ± 0.32
	Sex Ratio of Viable Fetuses
	Total Males/Total Females	1.1	1.0	1.1	1.0

Note: Preimplantation Loss = (Corpora lutea - implants) / Corpora lutea

       No statistically significant differences

Table: Summary of fetal examinations by litter

	Exposure Levels (ppm)
	0	1000	3000	9000
No. of Litters Evaluated	24	22	22	21
Soft Tissue Observations
No. Fetuses Evaluated	168^a	172	174	169
Visceral Malformations
Micropthalmia	0	1 (4.5%)	0	0
Soft Tissue Variations
Ureter(s)-Tortuous		1 (4.5%)	1 (4.5%)	0
Skeletal Observations
No. Fetuses Evaluated	160^a	162	162	156
Skeletal Malformations	2 (8.3%)	0	1 (4.5%)	1 (4.8%)
Total Skeletal Variations	23 (95.8%)	22 (100%)	21 (95.5%)	21 (100%)
Rudimentary Ribs	9 (37.5%)	15	13 (59.1%)	16
		(68.2%)		(76.2%)

^a One fetus from one control group litter was evaluated for both soft tissue and skeletal anomalies

Conclusions:

The maternal NOAEL and developmental NOAEL for unleaded gasoline vapor condensate were 23900 mg/m3 (analytical concentration). Under the conditions of this study, unleaded gasoline vapors did not produce evidence of developmental toxicity.
These findings do not warrant classification of the test material as a developmental hazard under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.

Executive summary:

Unleaded gasoline vapor condensate was administered once daily to pregnant rats on gestation days 6-19 via vapor inhalation at doses of 0, 2653, 7960, or 23900 mg/m3 (24 rats/dose) to assess for developmental toxicity. Maternal parameters (food consumption, body weight gain) monitored throughout gestation and at study termination (clinical chemistry, grossly visible abnormalities) were not adversely affected by treatment.  Reproductive parameters (number of implants, resorptions, or viable fetuses) were not adversely affected by administration of the test material at any of the dose levels tested. No evidence of abnormal development was observed during external, skeletal, or visceral examinations of fetuses from pregnant dams exposed. Thus, unleaded gasoline vapor condensate did not produce any maternal toxicity, fetal toxicity, or developmental effects in rats. Based on the study results, the maternal and developmental NOAELs were both 23900 mg/m3 (analytical concentration).

These findings do not warrant the classification of the test material as a developmental hazard under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations. 

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not applicable

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

Animal selection and care:
The test animals were Caesarean-originated Virus Antibody Free (VAF) Crl:CD-1 (ICR) BR outbred albino mice supplied by Charles River Laboratories, Inc.(Portage, MI). Sexually mature males were used for mating purposes only. Females were 12– 13 weeks of age and weighed 26–35 g at the start of mating.
Certified Rodent Diet, No. 5002; (Meal) (PMI Nutrition International, St. Louis, Missouri) was available ad libitum. Analysis of each feed lot used during this study was performed by the manufacturer. Water was available without restriction via an automated watering system. There were no known contaminants in the feed
or water to interfere with test results of these studies. Animals were without food and water while in the exposure chambers.

Housing and environmental conditions:
Animals were housed individually in suspended stainless steel wire mesh cages. A twelve hour light/dark cycle was controlled by an automatic timer. Temperature and relative humidity were maintained within the specified range (18-22°C, and 30–70%, respectively).

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

The experimental and control animals were placed into wholebody inhalation chambers operated under dynamic conditions for at least 6 h per day for GD 5–16 at 2000, 10,000, and 20,000mg/m3, or for GD 5–10 at 30,000 mg/m3 after target exposure levels were reached. The animals remained in the chambers for at least an additional 23 min (theoretical equilibration time) while the test atmosphere cleared. Females were exposed in 1.0 m3 stainless steel and glass chambers operated at a flow rate of approximately 12–15 air changes/hour. During exposure periods, animals were individually housed in stainless steel, wire mesh cages. Flow rate and slightly negative pressure was monitored continuously and recorded approximately every 30 min. Light (ca. 30–40 footcandles 1.0 m above the floor) and noise levels (<85 db) in the exposure room were measured pretest and at the beginning, middle and end of the study. Oxygen levels in the exposure chambers were maintained between 20.6 and 20.7%.
The control group was exposed to clean filtered air under conditions identical to those used for groups exposed to the test substance. The test substance was administered fully vaporized in the breathing air of the animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The chamber concentrations were measured in the breathing zone of the mice by on-line gas chromatography (GC). The chromatographic analyses were used to assess the stability of the test substance over the duration of the study.
Additionally, sorbent tube samples were collected once weekly and stored in a freezer for analysis by a detailed capillary GC method to compare component proportions of the test material atmosphere with the liquid test material. Homogeneity of the exposure system was validated prior to the start of each study. Particle size determination confirmed that exposures were to vapor only.

Details on mating procedure:

not described in study

Duration of treatment / exposure:

Day 5 to Day 17 (6 h/day)

Frequency of treatment:

daily

Duration of test:

18 days (animals sacrificed on Day 18)

Remarks:

Doses / Concentrations:
0, 2000, 10000, 20000 mg/m3/day
Basis:
nominal conc.

No. of animals per sex per dose:

25 females per dose

Control animals:

yes, concurrent vehicle

Maternal examinations:

Animals were examined for viability at least twice daily during the study. Body weights were recorded prior to selection and on GD 0, 5, 8, 11, 14, 17, and 18. Food consumption was measured for mated females on GD 5, 8, 11, 14, 17, and 18. A clinical examination of each female occurred prior to selection and daily during gestation. Additionally, group observations of the animals for mortality and obvious toxic signs while in the chambers were recorded at 15, 30, 45, and 60 min after initiation of the exposure and then hourly during each exposure. Body weights were recorded on GD 18, the day of necropsy. Dams were sacrificed by CO2 asphyxiation followed by exsanguination. A gross necropsy was performed on all confirmed-mated females.

Ovaries and uterine content:

Uterine weights with ovaries attached were recorded at the time of necropsy. Uterine contents were examined and the numbers and locations of implantation sites, early and late resorptions, and live and dead (live or dead in utero) fetuses were counted. Ovarian corpora lutea also were counted. The uteri of all apparently non-pregnant females were stained with 10% ammonium sulfide to confirm non-pregnancy status. Evaluations of dams during cesarean section were conducted without knowledge of treatment group in order to minimize bias.

Fetal examinations:

Fetal evaluations were conducted without knowledge of treatment group in order to minimize bias. Each fetus was weighed and examined externally for gross malformations and variations. Fetal sex was determined by external examination and confirmed internally only on those fetuses receiving visceral examinations. Fetuses were euthanized by hypothermia after the external examination and weighing. The viscera of approximately one-half of the fetuses of each litter
were examined by fresh dissection (Staples, 1974; Stuckhardt and Poppe, 1984) prior to decapitation of the fetus. The heads were preserved in Bouin’s solution for at least two weeks, then rinsed and subsequently stored in 70% ethanol. Free-hand razor blade sections of the Bouin’s-fixed fetal heads were examined for the presence of abnormalities. The remaining fetuses were eviscerated, processed by double staining with Alizarin red and Alcian blue, and examined for bone and cartilage development and any abnormalities. The fetal skeletons were preserved in glycerine with thymol after they were processed and stained.

Statistics:

Bartlett’s Test was performed to determine if the dose groups had equal variance followed by standard one-way analysis of variance after which parametric methods were employed when variances were equal. For continuous data, percentages were calculated and transformed by Cochran’s transformation, followed by the arc sine transformation. The raw percentages and the transformed percentages were both tested for statistical significance. For the parametric procedures, a standard one-way ANOVA using the F distribution to assess significance was used followed by Dunnett’s Test if significant differences in ANOVA were found. A standard regression analysis for linear response in the dose groups was performed, which also tested for linear lack of fit in the model. For the nonparametric procedures, equality of means was assessed by the Kruskal–Wallis Test followed by Dunn’s Summed Rank Test, if needed. Jonckheere’s Test for monotonic trend in the dose response was also performed. Bartlett’s Test for equal variance was conducted at the 1% level of significance. All other tests were conducted at the 5% and 1% level of significance. Data for non-pregnant females, dams delivering before GD 18, or from pups which were delivered early in those litters were not included in analyses. Means and standard deviations were calculated for animal, exposure and chamber environmental data. Body weight change and food consumption were evaluated for individual intervals and also for the pre-exposure, exposure, and post-exposure periods. The coefficient of variation was calculated when considered relevant. Fetal body weight was analyzed by a mixed model analysis of variance using the litter as the basis for analysis and the litter size as a covariate. The analysis of anomalies (malformations or variations) was based on a generalized estimating equation (GEE) application of the linearized model (Ryan, 1992).

Dose descriptor:

NOAEL

Effect level:

10 000 mg/m³ air

Basis for effect level:

body weight and weight gain

other: maternal toxicity - no adverse effects

Dose descriptor:

NOAEL

Effect level:

2 000 mg/m³ air

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

The no observed adverse effect levels for maternal/developmental toxicity in the Baseline gasoline vapor condensate (BGVC) study were 10,000/2000 mg/m3, whereby the result should be also applicable for the closely related substance 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear'.

Executive summary:

CD-1 mice were exposed to the closely related test substance Baseline Gasoline Vapor Condensate (BGVC). Inhalation exposures were 6 h/d on GD 5–17 at levels of 0, 2000, 10,000, and 20,000 mg/m3. Dams were evaluated for evidence of maternal toxicity, and fetuses were weighed, sexed, and evaluated for external, visceral, and skeletal anomalies. Exposure to 20,000 mg/m3 of BGVC produced slight reductions in maternal body weight/gain and decreased fetal body weight.

The no observed adverse effect levels for maternal/developmental toxicity in the BGVC study were10,000/2000 mg/m3.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

2 000 mg/m³

Study duration:

subchronic

Species:

mouse

Quality of whole database:

Study conducted in compliance with agreed protocols and has a klimisch score of 1 (reliable without restriction), with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done according to a valid guideline (OECD 414) and the study was conducted under GLP conditions.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

No specific developmental toxicity studies are currently available for 'Naphtha (Fischer-Tropsch), light, C4 -C10 - branched and linear'. However, evidence from pre-natal developmental studies (OECD 414) conducted with 'unleaded gasoline vapour condensate' in rats and 'baseline gasoline vapour condensate (BGVC)' in mice are available. The results should also be applicable for the closely related substance 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear'.

Unleaded gasoline vapour condensate:

Unleaded gasoline vapour condensate was administered once daily to pregnant rats on gestation days 6-19 via vapor inhalation at doses of 0, 2653, 7960, or 23900 mg/m3 (24 rats/dose) to assess developmental toxicity. Maternal parameters (food consumption, body weight gain) monitored throughout gestation and at study termination (clinical chemistry, grossly visible abnormalities) were not adversely affected by treatment. Reproductive parameters (number of implants, resorptions, or viable fetuses) were not adversely affected by administration of the test material at any of the dose levels tested. No evidence of abnormal development was observed during external, skeletal, or visceral examinations of fetuses from pregnant dams exposed. Thus, 'unleaded gasoline vapour condensate' did not produce any maternal toxicity, fetal toxicity, or developmental effects in rats.

Based on the study results, the maternal and developmental NOAELs were both 23900 mg/m3 (analytical concentration). BGVC (C4 -C11): CD-1 mice were exposed to the closely related test substance 'Baseline gasoline vapour condensate (BGVC). Inhalation exposures were 6 h/d on GD 5–17 at levels of 0, 2000, 10000, and 20000 mg/m3. Dams were evaluated for evidence of maternal toxicity, and fetuses were weighed, sexed, and evaluated for external, visceral, and skeletal anomalies.

Exposure to 20000 mg/m3 of BGVC produced slight reductions in maternal body weight/gain and 10000 mg/m3 decreased fetal body weight.

The no observed adverse effect levels for maternal/developmental toxicity in the BGVC study were 10000/2000 mg/m3.

Justification for classification or non-classification

Based on the available data of closely related substances, 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear' does not require classification for reproductive or developmental toxicity according to Regulation (EC) No 1272/2008.

However, 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear' is classified as reproductive toxicant (H361: Suspected of damaging fertility or the unborn child) since at least the Category 2 reproductive toxicant 'n-hexane' is present in the UVCB substance at or above the appropriate EU GHS generic concentration limit (≥ 3,0 %).