Asphalt, oxidized

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-06-25 to 2009-07-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study is classified as reliable without restrictions because it was GLP compliant and was conducted according to OECD 422 guidelines.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Not all details were provided

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Test Guidelines OPPTS 870.3650 Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Asphalt, oxidized

EC Number:

265-196-4

EC Name:

Asphalt, oxidized

Cas Number:

64742-93-4

Molecular formula:

Not applicable

IUPAC Name:

Asphalt, oxidized

Constituent 2

Reference substance name:

Aerosol from roofing asphalt fume condensate

IUPAC Name:

Aerosol from roofing asphalt fume condensate

Test material form:

other: fume condensate

Details on test material:

- Name of test material (as cited in study report): Roofing asphalt fume condensate (CAS number 64742-93-4)
- Substance type: Bitumen
- Analytical purity: Not reported
- Composition of test material, percentage of components: Not reported
- Expiration date of the lot/batch: August 2020
- Stability under test conditions: Stable
- Storage condition of test material: The test item was stored at -18°C except during use when it was kept at ambient room temperature and between daily dosing when it was kept at approx. 4 - 6°C.
- Density 0.8745 g/m
- Kinematic viscosity: 8.3616 cS at 100°F
- Refractive Index: 1.4831 at 25°C
See attachment for total hydrocarbon content

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: Approximately 10 weeks
- Weight at study initiation: Not reported
- Housing: Individually in polycarbonate cages except during mating
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 30% to 70%
- Air changes (per hr): About 15
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: From: 2008-07-15 To: 2009-01-21

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

nose only

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Direct flow nose only inhalation system
- Method of holding animals in test chamber: Tapered acrylic glass tubes with adjustable backstops
- Source and rate of air: Slowly flowing cool air stream
- System of generating particulates/aerosols: Fumes were regenerated using an free jet evaporation condensation generator.
- Temperature, humidity, pressure in air chamber: Temperature ranged from 21.9 to 23.3 degrees Celsius, humidity ranged from 44.8% to 61.8% in a slight overpressure
- Air flow rate: 35 to 47 litres per minute, increasing with increasing exposure concentration
- Method of particle size determination: Scanning mobility particle sizer
- Treatment of exhaust air: Into a laboratory hood


TEST ATMOSPHERE
- Brief description of analytical method used: Samples were collected on a combination glass fibre filter and a XAD absorption tube with a sample flow rate of approximately 2 litres per minute. Samples were then extracted and analyzed by IR spectroscopy and presented as mg of total hydrocarbons per cubic metre.
- Samples taken from breathing zone: Yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The average concentrations of the asphalt fume measured by chemical analysis approximately 2 hours after start of the exposure were 27.4 mg/m³, 90.2 mg/³ and 267.5 mg/m³ total hydrocarbon concentration (i.e., the absolute mass concentration) for the low-, mid- and high-dose group. The average concentrations measured continuously during the whole exposure period by aerosol photometers were 30.0 mg/m³, 100.1 mg/³ and 297.3 mg/m³ total hydrocarbon concentration for the low-, mid- and high-dose group.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: Vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Individually in polycarbonate cages with absorbent softwood bedding

Duration of treatment / exposure:

For the subchronic portion of the study animals were exposed for 28 days. Breeding females were exposed for 50 days.

Frequency of treatment:

6 hours a day, 7 days a week

Duration of test:

For the subchronic portion of the study animals were exposed for 28 days. Breeding females were exposed for 50 days.

Doses / concentrations

Remarks:

Doses / Concentrations:
30, 100, or 300 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

Twelve males per dose and 24 females per dose

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: Based on a range finding study
- Rationale for animal assignment (if not random): Randomly based on stratified body weight

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily, specifics were not reported

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for subchronic animals and in breeding females on gestation day 0, 7, 14 and 21 and postnatal day 0 and 4

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Weekly for subchronic animals and in breeding females on gestation day 0, 7, 14 and 21 and postnatal day 0 and 4

OTHER: Neurological tests, haematology, and clinical chemistry. Parameters examined in male parental generations: Testis weight, epididymis weight, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology

SACRIFICE
- Male animals: All surviving animals after 28 days of exposure
- Maternal animals: All surviving animals on postnatal day 4

GROSS NECROPSY
- Gross necropsy specifics were not provided.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Ovaries and uterine content:

Ovaries and uterus were weighed and examined microscopically. The number of implantation sites was determined, using ammonium sulphide staining in case of no macroscopically visible implantations. Corpora lutea were counted.

Fetal examinations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain

GROSS EXAMINATION OF DEAD PUPS: Yes, for gross abnormalities; possible cause of death was not determined for pups born or found dead.

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- It does not appear that these animals were examined.

Statistics:

Body weights, food and water consumption, and organ weight data were analyzed using analysis of variance (ANOVA). If the group means differed significantly according to this method, the means of the treatment groups were compared with the mean of the control group 1 using Dunnett's modification of the t test. Kruskall Wallis ANOVA (H-test) and Mann Whitney U test were applied in the case of non homogeneous data. Qualitative data were analyzed using the two tailed FISHER test with Bonferroni correction or Chi square test.

Indices:

Mating index; fertility index; birth index; live birth index; pregnancy index; survival index; viability index; lactation index

Historical control data:

No data reported.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: Organ weights

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): There were no treatment-related effects on clinical signs or mortality.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): There was a significant decrease in body weight gain and in food consumption in high-dose males, but there were no effects on body weight gain in females.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): There were no treatment-related effects on reproductive performance.

ORGAN WEIGHTS (PARENTAL ANIMALS): There was a statistically significant increase in lung weight in mid- and high-dose subchronic females and in high-dose breeding females.

GROSS PATHOLOGY (PARENTAL ANIMALS): There were no treatment-related effects on gross pathology.

HISTOPATHOLOGY (PARENTAL ANIMALS): In the nasal cavity, a statistically significant decrease of inflammatory cell infiltration was observed in the high-dose group as compared to the control group.

OTHER FINDINGS (PARENTAL ANIMALS): There were no other treatment-related findings.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEC

Effect level:

30 mg/m³ air (nominal)

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEC

Effect level:

300 mg/m³ air (nominal)

Basis for effect level:

other: other:

3

Dose descriptor:

NOAEC

Effect level:

300 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:
VIABILITY (OFFSPRING): There were no treatment-related effects on offspring viability.

BODY WEIGHT (OFFSPRING): There were no treatment-related effects of the body weight of the offspring.

GROSS PATHOLOGY (OFFSPRING): Gross pathology was only performed on dead offspring and did not reveal any effects.

Effect levels (fetuses)

Remarks on result:

other: see Details on embyotoxic/teratogenic effects

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Although the systemic NOAEC for the study was 30 mg/m3, based on a significantly increased relative lung weight in mid-dose subchronic females, there were no reproductive or developmental effects at concentrations up to 300 mg/m³ total hydrocarbon concentration.

Executive summary:

The animals were exposed to roofing asphalt fume condensate at target concentrations of 30, 100, and 300 mg/m³ total hydrocarbons, respectively, or clean air by nose only inhalation, 6 hours per day, 7 days per week. The average concentrations measured continuously during the whole exposure period by aerosol photometers were 30.0 mg/m³, 100.1 mg/m³ and 297.3 mg/m³ total hydrocarbon concentration for the low-, mid- and high-dose group. In the subchronic subgroup, males and females were exposed for 28 days. Breeding females were exposed daily for 2 weeks before mating and during the mating period (up to 2 weeks). After successful mating, females were exposed daily from gestational day 0 through 20. 

There were no treatment-related effects on clinical signs, mortality or neurological tests. Results of the study indicated that body weight gain and food consumption were significantly reduced in male rats in the 300 mg/m³ group. Results in females could not be assessed due to randomization errors. The absolute and relative lung weight was statistically-significantly increased in the high-dose males. Relative lung weight was statistically-significantly increased in the female 100 and 300 mg/m³ groups as well as in the 300 mg/m³ group of the breeding females. There were no treatment-related effects observed in haematology and clinical chemistry.

 

Treatment-related findings were observed in the nasal cavity and in the lungs. In the nasal cavity, a statistically significant decrease of inflammatory cell infiltration was observed in the high-dose group as compared to the control group. 

 

No adverse effects were seen in spermatology. No treatment-related effects were observed on reproduction (mating and pregnancy rates, pregnancy and postpartum body weight, food and water consumption, and clinical observations), as well as early postnatal development of the F1 offspring.

 

In summary, the low dose 30 mg/m3 was determined as the systemic NOAEL for the study, with clear (mild) effects in the high dose and based on a significantly increased relative lung weight in mid-dose subchronic females although, in the absence of any histopathological correlates. The results do not give any indication that the test item is a reproductive toxin or teratogen at concentrations up to 300 mg/m³ total hydrocarbon concentration.

 

This study received a Klimisch score of one and is classified as reliable without restrictions because it was GLP compliant and was conducted according to OECD 422 guidelines.