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

Repeated dose toxicity: oral

Administrative data

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.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

931-597-4

Molecular formula:

UVCB substance, not available. View remarks field.

IUPAC Name:

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

Details on test material:

- Name of test material: Mixed ash
- Physical state: powder
- Composition range of test material (% (w/w)): Aluminium (Al) 13.07, Calcium (Ca) 45.52 , Iron (Fe) 4.15 , Magnesium (Mg) 3.49, Phosphorus (P) 0.79 , Potassium (K) 2.56, Silicon (Si) 28.07, Sodium (Na) 1.08, Sulphur (S) 1.27.
- The critical minor components examined (mg/kg d.w.): Arsenic (As) 18, Barium (Ba) 670 Cadmium (Cd) 3.6, Copper (Cu) 410, Lead (Pb) 180 and Antimony (Sb) 22.
- Purity test date: the substance is UVCB substance
- Lot/batch No.: MIXED ASH 1-01032010
- Expiration date of the lot/batch: March 2011
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature under Argon.
- Other: Mixed Ash is named in dossier as Ash. Mixed in the substance name has meant that there has been several (mixed) fuels when producing ash.

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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.