Mixed LOF

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: According to or similar to OECD Testing Guideline 414 performed under GLP conditions on an analogue substance.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2001

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

unleaded gasoline vapor condensate (API 94-02)

IUPAC Name:

unleaded gasoline vapor condensate (API 94-02)

Details on test material:

A detailed description of test material deviation is available in the publication. In summary, unleaded gasoline (API 94–02) 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 in chilled vessels.

The chilled condensate was uniformly mixed, transferred to 5-gallon containers, and shipped to the testing laboratory where it was stored at ambient temperature until use. The unleaded gasoline vapor condensate (density 0.65) were characterized by gas chromatography using a modified ASTM D5134–92 procedure on a Hewlett Packard 5890 instrument.

Test animals

Species:

rat

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

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: The vehicle used was 99.98% nitrogen

Details on exposure:

Exposure apparatus was a 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:

Chamber exposure concentrations measured:
-Six times each day using infrared spectrophotometry
-Once daily by gas chromatography for the major airborne components ( 12 major components) of the test vapor

Details on mating procedure:

Females and males were placed in a 1:1 ratio nightly. Females were considered to have mated if on microscope observation sperm were observed following in a vaginal rinse (taken each morning), and/or a plug was observed in the vaginal opening. Day 0 of gestation was defined as the day on which evidence of mating occurred.

Mated females were randomly assigned to groups daily to provide an equal distribution among groups and equalize to the extent possible the gestation day 0 mean body weights between groups. The mean weight of mated females per group on day 0 of gestation was 261–264 g.

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

Daily from days 6 through 19 of gestation

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
2653
Basis:
analytical conc.
(~1000 ppm)

Doses / concentrations 2

Remarks:

Doses / Concentrations:
7960
Basis:
analytical conc.
(~3000 ppm)

Doses / concentrations 3

Remarks:

Doses / Concentrations:
23900
Basis:
analytical conc.
(~9000 ppm)

No. of animals per sex per dose:

24 females

Control animals:

yes, sham-exposed

Examinations

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:

Statistical analyses of interval data (i.e. body weight data, food consumption, litter information) was first evaluated for equal variance of means using Bartlett’s test at 1% significance, two-sided risk level, while other tests were conducted at 5% and 1% significance, two-sided risk level. If variance of means was equal, one-way ANOVA using the F distribution was used to assess significance. Dunnett’s test was used to determine which means were significantly different from control if the one-way ANOVA found a significant different. If Bartlett’s test indicated heterogeneity of variances the Kruskal-Wallis test was used to detect differences between groups. Dunn’s summed rank test to determine which treatments differed from control.
Testing for trend in the dose levels was used standard regression techniques with a test for trend and lack of fit where variances were equal or Jonckheere’s test for monotonic trend in nonparametric cases.
Statistical analyses for incidence data (i.e. females with resorptions, pregnancy rates, litters with resorptions, incidence of fetuses with malformations/variations, and incidence of litters containing fetuses with malformations/variations) used contingency tables. All ratios such as pre- and postimplantation loss indices were transformed via Bartlett’s transformation followed by arc sine transformation prior to analysis. Standard Chi-square analysis was used to assess whether proportion of indices differed between the groups tested. A 2x2 Fisher Exact Test was used to compare each treatment group to control, with correction by Bonferroni inequality to assure an overall test of the stated significance level. Assessment of linear trend was performed by Armitage’s test in the treatment groups. All tests were reported at the 5% and 1% levels of significance.

Indices:

Not reported in publication

Historical control data:

Not directly reported, but cited as: Middle Atlantic Reproduction and Teratology Association and Midwest Teratology Association (MARTA/MTA). Historical Control Data (1992–1994) for Developmental and Reproductive Toxicity Studies using the Crl:CD® BR Rat. Charles River Laboratories, Portage, MI: 1995. MARTA/MTA.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEC

Effect level:

>= 23 900 mg/m³ air (analytical)

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEC

Effect level:

>= 23 900 mg/m³ air (analytical)

Basis for effect level:

other: developmental toxicity

Results (fetuses)

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.The maternal NOAEL and developmental NOAEL for unleaded gasoline vapor condensate were 23900 mg/m3. 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.

Effect levels (fetuses)

Dose descriptor:

NOAEC

Effect level:

>= 23 900 mg/m³ air (analytical)

Basis for effect level:

other: developmental toxicity. No adverse effects on development

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The maternal NOAEL and developmental NOAEL for unleaded gasoline vapor condensate were 23900 mg/m3. 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 6 hours per day 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. 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.