Ashes (residues), coal

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and there is no or no significant human exposure

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
Regulation (EC) No. 1907/2006, Annex IX, 8.7.2. Column 1, states that a pre-natal developmental toxicity study (OECD 414), one species is required to fulfil the standard requirements for developmental toxicity, using the most appropriate route of administration, and having regard to the likely route of human exposure.
According to Regulation (EC) No 1907/2006, Annex X, 8.7. Column 2, a developmental toxicity study is not required if (i) the substance is of low toxicological activity, (ii) it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure and (iii) there is no or no significant human exposure.
Therefore, in accordance with Annex X, 8.7.2. Column 2, the registrant has considered the need to perform a developmental toxicity study. The summary below explains the rationale for the registrant’s conclusion that additional testing is not scientifically justified. In fact, additional testing is scientifically not justified based on (i) negligible systemic absorption via inhalation and also via the oral and dermal route of exposure, (ii) low toxicological hazard (including available data on reproduction and developmental toxicity) and (iii) the use of protective gear and adequate dust control measures are implemented to prevent significant human exposure.

Toxikokinetics
Ashes (residues), coal are a complex and heterogeneous mixture of metal and metalloid oxides in variable proportions. Ashes (residues), coal mainly consist of water insoluble compounds such as silicon dioxide (SiO2), aluminium oxide (Al2O3) and iron oxide (Fe2O3). The available information on the toxicokinetic behaviour of Ashes (residues), coal and the main components (SiO2, Al2O3, Fe2O3) indicate no relevant systemic absorption by any route of exposure.
Silicon dioxide and aluminium oxide are slightly soluble in body fluids (e. g. in the stomach) leading to the formation of silicic acid and aluminium chloride, respectively, which show low levels of absorption and rapid clearance via the kidneys (IARC, 1997; McEvoy, 1990). Therefore, a low level of absorption of Ashes (residues), coal via ingestion or after dermal contact is possible, but no relevant systemic bioavailability is expected. This is supported by studies on Ashes (residues), coal, available for acute oral and dermal toxicity. In all of these studies no mortalities occurred and no signs of systemic toxicity were observed (please refer to the study summaries in the respective chapters for more details). Furthermore, in a 3-year feeding study in cattle, the chemical analyses of milk, blood, urine and faeces indicated that no systemic absorption occurred after oral administration of 1850 mg/kg/day of fly ash (Herrmann, 1955).
The most relevant route of exposure for Ashes (residues), coal, taken into account the physicochemical properties of the substance and its uses, is by inhalation. About 3 mg fly ashes were inhaled by hamsters nose-only exposed to an aerosol with a concentration of 470 µg/L (Wehner et al, 1980). Approximately 2% of the particles were retained in the respiratory tract. After 99 days, about 90% of the fly ashes retained had been cleared from the lungs. Most of the fly ashes were recovered in the gastrointestinal tract and faeces (together ca. 85%). Some of the test material was recovered in the head (5.25%, combined external and internal deposition), pelt (4.43%) and carcass (2.25%). The latter value is probably due to external deposition on the extremities not removed on skinning of the animals. Little amounts (ca. 3% - which means 0.09 mg) were recovered in internal organs (liver, kidney) indicating a very low level of absorption.
In a further study, the pulmonary deposition and clearance of a coal fly ash were assessed in male Wistar rats exposed to coal fly ash aerosols at average exposure concentration of 10.4 mg/m3 for 7 h/day, 5 days/week during 1 month (Matsuno et al., 1985). The burden of fly ash was estimated by the measurement of aluminium contents in rat organs. The aluminium concentrations in the lungs of the exposed rats for each run were much higher than those of the controls, but they decreased with the increase of the clearance time. There is no statistical significance regarding the amount of fly ash deposited in lungs among the exposure groups. In the other organs (liver, kidney, spleen and blood), there were no significant differences of aluminium concentration between exposure groups and controls, indicating a low absorption of aluminium from coal fly ash.
Results of a 28-day inhalation study with fly ash derived from electrostatic precipitators of a thermal power station in male Wistar rats suggested that heavy metals derived from inhalation exposure to fly ash may be systemically absorbed and bioaccumulated in lung, liver and kidneys of rats (Mani et al., 2007). However, the metals investigated are all present in Ashes (residues), coal at concentrations < 0.1% (according to the substanc information profile (SIP)). Therefore, no concern and relevance for man can be expected due to the indirect exposure with heavy metals via the ashes. The information available on the main constituents SiO2, Al2O3 and Fe2O3 indicate that inhaled particles of these compounds deposit along the respiratory tract according to their aerodynamic diameter. Thus, small particles (< 5 µm) can reach the alveolar region. Following deposition on the surface of the lung, there is either a rapid mucociliary clearance if deposition is in the upper airways or phagocytosis by alveolar macrophages and slower clearance if deposition is in the alveolar region. Clearance by mucociliary mechanisms is generally considered to be efficient; clearance from the alveolar region is slow and incomplete and some of the particles may be carried by macrophages into the pulmonary interstitium and lymphoid tissues (ECHA, 2017; Friberg et al., 1986; IARC, 1972, 1997).
Mineralogical investigations showed that the constituent quartz in Ashes (residues), coal is embedded in glass (Borm, 1997; Nathan et al., 2009; Meij et al., 2000). Thus, based on the available information, it can be concluded that silicon dioxide in form of quartz is not freely available in the toxicologically relevant respirable fraction of Ashes (residues), coal, due to the fact that it is embedded in an amorphous glass matrix.
Taken together, Ashes (residues), coal as a whole and its main components are unlikely to be absorbed and systemically distributed to a relevant extent in humans. Moreover, none of these compounds is prone to undergo metabolic transformation. Therefore, Ashes (residues), coal will mainly be excreted within the faeces after oral exposure. Particles deposited in the respiratory bronchioles and proximal alveoli are cleared more slowly. Inhaled particles cleared from the lung as a result of mucociliary mechanisms will likely be swallowed and excreted via the gastrointestinal tract as well. Soluble material leaching from the primary particles and eventually being absorbed will most likely be excreted in the urine, as described for SiO2 and Al2O3 (Friberg et al., 1986; IARC 1997).

Animal studies covering developmental parameters
Information on developmental toxicity of Ashes (residues), coal is available from an oral (gavage) Reproduction / Developmental Toxicity Screening Test performed according to OECD guideline 421 and under GLP with Wistar rats (ČEZ Energetické produkty, 2008, key). The NOAEL was established at 1000 mg/kg bw/day for the parental animals and the pups, which was the highest dose tested.
In addition, there are no indications that the main components of Ashes (residues), coal, i.e. silicon dioxide (SiO2), aluminium oxide (Al2O3) and iron oxide (Fe2O3), induce toxic effects to reproduction / development in animals or humans (according to ECHA dissemination portal).

Additional toxicological data
The whole body of available data on the toxicological properties of Ashes (residues), coal points out the respiratory tract is the only target for potential toxic effects. No hazard, besides local effects in the lung, was observed in all available studies. The local effects in the lungs, observed in acute and repeated inhalation toxicity studies, are a direct result of deposition of fly ash within the lungs and thus, these are considered a natural response to inhaled particle deposition and not being specific to coal fly ash.
Human exposure
The use of protective gear and adequate dust control measures are implemented for all exposure scenarios related to Ashes (residues), coal to comply with the occupational exposure limits for respirable dusts of the different EU countries and thus, to prevent significant human exposure.

Conclusion
In conclusion, information with respect to developmental toxicity is limited to a Reproduction / Developmental Toxicity Screening Test performed according to OECD guideline 421. The results of this study did not show any potential for developmental toxicity in the examined animals. Based on the general toxicological profile, there is no hazard attributed to systemic availability of Ashes (residues), coal. Observed local effects in the lungs are a direct result of deposition of the test substance within the lungs and are considered an important natural response to inhaled particles, not being specific to Ashes (residues), coal. In addition and with respect to man, the use of protective gear and adequate dust control measures are implemented to prevent significant human exposure.
Based on the available information, there is no evidence for developmental toxicity to be expected from Ashes (residues), coal. Therefore, referring to Regulation (EC) No. 1907/2006, Annex X, 8.7. Column 2, and for animal welfare reasons, performing a developmental toxicity study is not scientifically necessary and, considering concerns regarding the use of vertebrate animals for experimental purposes, unjustified.

References:
Borm, P. J. A. (1997). Toxicity and Occupational Health Hazards of Coal Fly Ash (CFA). A review of data and comparison to coal mine dust. Ann Occup Hyg 41(6):659-676.
ECHA (2017). Guidance on information requirements and chemical safety assessment. Chapter R.7c: Endpoint specific guidance.
Friberg, L., Nordberg, G. F., Kessler, E. and Vouk, V. B. (eds.) (1986). Handbook of the Toxicology of Metals. 2nd ed. Vols I, II. Elsevier Science Publishers B. V.
IARC (1972). Monographs on the Evaluation of Carcinogenic Risks to Humans. Volume 1. Some Inorganic Substances, Chlorinated Hydrocarbons, Aromatic Amines, N-Nitroso Compounds and Natural Products. World Health Organization, International Agency for Research on Cancer.
IARC (1997). Monographs on the Evaluation of the Carcinogenic Risks to Humans. Volume 68. Silica, Some Silicates, Coal Dust and para-Aramid Fibrils. World Health Organization, International Agency for Research on Cancer.
McEvoy, G. K. (ed.) (1990). AHFS Drug Information 90. American Society of Hospital Pharmacists, Inc.
Meij, R., Nagengast, S. and te Winkel, H. (2000). The Occurrence of Quartz in Coal Fly Ash Particles. Inhalation Toxicology, 12 (suppl. 3), 109-116.
Nathan, Y., Metzger, A., Dvoracheck, M. and Pardo A. (2009). Occupational Health Aspects of Quartz in Bituminous Coal Fly Ash in Israel.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

10 Dec 1950 - 14 Apr 1954

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication meeting basic scientific principles

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Objective of study:

absorption

distribution

excretion

Principles of method if other than guideline:

Fly ash derived from coal combustion was fed at 0, 300 and 1500 g / animal / day to cows over a period of 2 years.
After two years, the chemical composition of milk, blood, urine and faeces samples were analysed.

GLP compliance:

no

Radiolabelling:

no

Species:

cattle

Strain:

other: Schwarzbunte Niederungsrasse

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Oldenburg, Germany (a geographical region which was assumed to be free of fly ash from coal combustion)
- Age at study initiation: 2.75 - 6.75 years
- Weight at study initiation: 535 - 710 kg
- Housing: Cattle pen; the animals were allowed to graze.
- Diet (e.g. ad libitum): fodder beed, hay and straw (ad libitum) supplemented with mash.
At their arrival the animals were carefully clinically examined. All animals were in good nutritional state and showed no symptoms of illness.
Some animals were seropositive for tuberculosis.

ENVIRONMENTAL CONDITIONS
All animals were kept on farm without contact to other cattle.
The cattle pen was always kept clean, dry and ventilated with fresh air.

IN-LIFE DATES:
Exposure period: From: December 10th, 1950 To: December 15th, 1953
Post exposure observation period: From: December 16th, 1953 To: April 14th, 1954

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed with the daily food.
Animals were fed twice a day in individual boxes to assure the intake of the individual portions.

DIET PREPARATION
In the high dose group, the fly ash was mixed with soaked fodder beed and mash, to improve the taste and to ascertain the daily intake of the high amounts of fly ash.

Duration and frequency of treatment / exposure:

2 years, twice daily

Dose / conc.:

0 other: g/animal/day (nominal in diet)

Dose / conc.:

300 other: g/animal/day (nominal in diet)

Dose / conc.:

1 500 other: g/animal/day (nominal in diet)

Remarks:

2 high-dose animals received temporarily up to 1800 g/animal/day

No. of animals per sex per dose / concentration:

3

Control animals:

yes, concurrent no treatment

Positive control reference chemical:

not applicable

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, milk
- Time and frequency of sampling: once on December 10, 1952

Statistics:

not applicable

Details on absorption:

no systemic absorption after oral administration

Analysis of milk samples collected on December 10^th, 1952:

(Average values in % determined from two different laboratories in parallel)

Animal	Treatment up to the sampling date	Dry residue	Ash	CaO	MgO	P2O5
1	concurrent no treatment	12.9	0.74	0.172	0.0144	0.212
2	concurrent no treatment	15.8	0.89	0.267	0.0222	0.276
3	concurrent no treatment	10.3	0.71	0.136	0.0155	0.185
4	300 g fly ash per day	11.2	0.78	0.173	0.0170	0.235
5	300 g fly ash per day	14.7	0.82	0.197	0.0194	0.299
6	300 g fly ash per day	14.1	0.71	0.152	0.0204	0.159
7	1500 - 1800 g fly ash per day	15.3	0.69	0.161	0.0161	0.220
8	1500 - 1800 g fly ash per day	14.1	0.73	0.155	0.0144	0.213
9	1500 - 1800 g fly ash per day	10.3	0.73	0.151	0.0153	0.224

Applicant's conclusion:

Two years of oral administration of two different doses of fly ash did not influence the composition of the milk regarding the analysed parameters.

 

Analysis of blood samples collected on December 10^th, 1952:

(Average values in % determined from two different laboratories in parallel)

Animal	Treatment up to the sampling date	Dry residue	Ash	CaO	MgO	P2O5	Fe2O3
1	concurrent no treatment	19.2	1.37	0.0127	0.0058	0.0374	0.0534
2	concurrent no treatment	19.9	1.03	0.0110	0.0060	0.0380	0.0544
3	concurrent no treatment	18.7	1.15	0.0116	0.0061	0.0371	0.0435
4	300 g fly ash per day	19.5	1.22	0.0133	0.0062	0.0396	0.0502
5	300 g fly ash per day	19.2	1.36	0.0094	0.0077	0.0333	0.0572
6	300 g fly ash per day	19.6	1.13	0.0101	0.0068	0.0350	0.0430
7	1500 - 1800 g fly ash per day	20.3	1.16	0.0105	0.0071	0.0359	0.0480
8	1500 - 1800 g fly ash per day	18.3	0.98	0.0112	0.0062	0.0362	0.0489
9	1500 - 1800 g fly ash per day	19.3	1.12	0.0116	0.0077	0.0345	0.0547

Applicant's conclusion:

Two years of oral administration of two different doses of fly ash did not influence the composition of the blood regarding the analysed parameters.

 

Analysis of urine samples collected on December 10^th, 1952

(Average values in % determined from two different laboratories in parallel)

Animal	Treatment up to the sampling date	pH	Glucose	Protein	Dry residue	CaO	MgO^a	P2O5
1	concurrent no treatment	8.14	neg.	neg.	6.84	0.0055	0.0170	0.0027
2	concurrent no treatment	8.20	neg.	neg.	5.18	0.0018	0.0221	0.0024
3	concurrent no treatment	8.18	neg.	neg.	5.41	0.0011	0.0261	0.0016
4	300 g fly ash per day	8.36	neg.	neg.	5.33	0.0066	0.0090	0.0013
5	300 g fly ash per day	8.26	neg.	neg.	4.53	0.00097	0.0080	0.0014
6	300 g fly ash per day	8.34	neg.	neg.	5.11	0.0043	0.0302	0.0031
7	1500 - 1800 g fly ash per day	8.08	neg.	neg.	3.85	0.0084	0.0100	0.0013
8	1500 - 1800 g fly ash per day	8.40	neg.	neg.	6.33	0.0041	0.0357	0.0022
9	1500 - 1800 g fly ash per day	8.38	neg.	neg.	5.80	0.0233	0.0411	0.0025

^aMgO was only analysed from one laboratorium

Applicant's conclusion:

Two years of oral administration of two different doses of fly ash did not influence the composition of the urine regarding the analysed parameters.

 

Analysis of faeces sampled on December 10^th, 1952

(Values in % of fresh faeces)

Animal	Treatment up to the sampling date	Ash	SiO2	Al2O3	Fe2O3	MgO	CaO	SO3	P2O5
1	concurrent no treatment	2.77	1.51	0.119	0.066	0.118	0.270	0.057	0.183
2	concurrent notreatment	2.23	1.11	0.086	0.054	0.150	0.250	0.044	0.164
3	concurrent no treatment	2.28	1.13	0.091	0.062	0.111	0.259	0.052	0.167
4	300 g fly ash per day	2.68	1.32	0.296	0.142	0.131	0.255	0.054	0.190
5	300 g fly ash per day	2.36	1.17	0.240	0.148	0.111	0.240	0.054	0.170
6	300 g fly ash per day	2.98	1.55	0.431	0.201	0.131	0.350	0.077	0.270
7	1500 - 1800 g fly ash per day	4.81	2.49	0.716	0.615	0.286	0.372	0.107	0.182
8	1500 - 1800 g fly ash per day	6.21	2.84	0.827	0.643	0.352	0.477	0.135	0.246
9	1500 - 1800 g fly ash per day	4.76	2.31	0.644	0.506	0.288	0.492	0.105	0.208

^aMgO was only analysed from one laboratorium

Applicant's conclusion:

Two years of oral administration of two different doses of fly ash influenced the ash and mineral contents of the faeces in a dose-dependent manner.

Analysis of trace elements in liver and bone samples:

At the end of the 3-year experiment duration, liver and bone samples of all animals were analysed for their contents of Arsenic, Manganese, Lead, Iron, Copper, Zinc and Cobalt. Bones were additionally analysed for their CaO and P₂O₅content.

In summary, there was no significant influence of the fly ash administration on the trace element contents in liver and bone samples.

Conclusions:

No bioaccumulation potential based on study results
The chemical analyses of milk, blood, urine and faeces indicate, that no systemic absorption after oral administration of 1850 mg/kg/day of the test substance occured.

Executive summary:

Fly ash derived from coal combustion in Scholven, Germany was fed at 0, 300 and 1500 g/animal/day to cows over a period of 3 years. After two years the control and intermediate dose groups were exchanged and therefore as far as possible only the results from this time point were taken into the present considerations.

Body weight development in cows were not disturbed by the oral administration of fly ash. Because treated animals showed a slightly higher milk production rate than control animals, the general condition of treated animals was considered to be better. Chemical analyses of milk, blood, urine and faeces samples after two years indicate that the test substance was practically not bioavailable for resorption.

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication which meets basic scientific principles.

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Objective of study:

absorption

distribution

excretion

Principles of method if other than guideline:

The pulmonary deposition, translocation and clearance of inhaled fly ash was determined in hamsters nose-only exposed to neutron-activated fly ash for 95 min and sacrificed and assessed at intervals over a period of 99 days post-exposure.

GLP compliance:

no

Radiolabelling:

yes

Remarks:

Coal fly ash was prepared for neutron activation in a Hanford's N-reactor. Radionuclides were induced by (n, gamma) reactions on the major and minor elements in the fly ash. The radionuclides 60Co, 46Sc and 59Fe were selected as fly ash tracers.

Species:

hamster, Syrian

Strain:

other: outbred LAK:LVG, CRL

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Lakeview Laboratories
- Weight at study initiation: 120-130 g

Route of administration:

inhalation: aerosol

Vehicle:

unchanged (no vehicle)

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only


GENERATION OF TEST ATMOSPHERE / CHAMPER DESCRIPTION
- Exposure apparatus: 20-L aerosol exposure chamber (Wehner et al., 1977. Food Cosmet Toxicol 15:213-224) constructed of Lucite with exposure ports for nose only exposures arranged in 7 tiers, each containing 10 exposure ports.
- Method of holding animals in test chamber: animals were placed in soft-drink bottles from which the bottom and part of the top had been removed in such a way that the noses of the animals were close to the open tops of the bottles. The bottles were inserted through neoprene stoppers into the ports of the exposure chambers. The animals were mantained in this position by wadding pushed against their posteriors, and by neoprene stoppers taped to the bottoms of, and sealing, the bottles.
- System of generating particulates/aerosols: The fly ash aerosol was generated by a Wright Dust Feed Mechanism (Wright, 1950. J Sci Instr 27:12-15) and then passed through a cyclone elutriator for removal of particles larger than 10 µm aerodynamic equivalent diameter (AED).
- Method of particle size determination: An Andersen cascade impactor (Andersen, 1966. Amer Ind Hyg Assoc J 27:160-165) positioned on tier 3 of the exposure chamber.


TEST ATMOSPHERE (if not tabulated)
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 3.53 +/- 0.33 µm; 2.77 +/- 0.05 µm

Duration and frequency of treatment / exposure:

95 min, single exposure

Dose / conc.:

470 other: µg/L air

Remarks:

+/- 39 µg/L air

No. of animals per sex per dose / concentration:

58 treated males
10 control animals, 5 of which were killed 2 days, the other five 102 days, after exposure

Control animals:

yes, concurrent no treatment

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, lungs (including mediastinal tissue), liver, kidney, skinned and decapitated carcass, pelt, head, gastrointestinal tract.
- Time and frequency of sampling: 15 min, 1, 3, 7, 14, 24, 51 and 99 days post-exposure.

Statistics:

The deposition in the liver samples from the exposed animals was compared to control animals at both sacrifice times by a two-tailed Mann-Whitney U test.

Details on absorption:

Based on the data reported, approximately 3 mg fly ash were inhaled in the 95-min exposure. Thereof, ca. 2% was retained in the lungs. After 99 days, 0.2% was still found in the lungs.

Details on distribution in tissues:

The overall amount of fly ashes found in tissue samples after the indicated time and expressed as percentage of the ca. 3 mg fly ashes initially inhaled was:
Liver: 0.39% (99 days)
Kidneys: 0.13% (99 days)
Carcass: 2.25% (99 days)
Gastrointestinal tract: 36.7% (14 days)
Head: 5.25% (7 days)
Pelt: 4.43% (7 days)

Details on excretion:

The overall amount of fly ashes found in excreta samples after the indicated time and expressed as percentage of the ca. 3 mg fly ashes initially inhaled was:
Faeces: 48.41% (3 days)
Urine: 0.58% (3 days)

Fly ash burdens in tissues and excreta as a function of time after exposure (expressed as mean values in µg)

	Lung			Liver			Kidney
	Sc	Fe	Co	Sc	Fe	Co	Sc	Fe	Co
15 min	65.8	63.2	40.1	0.52	1.2	3.4	0.12	0.4	0.94
1 day	48.8	48.4	23	0.41	2.7	1.9	0.18	0.51	0.49
3 days	55.7	55.4	25.6	0.62	3.1	1.6	0.25	0.43	0.43
7 days	27.9	29	11.5	0.35	2.5	1	0.13	0.65	0.38
14 days	34.1	37.3	14.1	0.53	4.1	0.86	0.19	0.85	0.28
24 days	20.7	24.2	8.3	0.48	2.9	0.79	0.08	1.2	0.18
51 days	12.2	14.5	5.2	0.59	2.2	0.48	0.09	1.3	0.2
99 days	6.4	7.65	2.4	0.36	2.6	0.21	0.21	2.3	0.17

 

	Carcass			GI tract			Head
	Sc	Fe	Co	Sc	Fe	Co	Sc	Fe	Co
15 min	26.9	28	66.9	814	792	767	102	103	120
1 day	10	11.4	14.9	259	228	270	20.3	21.8	46.5
3 days	6	0	8.8	57.6	59.6	67.6	6.1	9.2	25.7
7 days	1.7	4	2.4	4.3	5.3	11.3	2	4.7	22
14 days	1.5	5.7	2.2	4.7	5.7	5.8
24 days	0.82	4.5	1.3
51 days	0.61	3.6	0.67
99 days	0.7	4.3	0.54

 

	Pelt			Faeces			Urine
	Sc	Fe	Co	Sc	Fe	Co	Sc	Fe	Co
15 min	80.6	75.8	91.6	873	831	903	1	3.2	21.3
1 day	23.5	21.5	34.1	483	471	464	0.88	3.2	16.8
3 days	8.8	9.2	37.9	127	130	142	0.9	3.5	3.8
7 days	2.9	4.4	18
14 days
24 days
51 days
99 days

Fly ash burden estimates as determined by the radionuclides 46Sc and 59Fe were in good agreement for the majority of the samples analysed. Such close agreement indicated fly ash particulate levels in the lungs, carcass, head pelt, GI tract and faeces rather than leached radionuclides. Fly ash deposition estimates obtained with 60Co were lower for the lungs and higher at one or more sacrifice times for carcass, liver, head, pelt and urine samples. Thus, this indicates selective leaching of Co from fly ash deposited in the deep lung, translocation to other sites and excretion in the urine. Most of the 60Co urinary excretion took place within the first few days after exposure.

Approximately 63 µg fly ash (2% of the inhaled fly ash) was initially retained in the respiratory tract. The estimated biological half-times of the fly ash were 2.6 and 34.5 days, probably for the upper airways and for the deep lung, respectively. After 99 days, the mean lung burden had decreased to about 10% of the initial value. Extrapolating from the clearance data, it can be estimated that near-complete clearance of fly ash from the lung would have been achieved approximately 200 days post exposure.

Conclusions:

Low bioaccumulation potential based on study results.
The results of this study indicate that about 3 mg fly ash were inhaled by hamsters nose-only exposed to an aerosol with a concentration of 470 µg/L air. Approximately 2% of the particles were retained in the respiratory tract. After 99 days, about 90% of the fly ash retained had been cleared from the lungs. Most of the fly ash was found in the gastrointestinal tract and faeces (together ca. 85%). Some of the test material was found in the head (5.25%, combined external and internal deposition), pelt (4.43%) and carcass (2.25%). The latter value is probably due to external deposition on the extremities not removed on skinning of the animals. Little amounts were found in internal organs indicating a very low level of absorption.

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Objective of study:

absorption

distribution

Principles of method if other than guideline:

Study on pulmonary deposition and clearance of a coal fly ash aerosol by inhalation.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Not further data.

Route of administration:

inhalation: aerosol

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Respirable aerosols of fly ash were generated and transported to exposure chambers. The volume was 0.48 m3.
- System of generating particulates/aerosols: The system for the generation and exposure of fly ash aerosol was not reported in this publication (but in previous publicatoins from the same authors).
- Method of particle size determination: The size distribution of the aerosol was determined by using an Andersen cascade impactor. The mass median aerodynamic diameter (MMAD) and the geometric standard deviation of the fly ash particles were 3.0 µm and 1.8, respectively.

TEST ATMOSPHERE
- Brief description of analytical method used: The fly ash aerosol concentration in the exposure chamber was monitored continuously by a light-scattering method and was adjusted automatically by the on-off control of the screw feeder. The mass concentration of fly ash aerosol was measured gravimetrically at daily intervals by the isokinetic suction of air through a glass filter. The glass filters were weighed before and after the measurements. The size distribution of the aerosol was determined by using an Andersen cascade impactor.

Duration and frequency of treatment / exposure:

7 h/day, 5 days/week for 1 month
- 1st run: sacrifice directly after last exposure
- 2nd run: sacrifice after 6 months clearing time
- 3rd run: sacrifice after 10 months clearing time

Dose / conc.:

10.4 mg/m³ air

Remarks:

± 2.3

No. of animals per sex per dose / concentration:

Not specified.
In the table describing the aluminium concentration in rat organs there is stated: 1st run: 3 control and 5 exposure animals; 2nd run: 6 controls and 7 exposure animals; 3rd run: 4 controls and 7 exposure animals.

Control animals:

yes

Positive control reference chemical:

Not applicable.

Details on dosing and sampling:

In the 1st run, rats were sacrificed for analysis of aluminium in the visceral organs just after the exposure. In the 2nd and 3rd run, rats were exposed for 1 month and kept for the clearance time of 6 and 10 months respectively, before sacrifice. The control rats were also sacrificed at the same time. The burden of fly ash was estimated by the measurement of aluminium contents in rat organs.

Details on distribution in tissues:

The aluminium concentrations in the lungs of the exposed rats for each run were much higher than those of the controls, but they decreased with the increase of the clearance time. In the other organs, there were no significant differences of aluminium concentration between exposure groups and controls. Please also refer to Table 1.
The lung burden of fly ash was determined from the measured amounts of aluminium in the lungs, assuming 9.7% aluminium in fly ash. The lung deposition fraction was 5.1%, resulted in a lung burden of 0.7% (please also refer to Table 2). There is no statistical significance in the amount of fly ash deposited in lungs among the exposure groups.

The final body weights and the gains in body weight of the exposure and control groups were not significantly different. There were also no differences in organ weights between exposure groups and controls.

Table 1: Aluminium concentration in rat organs.

Run		Number of rats	Lung (µg/g ± SD)	Liver (µg/g ± SD)	Kidney (µg/g ± SD)	Spleen (µg/g ± SD)	Blood (ppm)
1	Control	3	1.9 ± 1.2	0.5 ± 0.3	2.1 ± 1.5	1.4 ± 1.6	ND
	Exposure	5	53 ± 22	0.5 ± 0.3	2.0 ± 1.0	1.5 ± 1.5	ND
2	Control	6	2.6 ± 0.5	0.6 ± 0.3	2.3 ± 1.3	1.4 ± 1.1	ND
	Exposure	7	27 ± 4	0.7 ± 0.4	2.4 ± 1.5	1.5 ±1.3	ND
3	Control	4	2.4 ± 0.4	0.7 ± 0.4	2.8 ± 0.5	1. 0 ± 0.2	ND
	Exposure	7	25 ± 7	0. 8 ± 0.5	3.1 ± 0.7	1. 1± 0.2	ND

ND: < 0.3 ppm

 

Table 2: Deposition of inhaled fly ash in rat lungs.

Run		Al content (µg ± SD)^a	Lung burden fly ash (mg ± SD)^b	Inhaled fly ash (m g± SD)^c	Apparent deposition fraction (% ± SD)
1	Exposure	70 ± 30	0.7 ± 0.3	13.6 ± 0.3	5.1 ± 2.0

^a: Net after subtraction of control values.

^b: Based on 9.7% Al concentration in fly ash.

^c: Calculated values.

Histopathological observations of the left lung and lymph nodes from rats showed no serious pathological changes in each run.

Conclusions:

Wistar male rats were exposed to coal fly ash aerosols at average exposure concentration of 10.4 mg/m3 for 7 h/day, 5 days/week for 1 month. Some rats were sacrificed just after the exposure, while others were kept for 6 or 10 months clearance time before sacrifice. There were no differences in body weight gain between fly ash exposure groups and controls.The burden of fly ash was estimated by the measurement of aluminium contents in rat organs. The aluminum concentrations in the lungs of the exposed rats for each run were much higher than those of the controls, but they decreased with the increase of the clearance time. There is no statistical significance in the amount of fly ash deposited in lungs among the exposure groups. In the other organs, there were no significant differences in aluminium concentration between exposure groups and controls. It was concluded that the clearance rate of fly ash deposited in rat lungs may be very slow. On the other hand, there were no serious changes revealed by histopathological examinations. Therefore, the authors thought that the cause of slow clearance is not pathological changes but may be due to the solubility of aluminium in fly ash aerosol. However, the clearance or behaviour of other components, except aluminium, in fly ash aerosol was not checked in this study.

Cross-reference 4

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication meeting basic scientific principles

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Objective of study:

absorption

distribution

Principles of method if other than guideline:

Study on biochemical and histomorphological changes in rat liver upon subacute inhalation exposure.

GLP compliance:

not specified

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Gheru Campus animal house facility, Industrial Toxicology Research Centre, Lucknow, India.
- Age at study initiation: 3 months
- Weight at study initiation: 150 +/- 20 %
- Housing: individually
- Diet (e.g. ad libitum): commercial pellet diet (Dayal Industries, Uttar Pradesh, India).
- Water (e.g. ad libitum):yes
- Acclimation period: yes, no data on period.

Route of administration:

inhalation: dust

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: glass whole body inhalation chambers: 21 L volume, internal diameter 30 cm.
The upper part was detachable, having arrangements for placing a thermometer.
The lower portion had four ports (one was connected with a fluid bed generator for introduction of the fly ash aerosol, the second was for collecting air samples from the chamber, the third was connected to a vacuum line and the fourth was closed airtight.

- Method of conditioning air: Filtered air through a compact air drier
- System of generating particulates/aerosols: Fly ash aerosol was generated by a fluid bed aerosol generator (Intox Products, USA).
- Temperature, humidity, pressure in air chamber: 22-25ºC, 50 - 55 %
- Air flow rate: 15 L/min
- Method of particle size determination: The particle size distribution was measured using a stainless steel cascade impactor (Intox Products, USA)

Duration and frequency of treatment / exposure:

4 h/day, 5 days/week, for 4 weeks

Dose / conc.:

14.4 mg/m³ air

Remarks:

+/- 1.77 mg/m³

No. of animals per sex per dose / concentration:

6

Control animals:

yes, sham-exposed

Positive control reference chemical:

none

Details on study design:

Exposure group 1: 6 male rats exposed to fly ash 4 h daily, 5 days a week for 4 weeks at 14.4 mg/m3 in a whole body inhalation chamber
Exposure group 2: 6 male rats exposed to fly ash 4 h daily, 5 days a week for 4 weeks at 14.4 mg/m³ in a nose only inhalation chamber
Exposure groups 3 and 4: control groups, exposed to compressed air

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: After termination of the study, blood, lungs, livers and kidneys were sampled.
- Time and frequency of sampling: once, after termination
- Other:
Serum ALP, GOT and GPT were analyzed.
Amounts of Cd, Cr, Cu, Mn and Pb were measured by inductively coupled plasma emission spectrophotometry in tissue samples.

GROSS PATHOLOGY:
No data
HISTOPATHOLOGY:
Lungs, liver and kidneys were fixed and stained with HE.

Statistics:

Student's t-test. p < 0.05

Details on distribution in tissues:

The results indicate a statistically significant accumulation of Cd, Cu, Mn and Pb in liver and kidneys after a 28 day inhalation exposure to fly ash.

Details on excretion:

not analysed

Metabolites identified:

not measured

Table 1

Effect of fly ash inhalation (4h/day, 5 days a week) for 28 days on organ weights (g)

(Data represent mean ± SE of the two control and the two exposed groups, comprising 6 rats in each group)

Group	Lungs	Liver	Kidney
Control	1.28 ± 0.15	6.73 ± 0.40	1.28 ± 0.09
Experimental	1.54 ± 0.12	7.80 ± 0.13*	1.14 ± 0.06

* p < 0.05

 

Table 2

Effect of 28 days fly ash exposure through whole body inhalation chamber on serum clinical enzymes

(Data represent mean of 6 rats in each group; no respective data for the nose only exposed group given)

Sl. no.	Serum enzymes	Control	Exposed
1	Alkaline phosphatase (KA units)	30.34	81.47*
2	Serum glutamate oxalacetate transaminase (units/mL)	15.35	15.81
3	Serum glutamate pyruvate transaminase (units/mL)	11.63	14.61*

* p < 0.05

 

Absorption and Distribution of Cd, Cr, Cu, Mn and Pb after inhalation exposure to fly ash:

Table 3

Distribution of metals in various organs after inhalation of fly ash for 28 days using whole-body inhalation chamber

(Data represent mean of 6 rats in each group)

Metals (μg/ g wet tissue)	Group	Lungs	Liver	Kidney
Cd	Control	0.18	0.06	0.17
	Experimental	0.95	0.12	0.34*
Cr	Control	0.62	0.17	0.70
	Experimental	1.72^*	0.26	0.71
Cu	Control	5.01	2.35	2.76
	Experimental	10.21	4.16*	12.22*
Mn	Control	1.75	0.73	0.46
	Experimental	4.05	3.14*	0.98
Pb	Control	4.00	2.76	3.02
	Experimental	4.29	2.58	7.53*

* p < 0.05

 

Table 4

Distribution of metals in various organs after inhalation of fly ash for 28 days using nose-only inhalation chamber

(Data represent mean of 6 rats in each group)

Metals (μg/ g wet tissue)	Group	Lungs	Liver	Kidney
Cd	Control	0.19	0.06	0.16
	Experimental	0.86*	0.23*	0.35*
Cr	Control	0.52	0.16	0.70
	Experimental	1.85*	0.25	0.73
Cu	Control	4.51	2.45	2.45
	Experimental	12.30*	3.26	10.49*
Mn	Control	1.72	0.66	0.37
	Experimental	2.65	1.21	0.51
Pb	Control	2.17	3.11	3.21
	Experimental	5.18*	4.14	6.49*

* p < 0.05

Conclusions:

Increased amounts of Cd, Cu, Mn and Pb were found in rat liver and kidneys after 28 days of inhalation exposure to fly ash.

Executive summary:

Fly ash derived from the electrostatic precipitators of Obra Thermal Power Station, Uttar Pradesh, India was used for inhalation exposure of 6 male Wistar rats for 28 days by whole body and nose only administration at a concentration of 14.4 mg/m3. Control animals were exposed to compressed air under identical conditions. After termination of the experiment, blood, lungs, livers and kidneys were sampled for serum enzyme analysis and metal content analysis of Cd, Cr, Cu, Mn and Pb. Organ samples were also histologically analyzed.

Serum ALP and GPT enzyme actitivities were increased in exposed rats. Bioaccumulation of Cd, Cu, Mn and Pb was observed in rat liver and kidneys. Necrotic changes in hepatocytes of the centrolobular areas were observed at histopathological examination.

The results indicate that heavy metals derived from inhalation exposure to fly ash were systemically absorbed and bioaccumulated in liver and kidneys of rats.

Cross-reference 5

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Mar - 12 Aug 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Lysolaje 15, 165 00 Praha 6, závod Koleč u Kladna, Czech Republic, RČH CZ 21760152
- Age at study initiation: (P) 6-7 weeks
- Weight at study initiation: (P) Males: 260.3-317.3 g; Females: 163.3-200.6 g
- Housing: Animals were housed in plastic cages (40x25x20cm) containing sterilised clean shavings of soft wood. Prior to mating: 2 rats of the same sex in one cage; during mating period: one male and one females in one cage; pregnant females: individually; offspring: together with mother.
- Diet (e.g. ad libitum): ad libitum, sterilised complete pelleted diet for rats in SPF breeding (ST 1 BERGMAN, manufacturer: Mlýn Kocanda, Výroba krmných směsí, Kocanda č.19, 252 42 Jesenice u Prahy)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): ca. 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 18 Mar 2008 To: 15 May 2008

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose in water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing suspensions were prepared daily prior to administration. The test material was suspended in 0.5% methylcellulose in water.

VEHICLE
- Concentration in vehicle: The test material concentration in vehicle was adjusted accounting for body weight in order to achieve a constant administration volume of 1 mL/100 g bw.
- Amount of vehicle (if gavage): 1 mL/100 g bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 7 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): single

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Days 1-14 of the study (before mating), males and females
Days 15-21 of the study (mating period), males and females
Days 22-41 of the study, males
Days 22-50 of the study, pregnant females (days 1-21 of pregnancy)
Days 22-53 of the study, non-pregnant females
Days 51-53 of the study, females in lactation period (days 1-3 of lactation)

Frequency of treatment:

daily

Dose / conc.:

160 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 male and 10 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Determination of Dose Levels:
The doses for this study were chosen with respect to the results of two studies performed at VUOS a.s. test facility before:

1) Study No. 46/07/1: Ashes (residues) - Acute Oral Toxicity - Acute Toxic Class Method, VUOS - CETA Study No. 0810, 2008.
The test substance administered at the dose of 2000 mg/kg caused no death of animals. No clinical signs of intoxication were observed in all six females. Macroscopic changes were not diagnosed during pathological examination in all females in both groups. According to the study results the value of LD50 of the test substance for female rats is higher than 2000 mg/kg of body weight.

2) Study No. 46/07/2: Ashes (residues) - Acute Toxicity (Dermal), VUOS - CETA Study No. 0820, 2008.
The study was performed as limit test: two groups of animals – 5 males and 5 females and the dose of 2000 mg/kg. The test substance was applied on the shaved skin of the test animals in delivered form (moistened with the smallest amount of water) for 24 hours. The test substance applied in the dose 2000 mg/kg of animal weight did not cause the death of animals. No clinical signs of toxicity were observed during the study in all animals. Macroscopic changes were not diagnosed during pathological examination in all animals.
According to the results of study the value of LD50 dermal of the test substance, Ashes (residues), for rats of both sexes is higher than 2000 mg/kg of animal weight.
Doses for the Reproduction/Developmental Toxicity Screening Test - 160, 400 and 1000 mg/kg/day were chosen on the basis of results of the quote study.

- Rationale for animal assignment: Random selection according to the internal rule SOP No. 42. At the beginning of the study the weight variation of animals in groups of each sex should not exceed + 20% of the mean weight.

Positive control:

none

Parental animals: Observations and examinations:

HEALTH CONDITION CONTROL: Yes
- daily - each time before application
- This observation was made in order to record possible clinical effects before and all changes in behaviour of animals. Animals were observed in natural conditions in their cages.

CLINICAL CONTROL OF MALES AND FEMALES: Yes
- daily (after administration)
- This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day. Animals were observed in natural conditions in their cages.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily (after administration) - males and females, as soon as possible after delivery and then daily - pups

BODY WEIGHT: Yes
- Time schedule for examinations:
males - the first day of administration and then weekly;
females - the first day of administration and then weekly,
mothers: during pregnancy: 0., 7th, 14th, 20th day;
mothers: during lactation: 0., or 1st, and 4th day;
pups (litters) - 0., or 1st and 4th

MORTALITY CONTROL: Yes
- All rats were examined for vitality or mortality changes daily.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- In the given day of every week the remainder of pellets of each cage was weighed, the new food was weighed out and the food consumption for the previous week was computed. The average values in groups were calculated for each week of the study. Food consumption for animal/day was calculated from average values of each group.

EXAMINATION OF VAGINAL SMEARS: Yes
- daily in mating period
- The vaginal smears were prepared from all the mated females each morning in the mating period. These smears were examined for presence of spermatozoa. The Day 0 of pregnancy was defined as the day the sperms were found.

Sperm parameters (parental animals):

OBSERVATION OF SPERM
In all males of all groups surviving to scheduled necropsy the sperm parameters were examined: sperm motility, sperm vitality and sperm morphology.

Litter observations:

CLINICAL OBSERVATION OF PUPS
Each litter was examined as soon as possible after delivery and then on the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies was recorded. A change of physical condition and behavioural abnormalities was recorded.

Postmortem examinations (parental animals):

NECROPSIES (all surviving animals)
- males – till 42nd day of study
- mothers – 4th day of lactation
- non-pregnant females – 54th – 56th day of study

PATHOLOGICAL EXAMINATION
Males and non-pregnant females were killed at the end of the application period and parental females were killed on the 4th day of lactation. Then they were macroscopically examined for any structural abnormalities or pathological changes with special attention to the organs of the reproductive system. All macroscopic abnormalities were recorded.


BIOMETRY OF ORGANS
Absolute weights of two testes, one epididymis, prostate gland and pituitary gland were recorded in males and absolute weight of ovaries; uterus (incl. uterine tube and cervix) and pituitary gland were detected in females or mothers. Afterwards the relative weight of organ were computed according to the following formula: relative weight of organ = absolute weight of organ x 100/ necropsy body weight. Necropsy body weight was measured just before euthanasia.


HISTOPATHOLOGICAL EXAMINATION
The following tissues and organs were collected from all males and females at necropsy and fixed in buffer 4% formaldehyde solution (v/v) for further histopathology evaluation: pituitary gland, coagulation gland, prostate gland, seminal vesicles, two testes and one epididymis (fixed in Davidson´s solution), cervix of uterus, ovaries, uterus and vagina.
Histopathological examination from all animals of the control and the high dose group were performed. The target organs at medium and low dose group were not investigated, because treatment-related changes at the high dose were not found.
For histopathological processing the routine histological paraffin technique with synoptic haematoxylin-eosin staining were used.

Postmortem examinations (offspring):

NECROPSIES
– 4th day of lactation

CLINICAL OBSERVATION OF PUPS
Each litter was examined as soon as possible after delivery and then on the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies was recorded. A change of physical condition and behavioural abnormalities was recorded.


PATHOLOGICAL EXAMINATION
Pups dead or pups killed before the day 4th day of lactation, were sexed and externally examined if practically possible, the stomach were examined for the presence of milk. All survivors pups were killed 4th day of lactation, they were sexed and subjected to external examination of the cranium, and macroscopic examination of the thoracic and abdominal tissues and organs. All macroscopic changes were recorded.

Statistics:

The ANOVA test - Analysis of Variance (a part of software QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of body weight, organs weight and some reproduction parameters: control group with vehicle was compared with three treated groups.

Reproductive indices:

For each of parental females the following parameters were counted:

Pre-implantation loss = (number of corpora lutea - number of implantations)
Post-implantation loss = (number of implantations – number of live births)
Post-natal loss = (number of live births – number of live at postnatal day 4)

For each dose group reproduction parameters will be counted:

Percentage of mating = (number of females mated / number of females paired) x 100
Fertility index = (number of pregnant females / number of females paired) x 100
Conception index: = (number of pregnant females / number of females mated) x 100
Gestation index: = (number of females giving birth to live pups / number of pregnant females) x 100

Offspring viability indices:

For each dose group reproduction parameters will be counted:
Percentage of postnatal loss (new-born death): = (number of dead pups on day 4 post partum / number of live pups at first check of litter) x 100
Viability index: = (number of live pups on day 4 post partum / number of pups born alive) x 100

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

HEALTH CONDITION CONTROL
Males
There were no changes in health condition during all the study.

Females/Mothers
There were no changes in health condition during all the study. (0 – no clinical changes).

CLINICAL CONTROL OF MALES AND FEMALES/MOTHERS
Males
There were no clinical changes during all the study. (0 – no clinical changes).

Females/Mothers
There were no clinical changes during all the study. (0 – no clinical changes).

MORTALITY
Males
There were no unscheduled deaths during all the study.

Females/Mothers
There were no unscheduled deaths during all the study.

FOOD CONSUMPTION
Males
At the all dose levels average food consumption was lower than in the control from the 1st week to the 2nd week of study. The changes were differentafter mating in the 4th and 6th weeks of study food consumption was higher of the dose level 400 and 1000 mg/kg/day. Dependence on dose level was observed.

Females
At all dose levels average food consumption was relatively well-balanced with the control. Dependence on dose level was not observed.

Mothers
Average food consumption during the gestation was well-balanced at treated groups compared to the control group. Only in the 20 days of gestation at the dose level 400 mg/kg/day food consumption was slightly increased. On the 4th day of lactation of the all treated mothers food consumptiondecreased. Dependence on dose level was observed at the end of study.

BODY WEIGHT
Males
Since the 1st week the growth curves of all treated animals was slightly under control growth curve. At the dose level 1000 mg/kg/day statistically significant difference of body weight (decreased about 28 grams- compared to control group) was recorded at the end of study.

Females
The body weight of females at the all dose levels was relatively well-balanced with the control. Statistically significant differences of body weight before mating were not recorded.

Mothers
The body weight of mothers at the all treated groups was relatively well-balanced with the control during the gestation and during the lactation. Of the body weight on the 4th day of lactation of treated mothers statistically significant differences were not recorded.

BODY WEIGHT INCREMENT
Males
Average body weight increment of males at the all dose levels were relatively well-balanced with the control animals.

Females
Average body weight increment of females at the all dose levels were relatively well-balanced with the control animals.

Mothers
Average body weight increment of treated mothers during the gestation and during the lactation was well-balance in comparison with control mothers during this period. Only 4th day of gestation the body weight increment of the dose level 1000 mg/kg/day was slightly under the control and others dose levels too, but not statistical significance. Dependence on the dose level was observed in body weight increment of mothers.

BIOMETRY OF REPRODUCTIVE ORGANS
Males
Absolute and relative weights of all observed organs were not balanced at the all dose levels compared to control group.
Animals at the dose level 1000 mg/kg/day showed increase of absolute and relative weight of all reproductive organs: testes, epididymis and prostate gland (without statistical significance). Only the absolute pituitary gland weight at this dose level showed decrease with statistical significance. At the dose levels 160 and 400 mg/kg/day slightly increased relative weight of testes, epididymis and prostate gland (without statistical significance). Slight (statistically no significant) decrease of pituitary gland was recorded also at the dose level 400 mg/kg/day.
Dependence on dose level was observed.

Mothers
In mothers of the dose level 400 mg/kg/day increase of absolute and relative weight of ovaries and uterus were detected. Weight of uterus was increased and the increase was statistically significant. Relative weight of ovaries at the dose level 400 mg/kg/day and relative weight of uterus at the doselevels of 160 and 1000 mg/kg/day was increased without statistical significance.
Relative weight of pituitary gland was relatively well-balanced in control and treated females, absolute weight was very slightly lower.

PATHOLOGY-MACROSCOPIC FINDINGS
Males
Incidence of affected animals is expressed in numeric form and ranged in sequence of dose levels 0-160-400-1000 mg/kg/day further in the text.
In 9-10-10-10 males no macroscopic changes were found out.
The following macroscopic affections in reproductive organs were detected only in one male of control group – it was atrophy of testes and epididymis.

Females
In 1-1-2-2 females no macroscopic findings were occurred.
Dilatation of uterus (horns with pellucid liquid) was recorded in 1-0-1-1 females.

Mother
In all mothers no macroscopic findings were occurred.

PATHOLOGY - MICROSCOPIC FINDINGS
Males
Incidence of affected animals is expressed in numeric form and ranged in sequence of dose levels 0-1000 mg/kg/day further in the text. Of dose levels 160 and 400 mg/kg/day was not examined.
Incidence of pathological affections of reproductive system in male genital tract was sporadic. All stage of spermatogenesis in detailed examination of spermiogenesis in testes in 9 -10 males were detected.
Diffuse atrophy of germinal epithelium of testes, Sertoli cells in testes tubules and epididymis dystrophy with occurrence of oligospermia was found out in 1-0 male and focal necrosis of testes was found in 0-1 male. Histopathological findings in prostate gland were more often: oedema 1-1; dystrophy 1-0 and focal inflammation of interstitium in 2-0 males. In the other organs - seminal vesicles and pituitary gland - pathological changes were not found. Dependence on dose level was not observed.

Females
Incidence of pathological affections of reproductive system in female genital tract the following affections were detected of ovary: follicular cyst in 0-1-0-0, degeneration of follicles 0-0-1-2 and Sertoli-like tubules in 0-1-2-0 females and in uterus: 0-0-1-0 hydrometra.

Mothers
Incidence of pathological affections of reproductive system in female genital tract the following affections were detected only of ovary: follicular cysts in 2-0-3-0, degeneration of follicles in 1-2-1-1 and Sertoli-like tubules in 2-2-1-3 mothers.

REPRODUCTION DATA OF MALES
No treatment related effects on sperm examination: sperm motility, sperm vitality and sperm morphology were observed.
The best sperm motility - fast, progressive motility and the best sperm vitality - fast were observed at the dose levels 160 and 400 mg/kg/day. In sperm morphology - major sperm abnormalities were observed in control males were flattened head, bent of tail and abnormal bent neck. The lowest number of abnormalities was found in males of the dose level 160 mg/kg/day. Marked dependence on dose level was not observed in any of changed parameters.

REPRODUCTION DATA OF MOTHERS
Mating
Number of paired females and accompanying number of mated females was identical. Number of pregnant females and accompanying number of mothers was identical too. Duration of pregnancy of treated groups was similar to control group. Marked dependence on dose level was not observed inany changed parameters.

Pregnancy
The differences among the groups were found in some parameters. Higher number of corpora lutea at the all treated mothers was recorded. Decrease in the number of implantation and increase of resorptions were found at the higher dose levels. Duration of pregnancy of treated groups was similar to control group.
At the all treated mothers the number of live pups the 1st and the 4th day was lower then in control mothers. The differences among the treated groups were very low.
At the number of live pups per litter a statistical analysis was performed using Mann-Whitney U-test (significance level 0.05). Statistically significant differences were not found.

Reproduction indexes
The differences of among the groups were found in some indexes, but these differences were very low. Percentage of post-implantation loss was slightly increased at the middle dose level 400 mg/kg/day. Slight decrease of percentage of mating was detected only at the dose levels 400 and 1000 mg/kg/day. Conception and gestation index were identical in all groups. Viability index and postnatal loss were well-balanced.

Dose descriptor:

NOEL

Effect level:

160 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects were noted in the parental animals at 160 mg/kg bw/day.

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related and adverse effects were observed in parental animals up to and including the highest tested dose level.

MORTALITY
- Only one pup death after birth at the dose level 400 mg/kg/day was observed.

BODY WEIGHT
- The body weight of litter in dose levels 160 and 400 mg/kg/day was lower as compared to the control and at dose level 1000 mg/kg/day was higher then control weight of pups.

DEVELOPMENT AND ABNORMAL
- No differences in development of pups were observed in treated groups compared with the control group.

PATHOLOGY OF PUPS
- Macroscopic examination of the thoracic and abdominal tissues and organs were carried out. All control pups and all treated groups had no macroscopic pathological changes.

TOTAL NUMBER AND SEX RATIO
- Average total number of pups was lover at dose level 1000 mg/kg/day than at the control group. Of the dose levels 160 and 400 mg/kg/day the total number of pups was similar to the control pups. The number of males and females per litter at all treated groups was similar to the control group. Statistical analysis at number of pups was performed using Mann-Whitney U-test (significance level 0.05). Statistically significant differences were not found (table No.27).

Dose descriptor:

NOEL

Generation:

F1

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects were observed in pups up to and including the dose level of 400 mg/kg bw/day.

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse and treatment-related effects were observed in pups up to and including the highest tested dose level

Reproductive effects observed:

no

      Table 27

Parameters of pups (mean of group)
Dose level	0	160	400	1000
Mortality	0	0	1	0
Clinical changes	0	0	0	0
Macro-changes	0	0	0	0
Abnormal pups	0	0	0	0
Weight of litter 1^stday	64.54	↓57.98	↓60.44	↓58.21
Weight of litter 4^thday	106.8	↓96.42	↓97.73	↓99.90
Weight of pups 1^stday	5.61	5.86	5.72	↑5.99
Weight of pups 4^thday	9.23	9.75	9.37	↑10.28
Number of pups 1^stday	11.50	9.89	10.57	↓9.71
Sex of live pups 1^stday	5.6M + 5.9F	5.6M + 4.3F	5.4M +5.2F	5.4M + 5.0F
Number of pups 4^thday	11.50	↓9.89	10.40	↓9.71
Sex of live pups 4^thday	5.6M + 5.9F	5.6M + 4.3F	5.1M + 4.6F	5.1M + 4.6F

       Note: M = male pups;  F = female pups.  

Conclusions:

Administration of the test substance Ashes (residues) did not affect mortality and health condition of males, females, mothers and pups; sperm motility, vitality, morphology of males, stage of the cell cycle in males spermatogenesis; body weight of mothers during gestation, development of mating and pregnancy; vitality and development of pups, distribution of sexes in litter, pathological examination of pups; macroscopic appearance and microscopic structure of reproduction organs of both sexes.
Statistically significant effects manifested in: decreased body weight in males (1000 mg/kg/day), but no influence on body weight gain observed; decrease in absolute weight of male pituitary gland (1000 mg/kg/day), but not in its relative weight; and increased weight of mothers uterus (400 mg/kg/day), but no dose-response observed.
Statistically non-significant effects manifested in: decrease in food consumption of mothers (1000 mg/kg/day), increased weight of reproduction organs in both sexes at all dose levels.
Negative effects on fertility manifested in: increased post-implantation loss at the dose levels 400 mg/kg/day and decrease of number of pups at dose level 1000 mg/kg/day, but no statistical significance and no dose-response observed.
Based on the study results, no effects were noted in the parental animals at 160 mg/kg bw/day, which was therefore considered a No-Observed-Effect-Level (NOEL). At the dose levels 160 and 400 mg/kg bw/day no negative effects were detected in pups. Thus, 400 mg/kg bw/day was considered a foetal NOEL. Changes observed in the parental animals and pups at 400 and 1000 mg/kg bw/day were not consistent enough and/or not statistically significant in order to be considered as adverse to reproduction. Therefore, the No-Observed-Adverse-Effect-Level was established at 1000 mg/kg bw/day.

Materials and methods

Test animals

Species:

rat

Results and discussion

Results (fetuses)

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified