The product from the burning of a combination of carbonaceous materials.

Endpoint summary

Description of key information

In the 28-day oral toxicity study in rats, Ash administered at 500, 1000 or 2000 mg/kg bw/day for 28 consecutive days induced no mortality, no clinical sign, no neurobehavioural alterations and did not affect body weight development or food consumption. No target organs could be defined. The only treatment-related effect was temporarily increased serum urea nitrogen levels in males only at doses of 1000 mg/kg and above. This effect was reversible and not associated with histopathological alterations. In addition, the urea nitrogen levels even the highest dose level were within the both within the normal limits of the laboratory (Centre de Recherches Biologiques) and the normal value data provided by Harlan for male Sprague Dawley rats. Thus, this change was not toxicologically relevant, and accordingly the NOAEL was 2000 mg/kg bw/day.
In inhalation toxicity studies with CFA the effects on lungs were largely similar to those induced by concentrated ambient particles (mild neutrophil / mononuclear cell inflammation, focal alveolar septal thickening, increased cellularity, slight fibrosis). Based on the observed differences between exposed and control rats, these effects were not considered to constitute an adverse response.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between 19 May 2010 and 30 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, France.
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: males 151.4 g and 181.1 g, females 131.9 g and 151.6 g
- Fasting period before study: -
- Housing: 5 rats of one sex per cage
- Individual metabolism cages: no
- Diet (e.g. ad libitum): RM1 (E)-SQC SDS/DIETEX feed
- Water (e.g. ad libitum): available ad libitum in polycarbonate bottles with a stainless steel nipple
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): relative humidity between 45% and 65%
- Air changes (per hr): approximately 10 times
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness with light on at 7.30 a.m.

IN-LIFE DATES: From: May 19, 2010 To: Jul. 30, 2010

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1%

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: MIXED ASH was weighed in a brown glass flask previously tarred. The vehicle was added. The suspension was ground using a homogeniser without agitation until homogenisation was completed. Samples were taken under magnetic stirring. The test item formulations were prepared daily before administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): homogeneity of dosing suspensions
- Concentration in vehicle: 0, 50, 100 or 200 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required): SIGMA-Aldrich, Batch No. 044K0101, Expiry date: Oct 2010.

HOMOGENEITY AND STABILITY OF TEST MATERIAL: based on chemical analysis of Pb, Cr and Cd; dosing suspension prepared daily immediately before dosing.

Details on analytical verification of doses or concentrations:

Based on analysis of Pb, Cr and Cd in dosing suspensions.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
0, 500, 1000 or 2000 mg/kg bw/day (m/f)
Basis:
actual ingested

No. of animals per sex per dose:

- 40 animals in the main groups i.e. 4 groups of 5 males and 5 females (3 doses and a control group)
- 20 animals in the recovery groups i.e. 2 groups of 5 males and 5 females into the control and the highest dose group to investigate delayed occurrence, persistence or reversibility of any findings.

Control animals:

other: 5 males and 5 females

Details on study design:

- Dose selection rationale: A dose range-finding study
- Rationale for selecting satellite groups: Recovery groups: control and high dose are the most relevant; Satellite groups for toxicokinetics: control and all dose levels are necessary for the assessment of systemic exposure.
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked in table No.7 (see attached report) were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly, on Days 1, 7, 14 and 21.

BODY WEIGHT: Yes
- Time schedule for examinations: Once weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Days 29 and 43
- Anaesthetic used for blood collection: Yes; isoflurane
- Animals fasted: Yes
- How many animals: 40 (main study) + 20 (recovery)
- Parameters checked in table No.3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Days 29 and 43
- Animals fasted: Yes
- How many animals: 40 (main study) + 20 (recovery)
- Parameters checked in table No.4 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Days 29 and 43
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No.5 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before the first dosing and during the last week
- Dose groups that were examined: All main study and recovery groups
- Battery of functions tested: see tables 8-9 in attached report.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 6)
HISTOPATHOLOGY: Yes (see table 6)

Other examinations:

Toxicokinetics

Statistics:

Results of functional and neurobehavioural tests, full clinical observations, general observations and mortality were expressed as incidence of the various clinical signs within each group. The effects of Ash on the incidence of the various clinical signs were compared with those of the vehicle using a Fisher’s test at each measurement time. Only clinical signs exhibited were tabulated.

Results of body weights were expressed as absolute values and percentage of variation calculated in relation to predose values. Homogeneity of predose values were tested by an analysis of variance. The effects of Ash on body weight changes from D1 up to D28 were compared with those of the vehicle using an analysis of variance for repeated measurements with a Dunnett’s test in case of significance (P≤0.05).

Organ weights were expressed as absolute values (g) and relative values (g per 100 g of body weight measured on the day of necropsy and g per 100 g of brain weight). The effects of Ash on organ weights, body temperature, quantitative urinalysis and quantitative mean clinical pathology results (haematology and blood chemistry) were compared with those of the vehicle using an analysis of variance with a Dunnett’s test in case of significance (P≤0.05).

Since changes in basophilic polymorphonuclear cells and monocytes can theoretically be considered, in terms of probability, as rare events (low percentages, often close to zero), their results were discussed but not analysed statistically.

Urinary volume and osmolality were expressed as median values. The effects of the test item on urinary volume and osmolarity were compared with those of the control group using a Dunnett’s test in case of significance (P≤0.05).

Statistical analysis were performed separately for each sex.

Results of food consumption (per cage), semi-quantitative urinalysis, macroscopic findings of organs on the day of sacrifice and histopathology findings were discussed but not analysed statistically.

Clinical signs:

no effects observed

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:

effects observed, treatment-related

Description (incidence and severity):

a statistically significant higher eosinophils in females, not dose dependent, not observed on day 43

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

a statistically significant, dose dependent higher serum urea levels in males, not observed on day 43

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Mortality: There was no mortality in males and females dosed with the vehicle (1% CMC) and the test item whatever the dose.
Clinical findings: There was no clinical sign in animals dosed with the vehicle (1% CMC). There was no significant clinical sign in animals dosed with the test item whatever the dose.

BODY WEIGHT AND WEIGHT GAIN
The body weight of animals dosed with the vehicle was normal. There was no effect on body weight of animals dosed with the test item whatever the dose when compared with the vehicle.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The food consumption of animals dosed with the vehicle was normal. There was no effect on food consumption of animals dosed with the test item whatever the dose when compared with the vehicle.

FOOD EFFICIENCY
Not examined

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Not examined

OPHTHALMOSCOPIC EXAMINATION
Not examined

HAEMATOLOGY
The haematology and coagulation parameters of animals dosed with the vehicle were normal. There was a significantly higher eosinophils count not dose dependent, in females dosed at each dose on D29 when compared with the vehicle group (+ 300%, +1700% and +600% at 500, 1000 and 2000 mg/kg, respectively), not observed at the end of the withdrawal period (D43) at the highest dose of 2000 mg/kg (0% vs vehicle). There was no other change on haematology and coagulation parameters of animals dosed with the test item when compared with the vehicle.

CLINICAL CHEMISTRY
Blood chemistry parameters: The blood chemistry parameters of animals dosed with the vehicle were normal. Except a statistically significant higher urea level on D29 in males dosed at 1000 and 2000 mg/kg on D29 (+22% and +31%, respectively) there was no other relevant effect on haematology and coagulation parameters of animals dosed with the test item when compared with the vehicle.

URINALYSIS
The urinary parameters of animals dosed with the vehicle were normal. There was no effect on urinary parameters of animals dosed with the test item whatever the dose when compared with the vehicle.

NEUROBEHAVIOUR
No effects

ORGAN WEIGHTS
No toxicologically relevant changes.

GROSS PATHOLOGY
No toxicologically relevant changes

HISTOPATHOLOGY: NON-NEOPLASTIC
no effects

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
no effects

HISTORICAL CONTROL DATA (if applicable)

OTHER FINDINGS
Body temperature: The body temperature of animals dosed with the vehicle was normal. There was no effect on body temperature of animals dosed with the test item whatever the dose when compared with the vehicle.

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

not specified

Basis for effect level:

other: other: no toxicologically relevant alterations.

Critical effects observed:

not specified

See tables in attached report.

Table 26 Effect on haematology and coagulation parameters - Male - D29 (mean values)

Table 27 Effect on haematology and coagulation parameters - Female - D29 (mean values)

Table 28 Effect on haematology and coagulation - Male - D43 (mean values)

Table 29 Effect on haematology and coagulation - Female - D43 (mean values)

Table 31 Effect on clinical chemistry parameters - Male - D29 (mean values)

Table 32 Effect on clinical chemistry parameters - Female - D29 (mean values)

Table 33 Effect on clinical chemistry parameters - Male - D43 (mean values)

Table 34 Effect on clinical chemistry parameters - Female - D43 (mean values)

Conclusions:

No serious toxic effects were observed and no target organs could be defined.

Executive summary:

In the 28-day oral toxicity study in rats Ash administered at 500, 1000 or 2000 mg/kg bw/day for 28 consecutive days induced no mortality, no clinical signs, and no neurobehavioural alterations and did not affect body weight development or food consumption. In clinical chemistry, a dose-dependent increase in serum urea was observed in males at 1000 and 2000 mg/kg bw/day (NOAEL 500 mg/kg bw/day). This change was not observed at the end of the recovery period. In histopathology, no treatment-related changes were observed. No other alterations of toxicological relevance were observed. The results do not indicate any serious toxic effects, and no target organs could be defined.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP compliant, non-guideline experimental investigation. Study published in scientific, peer reviewed journal.

Principles of method if other than guideline:

Rats were exposed to respirable (mass median aerodynamic diameter MMAD about 2 μm) aerosols of size-classified power plant fly ash at average concentrations of up to 4.2 mg m(3) for 8 h per day for up to 180 consecutive days. The aerosols were characterized with respect to both physical and chemical properties. Sampling time points were after exposure for 7, 50, 90 or 180 days. Immediately after exposure, the animals were evaluated biochemically, morphologically and with other sensitive biological tests to detect effects associated with fly ash inhalation when compared with unexposed controls.

GLP compliance:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

The rats were 70 days old Hilltop chronic respiratory disease (CRD) –free male rats, During exposure the rats were maintained in pairs in stainless steel wire mesh cages. Conditions in exposure chambers: temperature 21 ± 2 ºC, relative humidity 50 ± 20%.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: 1.77 / 1.52

Details on inhalation exposure:

see Fig. 1

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Royco 225 light-scattering particle counter (Royco Instruments, Menlo Park, CA, USA). Comparison measurements were made using a Climet 208 light-scatter particle counter (Climet Instruments, Redlands, CA, USA). Particle size distribution data were collected with these instruments operated in conjunction with a multichannel pulse height analyzer.
see attached Figure

Duration of treatment / exposure:

Two series of experiments were carried out with the sampling time points after 7, 50 or 90 days of exposure and after 90 or 180 days of exposure, respectively.

Frequency of treatment:

Daily 8 h/day

Remarks:

Doses / Concentrations:
4.2 mg/m3
Basis:

No. of animals per sex per dose:

9 - 32

Control animals:

yes

Positive control:

No

Observations and examinations performed and frequency:

Body weight in the beginning and at the end of exposure.

Sacrifice and pathology:

Morphological evaluation of lungs: Lung volume, histopathology (light microscopy, HE staining), scanning electron microscopy (SEM), analytical electron microscopy, back-scattered electron (BSE) image for identification of individual fly ash particles.

Biochemical evaluation of lungs: DNA, RNA, protein and hydroxyproline content, mucus glycoprotein synthesis and secretion rates.

Cellular studies on lungs: alveolar macrophage and haematopoietic progenitor cell kinetics, macrophage functional assays (mobility, phagocytic indices), bone marrow granulocyte-monocyte progenitors.

Other examinations:

Lung burden measurements (aluminum) and calculation of deposited fraction in lungs.

Statistics:

Student’s t test, Mann-Whitney rank-sum test

Clinical signs:

no effects observed

Description (incidence and severity):

No mortality or clinical signs reported

Mortality:

no mortality observed

Description (incidence):

No mortality or clinical signs reported

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No effect on lung weight, no data for other organs

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Histopathology: non-neoplastic
CFA exposed animals had higher concentration of alveolar macrophages in alveolar lumens and refractile brownish pigment (presumably fly ash) was visible in these cells. Alveolar macrophages were larger than in lungs of control animals. Alveolar septal walls contained occasionally cellular aggregates consisting of some mononuclear leucocytes admixed with brownish particulate material (ash). These differences between CFA exposed and control animals had minimal impact, if any, on health status of the animals.

Lung burdens of up to 4 mg of ash were found, macrophages lavaged from lungs of exposed rats were more numerous and yielded more progenitor cell colonies in culture than from controls, tracheal mucous glycoprotein secretion was decreased at 7, 50 and 90 days of exposure and increased at 180 days and clumping of fly ash in cells in the lungs was observed. Despite of this, there were no apparent untoward health effects from inhaled fly ash, since the observed differences between exposed and control rats did not constitute an adverse response.

Remarks on result:

not measured/tested

Remarks:

Measured parameters defined in method description

Critical effects observed:

not specified

Lung burdens of up to 4 mg of ash were found. Macrophages lavaged from lungs of exposed rats were more numerous and yielded more progenitor cell colonies in culture (at the 10% confidence level based on the Mann-Whitney rank-sum test) than from controls. Tracheal mucous glycoprotein secretion was decreased at 7, 50 and 90 days of exposure and increased at 180 days. Clumping of fly ash in cells in the lungs was observed. There were no apparent untoward health effects from inhaled fly ash, since the observed differences between exposed and control rats did not constitute an adverse response.

Conclusions:

There were no apparent untoward health effects from inhaled fly ash, since the observed differences between exposed and control rats did not constitute an adverse response.

Executive summary:

Respiratory disease-free rats were exposed in two experiments to respirable (mass median aerodynamic diameter MMAD about 2 μm) aerosols of size-classified power plant fly ash at average concentrations of up to 4.2 mg m(3) for 8 h per day for up to 180 consecutive days. The aerosols were characterized with respect to both physical and chemical properties. Immediately after exposure, the animals were evaluated biochemically, morphologically and with other sensitive biological tests to detect effects associated with fly ash inhalation when compared with unexposed controls. Lung burdens of up to 4 mg of ash were found. Macrophages lavaged from lungs of exposed rats were more numerous and yielded more progenitor cell colonies in culture (at the 10% confidence level based on the Mann-Whitney rank-sum test) than from controls. Tracheal mucous glycoprotein secretion was decreased at 7, 50 and 90 days of exposure and increased at 180 days. Clumping of fly ash in cells in the lungs was observed. There were no apparent untoward health effects from inhaled fly ash, since the observed differences between exposed and control rats did not constitute an adverse response.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

4.2 mg/m³

Study duration:

chronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Remarks:

Doses / Concentrations:

Basis:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity data are based on a 28-day oral toxicity study with Ash in rats and on a published inhalation toxicity studies with CFA in rats. No serious toxic effects were observed and no target organs could be defined.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP compliant guideline study.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
The lowest NOAEC observed.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Repeated dose dermal toxicity is evaluated on the basis of oral 28-day toxicity study with the assumption that systemic absorption from the dermal route is not higher than absorption after oral ingestion.

Justification for classification or non-classification

Justification is based on the outcome from the 28-day oral toxicity study.