Ashes (residues), coal

Toxicity to reproduction

Administrative data

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because (i) the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), (ii) 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 (iii) there is no or no significant human exposure

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING

Regulation (EC) No. 1907/2006, Annex X, 8.7.3 Column 1, states that an extended one-generation reproduction toxicity study (EOGRTS, OECD 443, standard configuration) is required to fulfil the standard requirements for reproductive 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, an extended one-generation toxicity study for assessment of reproductive toxicity 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, 8.7.3. Column 2, the registrant has considered the need to perform an extended one-generation reproduction 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 due to the rapid clearance. 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 bw/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 in aluminium concentration between exposed groups and controls, indicating a low absorption of the 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 reproductive and fertility parameters
Information on reproduction 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 an earlier study where fly ash derived from coal combustion was fed to cows over a period of 3 years, pregnancy rates, conception, parturition and body weight development were not disturbed by the oral administration (Herrmann, 1955). Because treated animals showed a slightly higher milk production rate than control animals, the general condition of treated animals was even considered to be better than that of the controls. Necropsy results did not reveal major pathological changes due to fly ash administration.
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 for 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 reproduction and fertility is available from a Reproduction / Developmental Toxicity Screening Test performed according to OECD guideline 421 and a 2-year feeding study with cows over a period of 3 years. The results of these 2 studies did not show any potential for reproduction 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 reproduction 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 an extended one-generation reproduction toxicity study (standard configuration or with additional modules) 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.

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

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

Reproductive effects observed:

not specified

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

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.

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

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Species:

rat

Abnormalities:

not specified

Developmental effects observed:

not specified

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

developmental toxicity

Remarks:

non-rodent species

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 2, states that a pre-natal developmental toxicity study (OECD 414) shall be initially performed on one species. A decision on the need to perform a study at this tonnage level or the next on a second species should be based on the outcome of the first test and all other relevant available data.
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 generally considered the need to perform a developmental toxicity study (both for rodent and non-rodent species). 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 exposed 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 SiO2and 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 for 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 that 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 (both with rodent and with non-rodent species) 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.

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

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Species:

rabbit

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ashes (residues), coal

EC Number:

931-322-8

Cas Number:

68131-74-8

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Ashes (residues), coal

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Details on results (P0)

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.

Effect levels (P0)

1

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.

2

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.

Results: F1 generation

Details on results (F1)

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).

Effect levels (F1)

1

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.

2

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

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

      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.  

Applicant's summary and conclusion

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.