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Toxicological information

Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This endpoint is fulfilled by read-across based on grouping of substances (category approach), the test substance was also changed from that indicated in the originally submitted test proposal. Justifications for both of these items are given briefly below and in more detail in "Bitumen category approach and read-across Dec 2018".

Change in Tested Substance
Once setup work on test material generation began, it became apparent that the original CAS No. selected was not suitable for testing for a number of reasons (outlined below) and the decision to switch to a more appropriate test material was communicated to ECHA on 8th February 2016.
Rationale for change of CAS No.:
- Worker exposure: the original CAS No. represents less than 5% of the total volume of bitumen used in Europe. Moreover, the manufacturing route for this CAS number produces a “hard” bitumen that is only used as a blending component. Therefore, workers are not exposed to this material, i.e. the selected worse case sample, but rather to a blend with a less severe PAH profile.
- Validation of fume condensate: composition of the condensate collected at the manufacturing site should be validated against actual workplace fume samples through a workplace monitoring campaign to ensure that the tested material is representative of real-life exposures. Due to the lack of worker exposure (see above) it was not possible to adequately validate samples of CAS 92062-05-0.
- Technical limitations: under normal conditions of use with CAS No. 92062-05-0 it proved impossible to generate the required volume of fume condensate in an acceptable timeframe (> 2000 years to generate 10 kg of fume at 180°C).

A sample with CAS No. 8052-42-4 was therefore used for workplace monitoring and fume condensate collection. This was selected based on:
- PAH profile (PAH profile of registered substance (see attached documents); total PAH content of EPA-16 PAHs plus triphenylene and benzo[e]pyrene was 70.6 mg/kg),
- greater worker exposure (> 10-fold increase in tonnage used for paving applications compared to CAS 92062-05-0) and
- ability to produce sufficient quantities of fume condensate (due to lower boiling profile).


REPORTING FORMAT FOR THE CATEGORY APPROACH
[Please provide information for all of the points below addressing endpoint-specific elements that were not already covered by the overall category approach justification made available at the category level. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The Concawe Bitumen category is composed of similar hydrocarbon UVCB substances derived by refinery processes from crude oil and grouped within established production boundaries based on phys-chem properties and hydrocarbon type (a full justification for this grouping is given in the Concawe Bitumen Category Justification document attached to the category object and in Section 13 of the IUCLID dossier); substances within the category have qualitatively similar properties. The prediction for read-across in the category will be based on what is considered to be the worst-case substance.

More details of the proposed read-across and category justification are given in the attached document ("Bitumen category approach and read-across Dec 2018").

CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL)
See detailed discussion of available in vitro and in vivo data on UVCB hydrocarbon classes as well as work currently in progress to help underpin the justifications for category read-across for toxicity to reproduction in "Bitumen category approach and read-across Dec 2018" document attached below.
• <3 ring polycyclic aromatics: no effects on reproductive organs and no selective developmental effects.
• ≥4 ring polycyclic aromatics (mainly 4-7 ring PACs) with specific structures (not necessarily present in gas oils) are associated with systemic toxicity (effects on liver, thymus and blood forming organs but not reproductive organs) and are potentially mutagenic and dermal carcinogens. In developmental studies they produce foetal death and resorption. Recent in vitro work suggests that ≥3 ring polycyclic aromatics can alter embryo development.

Based on recent analytical research work on representative samples of category members as well as on expected worker exposure and practical considerations for test item generation; the following substance has been chosen as a worst-case based on having the highest level of 4-7 ring PAH

- Name of the substance on which testing is proposed to be carried out
Asphalt (CAS 8052-42-4, EC 232-490-9)

- Name of the substances for which the testing proposal will be used [if different from tested substance]
Asphalt (CAS 8052-42-4, EC 232-490-9)
Residues (petroleum), vacuum (CAS 64741-56-6, EC 265-057-8)
Residues (petroleum), hydrodesulfurized vacuum (CAS 64742-85-4, EC 265-188-0)
Residues (petroleum), thermal cracked vacuum (CAS 92062-05-0, EC 295-518-9)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
OECD Guideline 414 (22 January, 2001)
Deviations:
yes
Remarks:
Staining technique for fetal skeletons changed by study amendment 2 following technical difficulties (see below)
Principles of method if other than guideline:
Details on staining technique:
Initially, some of the fetuses were fixed in 70 % ethanol and subsequently eviscerated. After that double staining with alizarin red and alcian blue was performed and fetuses were examined for skeletal anomalies and ossification defects as indicators of a possible develop-mental retardation.

However, due to technical problems with the double staining this proce-dure was changed by study plan amendment no. 2. According to this, the remaining fetuses were transferred and fixed in 99 % ethanol and subsequently eviscerated. After that bone staining with alizarin red was performed. This still allowed examination of both bone and carti-lage structures (bones stained red, cartilage can be investigated under the microscope due to a different light refraction). The staining technique was performed equivalently across exposure groups.

In a few fetuses some structures were missing for skeletal examination due to technical problems with intended double staining. These structures are reported in the study appendices and were not expected to affect the outcome or integrity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: mix of aerosol and vapour
Details on test material:
- Name of test material (as cited in study report): Bitumen fume
- Substance type: Bitumen
- Source: The condensate used for the study is characterized (using the methods described in “Bitumen (CAS 8052-42-4): Fume collection and validation”). A paving grade bitumen with CAS No. 8052-52-4 (EC No. 232-490-9) was considered to be a worst case representative regarding the PAH content (total EPA PAH per mass) and was therefore selected for testing.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: The condensate used for the study is characterized (using the methods described in “Bitumen (CAS 8052-42-4): Fume collection and validation”). A paving grade bitumen with CAS No. 8052-52-4 (EC No. 232-490-9) was considered to be a worst case representative regarding the PAH content (total EPA PAH per mass) and was therefore selected for testing.
- Lot/batch no.: A010/16

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored at -18 °C under nitrogen atmosphere before use.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: Wistar (Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Males: 10 weeks of age at delivery, 15 weeks of age at mating; Females: 6 weeks of age at delivery, 11 weeks of age at mating
- Weight at study initiation: Female (at first mating): 195.7-201.4 (group mean values)
- Housing: except for mating periods, animals were housed individually in polycarbonate (MakrolonR) cages type III. Absorbent softwood Lignocel BK 8-15 (Rettenmeier Holding AG, Wildburgstetten, Germany), was used as bedding material. For enrichment nesting material as well as polycarbonate rat retreat tubes (red coloured) were added to the cages. Cages and bedding material were changed twice a week or more often, if necessary.
- Diet: closed formula diet in pellet form identified as R/M-Z (ssniff Spezialdiaeten GmbH, Soest, Germany) available ad libitum
- Water: tap water available ad libitum
- Acclimation period: 4 weeks. Starting
in the first week of the acclimatization period, a training program was performed to train the animals to the exposure tubes for increasing periods of time. During this time, clinical observations were made once a day.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 degrees C
- Humidity (%): 55 +/- 15%
- Photoperiod (hrs dark / hrs light): 12hours dark/light

Study Dates:
Initiation: 14/10/2016
Experimental start (first mating): 24/10/2016
Experimental completion: 03/05/2016

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
nose only
Remarks:
rats were exposed to an aerosol and vapour mixture of bitumen fumes in a direct flow nose-only inhalation exposure system
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: The mean mobility diameter was 300 (1.59), 300 (1.59), 350 (1.69) nm for the low-, mid- and high-dose groups respectively (geometric standard deviation in brackets).
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: direct flow nose-only inhalation exposure system
- Method of holding animals in test chamber: tapered acrylic glass tubes with adjustable backstops
- Source and rate of air: Flow resistors controlled the flow of bitumen fume to each inhalation unit with flow rate maintained by keeping a constant pressure difference between the inhalation units and the generator by controlling the flow rate of the cooling air in the generator. The final concentrations were achieved by mixing the bitumen fume with dilution air, regulated by mass flow controllers
- System of generating particulates/aerosols: Free jet evaporation-condensation generator
- Temperature, humidity, pressure in air chamber: mean temperatures for the control and three test groups (low to high) were 21.4, 22.1, 21.7 and 21.8 +/- 0.3 degrees C. Mean humidity was 57.9%, 60.6%, 65.2% and 61.5%
- Air flow rate: approx. 60 L/min
- Method of particle size determination:
- Treatment of exhaust air: into a laboratory hood

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 approximately 2 L/min with an average of two samples taken per week. Samples were analysed by IR spectroscopy to give concentrations in total hydrocarbon content (THC) in mineral oil (ME) equivalents expressed as mg THC ME/m3. Comparison with calibration curves from defined condensate samples and measurement of UV fluorecence in mg/kg (as DPA) were also conducted.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Mean total organic matter (TOM) for the three doses were 51.6 +/- 6, 151 +/- 20 and 493 +/- 34 mg/m3 showing good agreement with target nominal concentrations

The UV fluorescence response measured as DPA equivalent was 510 mg/kg, 440 mg/kg, and 391 mg/kg for the low, mid and high dose group indicating that the composition of the atmosphere was similar in all exposure chambers. The average total PAH content in the exposure chambers were 24 μg/m³, 67 μg/m³ and 194 μg/m³ for the low, mid and high dose group. Normalised to the total organic matter an average value (over all dose groups) of 454 μg PAH/g TOM was determined.

These results confirmed that the test material represented a conservative worst case sample when compared to workplace exposure samples.

Additional details on analytical verification are available in "Appendix K1 Overview of Analytical Data" attached in background data below.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male: 2 female
- Length of cohabitation: 7 nights or until successful mating from proof of pregnancy
- Proof of pregnancy: sperm and/or vaginal plug referred to as day 0 post conception (p.c.). 96 sperm positive females were included into the study in four groups.
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
Daily
Duration of test:
Days 1 to 19 post conception
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/m³ air (nominal)
Dose / conc.:
50 mg/m³ air (nominal)
Dose / conc.:
150 mg/m³ air (nominal)
Dose / conc.:
500 mg/m³ air (nominal)
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses were selected based on findings of significant decreases in both total and net body weight gain at 500 and 1000 mg/m³ in the “Dose Range Finding Study for Prenatal Developmental Toxicity Study with Bitumen Fumes in Crl:Wistar (Han) Rats” (Fraunhofer ITEM study 12N15530). Therefore the top dose was selected with the expectation of decrease in body weight gain and the low dose with the intention to induce no parental or embryo-foetal toxicity.
- Rationale for animal assignment (if not random):
A unique consecutive individual identification number was assigned to each animal in the study, after successful mating and randomization of the females they were assigned a study specific
animal number. All data collected from an animal were filed under this study specific individual number

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily, immediately after exposure during treatment period (more frequently during the first few days of dosing to establish optimum time)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once per week - this included inspection of skin, fur, eyes, visible mucous membranes, examination for pathomorphological changes (e.g. unusual breathing pattern, masses, nodules), abnormal behaviour and central nervous symptoms (e.g. changes in gait, posture or grooming activity, unusual response to handling, secretion/excretion abnormalities, clonic/tonic movements, stereotypies) and/or other clinical abnormalities.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 18 and 21 p.c.

FOOD CONSUMPTION: Yes
- Individual food consumption was recorded to the nearest 0.1 g by the difference between initial and remaining food on days 0, 3, 6, 9, 12, 15, 18 and 21 p.c.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 21 post conception by CO2 overdose and subsequent exsanguination
- Organs examined: Macroscopic changes recorded. Absolute and relative weight of lungs determined.
Histopathological examination of the respiratory tract performed
Tissues for histological examination [lungs, larynx, lung associated lymph nodes (tracheobronchial), nasal and paranasal cavities, trachea and pharynx] were fixed for at least one week in 10% neutral buffered formalin, embedded in paraffin, sectioned at 3 - 4 μm, and stained with hematoxylin and eosin.

OTHER:
For 10-12 animals per group, lung associated lymph nodes (LALN) were not fixed in a separate capsule and were, therefore not available for histopathological examination (study plan deviation no. 1). Still, this deviation had no impact on the validity of the study, since LALN are important in case of particulate test items, which was not the case in this study.
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 (after ammonium sulfide staining in uteri without visible implantations)
- Number and position of early resorptions: Yes
- Number and position of late resorptions: Yes
- Number and position of live and dead fetuses
- Sec, position and individual weight of live fetuses
- Individual placental weight of live fetuses
- Other:
An animal was considered "non pregnant" only if no implantations sites could be determined after ammonium sulfide staining.
Fetal examinations:
- External examinations: Yes: [all per litter]
All fetuses were carefully inspected for external anomalies (incl. hematomas and position anomalies of the limbs).
- Soft tissue examinations: Yes: [half per litter]
fixed in BOUIN'S fixative and examined for soft tissue anomalies using WILSON's sectioning technique. At least 1 day before examination, the fetuses were placed in ethanol to remove BOUIN's fixative. Immediately before preparation of sections, fetuses were placed on dry ice in order to improve the quality of the sections
- Skeletal examinations: Yes: [half per litter]
Initially, some of the fetuses were fixed in 70 % ethanol and subsequently eviscerated. After that double staining with alizarin red and alcian blue was performed and fetuses were examined for skeletal anomalies and ossification defects as indicators of a possible develop-mental retardation. However, due to technical problems with the double staining this procedure was changed by study plan amendment no. 2. According to this, the remaining fetuses were transferred and fixed in 99 % ethanol and subsequently eviscerated. After that bone staining with alizarin red was performed. This still allowed examination of both bone and carti-lage structures (bones stained red, cartilage can be investigated under the microscope due to a different light refraction). The staining technique was performed equivalently across exposure groups.
Statistics:
Statistical comparison was performed at the level of 0.05
Body weights, food consumption data, uterine, fetal and placental weight were analyzed using analysis of variance
If the group means differed significantly, 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 and the Mann-Whitney U-test was applied in the case of non-homogeneous data.
Qualitative data were analyzed using Chi square test or FISHER test with Bonferroni correction.

In all instances, the dam or litter was used as the basic statistical unit. For assessment of incidence data, additional comparisons were performed based on fetuses. Data of females without implantation sites were excluded from statistical analysis. All statistical analyses were run using Provantis.
Indices:
Preimplantation loss [%], postimplantation loss [%]

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
red discharge from the vagina seen on one day for each of three animals (1 in low dose and 2 in medium dose groups) between days 12 and 14 p.c. No effects were seen for these animals on body weight gain during this period and no observations connected with this finding were noted during caesarian section. The low dose animal showed 2 early resorptions, one of the medium dose animals had 1 early resorption (the other had no postimplantation loss). Due to lack of dose response this finding was not considered treatment related.
Mortality:
no mortality observed
Description (incidence):
no mortality was observed in the course of this study
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight was statistically significantly decreased in the high dose group from day 6 p.c. onward, and in the low dose group 2 from day 9 p.c. onwards. Both the terminal body weight and the net body weight gain were also significantly decreased for these groups with the effects again strongest in the high dose group. No such effects were observed in the medium dose group and no statistically significant differences from the control group were found for the medium dose group at any time point of the study.

Weight gain was decreased in all exposed groups at several intervals of the study and total weight gain was also significantly decreased in all exposed groups (by approximately 16 %, 13 % and 26 % of control for low, medium and high dose groups respectively). The effects were most pronounced in the high dose group at the beginning of the exposure (day 3 to 6 p.c.). However, there is no dose-response relationship for the other dose groups, with effects more pronounced in the low dose group compared to the medium dose group.

Clear exposure related effects on body weight were observed in the high dose group. Due to the lack of any dose response relationship for the low and medium dose group, an exposure related effect on body weight for these groups is considered unlikely, but cannot be ruled out with certainty.

See Table 1 below for group mean bodyweights.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption was decreased in all exposed groups at several intervals of the study. Total food consumption over the exposure period was decreased in low and high dose groups (by 7 % and 14 % of controls, respectively), but no effect was indicated in the medium dose group.

Effects observed in the high dose group were considered exposure related, but due to the lack of any dose response relationship for the low and medium dose group effects on total food consumption in these groups are not considered exposure related.
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):
no effects observed
Description (incidence and severity):
no behaviour related abnormalities were noted as part of the general clinical observations
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative lung weights were increased in the high dose group (statistical significance 1% Dunnetts test). No statistically significant effects were seen in medium and low dose groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathology lesions were found in any adult females
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Findings related to the inhalation of the test item were detected within the larynx and the lung.

In the larynx very slight focal epithelial alteration was seen in 8/24, 11/24 and 15/24 animals of the low, medium and high dose groups respectively. Additionally, very slight focal squamous cell metaplasia was visible in 2/24 animals of the mid dose and in 3/24 animals of the high dose group.
Other findings in the larynx such as focal, very slight, subepithelial mixed inflammatory cell infiltration and (multi)focal, very slight/slight, subepithelial mononuclear cell infiltration were interpreted as incidental findings and unrelated to the exposure.

In the lung exposure-related findings were represented by a a very slight multifocal accumulation of pigment-laden macrophages (characterized by a pale yellowish col-or within the cytoplasm of the alveolar macrophages) in 9/24, 23/24 in low and medium dose groups respectively. The lesion was visible in all high dose group animals (17/24 with a very slight and 7/24 with a slight grade).
Furthermore, there was a very slight increase of the alveolar macrophage amount (alveolar histiocytosis), apparent in 2/24 and in 11/24 low and medium dose animals respectively. Alveolar histiocytosis occurred in all animals of the high dose group (12/24 very slight; 12/24 slight). The alveolar macro-phages exhibited a dose-dependent foamy appearance. Furthermore, there was a very slight alveolar infiltration of granulocytic cells in 17/24 animals of the high dose group.

A mixed inflammatory cell infiltration was visible perivascular in many animals of all groups. These inflammatory cells consisted mainly of granulocytes. The occurrence of the inflammatory cell infiltration was elevated significantly in the high dose group (18/24 very slight and 2/24 slight) as compared to the control group (11/24 very slight). Furthermore, the mixed inflammatory cell infiltration was visible in single animals within the alveoli or generally in the lung tissue.

All other findings in the lung or in other tissues of the respiratory tract were interpreted as incidental findings unrelated to the treatment.

Conclusion
Exposure-related findings were detected within the larynx and the lung in all exposed groups.

Of these, the alveolar granulocytic cell infiltration within the lung in 17 out of 24 animals of the high dose group and the squamous cell metaplasia in the larynx in 2 of 24 medium dose and 3 out of 24 animals of the high dose group were considered to be adverse treatment related findings.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No other effects observed but systemic exposure was confirmed during the range-finding study when PAH urine metabolites were found at all dose levels

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
See Table 2 below (in "any other information on results" section)
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
See Table 2 below (in "any other information on results" section)
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
See Table 2 below (in "any other information on results" section)
Early or late resorptions:
no effects observed
Description (incidence and severity):
See Table 2 below (in "any other information on results" section)
Dead fetuses:
no effects observed
Description (incidence and severity):
See Table 2 below (in "any other information on results" section)
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
In general, there was a slight decrease in the number of Corpora lutea in all exposed groups, without reaching statistically significance. This finding was concluded not to be exposure related, since ovulation takes place before start of the treatment.

However, this finding led to decreased numbers of implantation sites, which was statistically significant, and consequently decreased gravid uterine weight in the low dose group 2 (due to a decreased number of foetuses). Since this effect was not observed in the high and medium dose groups, it was not considered treatment related (decreased gravid uterus weight in the high dose group was considered to be the result of low foetal weights).

See Tables 1 and 2 below (in "any other information on results" section)
Details on maternal toxic effects:
Based on these findings, the maternal NOAEL was determined to be the low dose group, 50 mg/m³. Observations at 500 mg/m³ included effects on body weight, food consumption, lung weight and histopathological changes in the lung and larynx. At 150 mg/m³ observations were restricted to histopathological changes in the larynx.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/m³ air
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group, fetal weight was decreased in both sexes (about 6 % (males and females combined) compared to the control group), this was considered to be a treatment related effect, perhaps as a consequence of decreased maternal feed consumption, body weight gain and body weights. No effects were found on fetal weights in the medium and low dose groups.

No statistically significant differences from the control group were observed for placental weights in any of the exposed groups

See Tables 1 and 2 below (in "any other information on results" section)
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
See Table 2 below (in "any other information on results" section)
Changes in sex ratio:
no effects observed
Description (incidence and severity):
See Table 2 below (in "any other information on results" section)
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One case of Polyhydramnios was observed for one animal in the medium dose group, with an absent tail found in another animal in the same group. In the low dose group, fused placentas were found in one animal. Subcutaneous hemorrhage was observed sporadically in single fetuses of all groups ex-cept the low dose group.
None of these external anomalies were considered exposure related.

Please see "Bitumen PNDT Malformation Tables Report 12G16011.pdf" attached below for more details
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Findings consisted of incompletely ossified or unossified forepaw phalanx, metacarpals hindpaw phalanges, wavy ribs, detached ribs, incomplete ossification of hyoid, interparietals, nasal, parietal, squamosal, supraoccipital, asymmetric, incompletely ossified, fused, misshapen or misaligned sternebrae, split xiphoid cartilage, supernumerary cervical or thoracolumbar ribs, unossified or incompletely ossified cervical centra, dumbbell shaped, bipartitely ossified thoracic centra, dumbbell shaped lumbar centra, unossified or incompletely ossified caudal arches and centra.

None of the findings showed any dose response relationship, with only one exception, incomplete ossification of the 5th sternebra. However, this does not reach statistical significance in any of the exposed groups, it represents a common observation in this rat strain and is by no means a structural defect but a rather an expression of some retarded ossification. This is most unlikely to be exposure related, since no other bone structures show such an effect (which would be likely the case if it were an expression of an exposure related generally delayed ossification).

Moreover, all anomalies (including incomplete ossification of the 5th sternebra) were observed in all exposed and control groups, and thus none of them were considered exposure induced.

Please see "Bitumen PNDT Malformation Tables Report 12G16011.pdf" attached below for more details
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
When, in exceptional cases, subcutaneous haemorrhage was observed in fetuses and this was not detected at external examination, these findings were considered artifacts occurring during processing of the fetuses and where not entered into the system.

The only findings consisted of dilated renal pelvis, dilated ureter, fluid- (blood) filled abdomen and thorax. The latter two findings may also be induced artificially during processing of the fetuses. All these finding are considered minor anomalies (variations), occurring sporadically in this strain. They were observed in all exposed and control groups, and none of them were considered exposure induced.

Please see "Bitumen PNDT Malformation Tables Report 12G16011.pdf" attached below for more details
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
The developmental NOAEL was the medium dose group, 150 mg/m³, due to reduced fetal weight at 500 mg/m³. Nose-only exposure to bitumen fumes in concentrations up to 500 mg/m³ from p.c days 1 to 19, did not induce any fetal anomalies.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/m³ air
Based on:
test mat.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
150 mg/L air
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 1: Group mean bodyweight data (+/- SD) n = 21-24

 

Dose (mg/m3) (mg/m3) 0 100 500 1000
Parameter          
Bwt Day 0 p.c. (g)   199 ± 13 196 ± 11 201 ± 11 197 ± 17
Bwt Day 21 p.c. (g)    315 ± 22  293 ± 20**  303 ± 19  283 ± 24**
Bwt gain Day 3 to 6 p.c. (g/day)   2.6 ± 1.4  1.4 ± 0.7** 1.7 ± 0.9**  0.2 ± 0.9**
Bwt gain Day 0 to 21 p.c. (g/day)   116 ± 14 98 ± 17** 101 ± 19** 86 ± 13**
Gravid Uterus (g)   80.2 ± 10.2 67.6 ± 17.2* 71.1 ± 15.7 66.6 ± 13.4**
Net bwt gain (g)   36 ± 8  30 ± 7* 30 ± 8 19 ± 9**

* - Test: Dunnett 5% significance level

** - Test: Dunnett 1% significance level

Table 2 Ceasaren Section Data

Generalised Results - Group Summary by Mixed Parameter / Time

Dose

(mg/m3)

0

100

500

1000

Parameter

 

 

 

 

 

Number of Females Pregnant

 

23

21

24

21

Dams with Viable Fetuses

 

23

21

24

21

No. Corpora Lutea/Grp

  309 260 305 268
No. Corpora Lutea/Animal Mean 13.4 12.4 12.7 12.8
SD 1.7 1.3 2 1.7
No. Implantations/Group   286 227 271 240
No. Implantations/Animal Mean 12.4 10.8* 11.3 11.4
SD 1.4 2.5 2.3 1.6
Preimplantation Loss   23 33 34 28
Dams with Preimp Loss N 14 15 16 16
Dams with Preimp Loss > 2 N 3 3 5 4
Preimplantation Loss/Animal Mean 1 1.57 1.42 1.33
SD 1.04 2.01 1.41 1.06
% Preimplantation Loss Mean 7.02 13 11.61 10.21
SD 7.02 16.56 12.98 8.25
No. of Live Fetuses/Group   266 209 251 214
No. of Live Fetuses/Animal Mean 11.6 10 10.5 10.2
SD 1.6 2.7 2.4 2.2
No. of Live Male Fetuses/Group   146 110 113 106
% of Live Male Fetuses in Litter Mean 54.62 51.03 43.39 50.27
SD 14.68 21.09 16.22 13.33
No. of Live Female Fetuses/Group   120 99 138 108
% of Live Female Fetuses in Litter Mean 45.38 48.9 56.61 49.73
SD 14.68 21.09 16.22 13.33
Postimplantation Loss   20 18 20 26
Dams with Postimplantation Loss N 12 13 14 11
Dams with Postimplantation Loss > 2   1 c 0 1 5
Postimplantation Loss/Animal Mean 0.87 0.86 0.83 1.24
SD 1.18 0.79 0.87 1.61
% Postimplantation Loss/Animal (%(UD)) Mean 6.87 9.13 7.44 10.89
SD 8.62 9.71 7.86 13.9
No. Dead Fetuses/Group   0 0 0 0
No. Early Resorptions/Group   20 15 19 23
No. Early Resorptions/Animal Mean 0.9 0.7 0.8 1.1
SD 1.2 0.8 0.8 1.5
% Early Resorptions/Animal Mean 6.87 7.96 6.92 9.65
SD 8.62 10 7.02 12.76
Mean Male Fetal Weight (g) Mean 5.341 5.224 5.287 4.955**
SD 0.252 0.283 0.251 0.25
N 23 19 23 21
Mean Female Fetal Weight (g) Mean 5.051 4.945 4.997 4.809**
SD 0.279 0.318 0.213 0.181
N 23 21 24 21
Mean Combined Fetal Weight (g) Mean 5.212 5.087 5.129 4.878**
SD 0.248 0.28 0.197 0.205
N 23 21 24 21
Live Male Fetuses Placental Weight (g) Mean 0.596 0.576 0.576 0.559
SD 0.064 0.07 0.068 0.068
N 23 19 23 21
Live Female Fetuses Placental Weight (g) Mean 0.566 0.565 0.552 0.517
SD 0.06 0.102 0.078 0.043
N 23 21 24 21
Live Combined Fetuses Placental Weight (g) Mean 0.583 0.573 0.567 0.538
SD 0.056 0.088 0.075 0.056
N 23 21 24 21

* Test: Dunnett 5% significance

c Group Factor Test: Chi-squared p < 0.05

**  Test: Dunnett 1% significance

 

Applicant's summary and conclusion

Conclusions:
The maternal NOAEL was determined to be the low dose group, 50 mg/m³. Observations at 500 mg/m³ included effects on body weight, food consumption, lung weight and histopathological changes in the lung and larynx. At 150 mg/m³ observations were restricted to histopathological changes in the larynx.

The developmental NOAEL was the medium dose group, 150 mg/m³, due to reduced fetal weight at 500 mg/m³.

Importantly, nose-only exposure to bitumen fumes in concentrations up to 500 mg/m³ from p.c days 1 to 19, did not induce any fetal anomalies
Executive summary:

The maternal NOAEL was determined to be the low dose group, 50 mg/m³.  Observations at 500 mg/m³ included effects on body weight, food consumption, lung weight and histopathological changes in the lung and larynx.  At 150 mg/m³ observations were restricted to histopathological changes in the larynx.

The developmental NOAEL was the medium dose group, 150 mg/m³, due to reduced fetal weight at 500 mg/m³.

Importantly, nose-only exposure to bitumen fumes in concentrations up to 500 mg/m³ from p.c days 1 to 19, did not induce any fetal anomalies