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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2009-06-25 to 2009-07-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified as reliable with restrictions because it was conducted in compliance with OECD Test Guideline 474.
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
Oxidized asphalt fume condensate (CAS # 64742-93-4)
IUPAC Name:
Oxidized asphalt fume condensate (CAS # 64742-93-4)
Details on test material:
- Name of test material (as cited in study report): roofing asphalt fume condensate (RAFC), API #06 01, CAS# 64742-93-4
- Density: 0.8745 g/mL
- Expiration date: August 2020
- Kinematic viscosity: 8.3616 centi stokes at 100 degrees F

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 7 weeks
- Assigned to test groups randomly: yes, via a computer generated randomization program (PROVANTIS)
- Housing: Animals were individually housed in Makrolon® Type III cages. Absorbent softwood was
used as bedding material in the cages.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 3 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 degrees C
- Humidity (%): 55% + 15%.
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
- Vehicle(s)/solvent(s) used: none
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: free jet evaporation condensation generator
- Method of holding animals in test chamber:
- System of generating particulates/aerosols: In the free jet evaporation condensation generator, a well defined mass flux of vaporized
liquid asphalt fume condensate is issued together with a carrier (nitrogen) at high velocity
through a nozzle into a stream of slowly flowing cool air. An expanding turbulent jet is
formed, as a result of the surrounding air mixing with the vapor stream. The asphalt vapor is
cooled and diluted downstream of the nozzle, as the jet developes.
- Temperature, humidity, pressure in air chamber: nozzle was operated
with heated nitrogen (160 °C), droplets fed into tube heated at 220 °C, where droplets evaporated
- Air flow rate: 5 L/min
- Method of particle size determination: determined using a scanning mobility particle sizer
(SMPS, TSI - Inc.) which gave a number size distribution in a size range of about 7 - 300 nm


TEST ATMOSPHERE
- Brief description of analytical method used: The fume concentration was determined by sampling from the nose only units using a
combination of a glass fiber filter and a XAD absorption tube with a sample flow rate of
approx. 2 l/min. Samples were taken from each exposure unit. An average of two samples
per unit a week was taken. The material collected on the filter and the XAD tube was
extracted and analyzed separately by IR spectroscopy according to BIA guideline #6305.
The concentrations are given in mg Total Hydrocarbons (THC)/m³ aerosol and vapor phase.
- Samples taken from breathing zone: yes, from sampling the nose only units
Duration of treatment / exposure:
28 days
Frequency of treatment:
6 hours per day, 7 days per week
Post exposure period:
The bone marrow micronucleus assay in vivo was performed in five male and five female
animals per group of the subchronic subgroup, alive at the moment of terminal
sacrifice
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/m3 THC (total hydrocarbons)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
30 mg/m3 THC (total hydrocarbons)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
100 mg/m3 THC (total hydrocarbons)
Basis:

Remarks:
Doses / Concentrations:
300 mg/m3 THC (total hydrocarbons)
Basis:

No. of animals per sex per dose:
5 animals per sex, per dose
Control animals:
yes, concurrent no treatment
Positive control(s):
Cyclophosphamide monohydrate (CP; SIGMA, Germany, lot 076K1050)
- Route of administration: oral, aqueous solution (once)
- Doses / concentrations: 10 mL/kg body weight (concentration of cyclophosphamide monohydrate: 60 mg/10 mL)
Administered once, 24 hours before final sacrifice.

Examinations

Tissues and cell types examined:
One femur of each rat was collected and cleaned from the surrounding muscle
tissue
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
The ends of each femur were cut off and the bone marrow of each animal was washed out with 5 mL of foetal calf serum and transferred into a tube. Cell suspension was pulled gently up and down until a fine cell suspension was observed. This resulting bone marrow suspension was subsequently cleaned from nucleated cells by a cellulose column procedure (adopted from Sun et al., 1999) to facilitate the erythrocyte counts and to eliminate granulocytes which might have led to artefacts with scoring of micronuclei in erythrocytes. On the day of necropsy, the bone marrow suspensions were pre filtered through microscope cleaning paper, carefully loaded onto the cellulose columns, and allowed to drain into 15 mL centrifuge tubes. The cleaned bone marrow was then centrifuged and most of the supernatant was discarded. The cell pellet was carefully re-suspended in a very small volume of foetal calf serum, resulting in about 2 drops of bone marrow cell suspension per animal. From this suspension two smears (A and B) were prepared on defatted slides. The smears were fixed for 10 min in absolute methanol and subsequently stained according to Pappenheim with May Grünwald and Giemsa solution.

METHOD OF ANALYSIS:
The slides were analyzed microscopically under 630 - 1000 x magnification. For each animal, the incidence of micronucleated cells per 2.000 polychromatic erythrocytes (PCE) of the bone marrow was determined. In addition, the ratio of PCE (immature) to normochromatic erythrocytes (NCE, mature) was calculated by counting the number of PCE per 500 red blood cells (RBC), as any toxic effects of the test item on immature nucleated erythrocytes may lead to a reduction in cell division.
Evaluation criteria:
If the ratio of PCE to NCE in the bone marrow is determined and is found to be significantly les than the control value, this is taken as an indication of toxicity. A reduction in the proportion of immature among total erythrocytes thus indicates that the test item or its metabolites reached the bone marrow. In the mammalian erythrocyte micronucleus test a genotoxic effect is claimed if a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant increase in the number of micronucleated polychromatic erythrocytes in a single dose group at asingle sampling time is observed
Statistics:
Statistical comparison of groups were performed at the level of alpha =0.05.
Kruskall Wallis ANOVA (H-test) and Mann Whitney U test were applied in the case of non homogeneous data (e.g. micronucleus test).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 mg/m3 THC
- Results: blood formation was significantly depressed in female animals, thus indicating that RAFC or components of RAFC are able to reach the bone marrow as target organ for the mammalian erythrocyte micronucleus test.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no tendency towards repression of red blood cell (RBC) formation in both male
and female animals, as compared to controls
- Ratio of PCE/NCE (for Micronucleus assay): see table
- Statistical evaluation: There was no evidence of a significantly enhanced mean frequency of micronucleated erythrocytes (MN per 2000 PCE) due to RAFC exposure, as compared to the respective clean air groups.

Any other information on results incl. tables

Group

Treatment Group

Concentration

PCE per 500 RBC

PCE/NCE

MN per 2000 PCE

% MN in PCE

Number

 

 

5m/5f

5m/5f

5m/5f

5m/5f

1

Negative control (clean air)

-

M: 250

M: 1.00

M:1.0

M: 0.05

F: 247

F: 0.98

F: 1.0

F: 0.05

2

Test item (RAFC)

30 mg/m3THC

M: 252

M: 1.02

M: 0.6

M: 0.03

F: 247

F: 0.98

F: 1.8

F: 0.09

3

Test item (RAFC)

100 mg/m3THC

M: 250

M: 1.00

M: 1.4

M: 0.07

F: 247

F: 0.98

F: 0.6

F: 0.03

4

Test item (RAFC)

300 mg/m3THC

M: 253

M: 1.03

M: 1.4

M: 0.07

F: 252

F: 1.01

F: 1.4

F: 0.07

5

Positive control (CP)

60 mg/kg b.w.,

p.o.

M: 248

M: 0.99

M: 15.0**

M: 0.75

F: 251

F: 1.01*

F: 12.8**

F: 0.64

polychromatic erythrocyte

RBC: red blood cell

NCE: normochromatic erythrocyte

MN: micronuclei

* : P < 0.05 Mann-Whitney U-Test

**: P < 0.01 Mann-Whitney U-Test

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the restrictions of the micronucleus assay and the chosen target cells, roofing asphalt fume condensate is considered non mutagenic in immature bone marrow erythrocytes of rats.
Executive summary:

A read across in vivo micronucleus test on oxidised bitumen was negative for genotoxicity. In addition chronic inhalation studies with oxidised bitumen, together with comparative fume compostion information, indicate that read across to the bitumen category, is appropriate.

In a Wistar rat bone marrow micronucleus assay, (5 rats/sex/dose) were treated via inhalation with Roofing asphalt fume condensate (CAS No. 64742-93-4) at doses of 0, 30, 100, or 300 mg/m3 THC (total hydrocarbons). Bone marrow cells prepared from the femur of each rat into a suspention. From this suspension two smears (A and B) were prepared on defatted slides. The slides were then analyzed microscopically

 

In summary, the low dose 30 mg/m3 was determined as the overall NOAEL for systemic toxicity in the study. After 28 days of exposure the test item mediated no tendency towards repression of red blood cell formation in both maleand female animals, as compared to controls. This does not mean that RAFC or components of RAFC were not systemically available, because in a range finding study using a concentration of 1000 mg/m3THC blood formation was significantly depressed in female animals, thus indicating that RAFC or components of RAFC are able to reach the bone marrow as target organ for the mammalian erythrocyte micronucleus test. There was no evidence of a significantly enhanced mean frequency of micronucleated erythrocytes (MN per 2000 PCE) due to exposure, as compared to the respective clean air groups. The positive controls indicated the desired results.

 

This study received a Klimisch score of 1 and is classified as reliable without restriction because it was conducted in compliance with OECD Test Guideline 474.

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