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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Reference 1

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

Reference 2

Endpoint:

extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 3

Endpoint:

extended one-generation reproductive toxicity - with both developmental neuro- and immunotoxicity (Cohorts 1A, 1B without extension, 2A, 2B, and 3)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

The conditions according to ANNEX IX and X 8.7.3. , Column 2, requiring to perform this test are not fulfilled:


An Extended One-Generation Reproductive Toxicity Study including cohorts 2A/2B (developmental neurotoxicity) and/or cohort 3 (developmental immunotoxicity) are not required as particular concerns on (developmental) neurotoxicity or (developmental) immunotoxicity cannot be justified by any of the following: — existing information on the substance itself derived
from relevant available in vivo or non-animal approaches (e.g. abnormalities of the CNS, evidence
of adverse effects on the nervous or immune system in studies on adult animals or animals exposed prenatally), or — specific mechanisms/modes of action of the substance with an association to (developmental) neurotoxicity and/or (developmental) immunotoxicity (e.g. cholinesterase inhibition or relevant changes in thyroidal hormone levels associated to adverse effects), or —existing information on effects caused by substances structurally analogous to the substance being studied, suggesting such effects or mechanisms/modes of action. [No such concerns were discovered for Ash.]

Reason / purpose for cross-reference:

data waiving: supporting information

Remarks:

Thyroidal hormones measured in OECD 414 Study

Reference 4

Endpoint:

reproductive toxicity, other

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not reported

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Pastures and hay production areas for sheep treated with electrofilter ash from coal power plants were experimentally cultivated and effects on sheep health and breeding studied.

GLP compliance:

no

Limit test:

no

Species:

other: sheep

Strain:

other: Local Slovenian sheep breed

Sex:

male/female

Details on test animals or test system and environmental conditions:

Group size: 10 older ewes and 10 one year old ewes. Detailed examination of health status, hematology and clinical chemistry, and dehelminthzation were carried out prior to study.

Route of administration:

oral: feed

Details on exposure:

Exposure via grass grown on electrofilter ash treated pastures. Ash deposits were spread out in dumping areas in the depth of 2-4 meters followed by several months of watering and compression to stabilize the ground. The second phase included conveying, spreading, compression and preparation of earth deposit at the depth of 0.5 meters, seeding of grass and corresponding one year maintenance to make the pasture suitable for grazing and production of hay for winter nutrition.

Details on mating procedure:

Mating of all ewes at the end of first experimental year by the same ram.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of Pb, Cd, Cu and As, and radionuclides K40 and Cs137 in muscle, liver and kidney. Analyses carried out according to AOAC 1995 (Official methods of analysis of the AOAC, 16th edition, AOAC, Washington, D.C.

Duration of treatment / exposure:

2 years, mating at the end of the first experimental year

Frequency of treatment:

Daily via feed

Details on study schedule:

Groups of 20 ewes were maintained on pastures for one year after which two ewes from each group were euthanized for analysis. The rest of the ewes were mated and the body weight development of the offspring were monitored for 75 days. At the end of the second year three ewes from each group were euthanized and analysed. The follow-up of the remaining animals was published separately (Juntes et al., 2007).

Remarks:

Doses / Concentrations:
Control pasture and electrofilter ash treated pasture
Basis:
other: Control pasture and electrofilter ash treated pasture

Control animals:

yes

Positive control:

no

Parental animals: Observations and examinations:

Veterinary examination was carried out 4 times per year and included clinical examination, body weight, hematology and clinical chemistry.

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

No data

Litter observations:

Body weights at birth and at the age of 30, 45 and 75 days.

Postmortem examinations (parental animals):

Body weight , hematology, clinical chemistry, analysis of Pb, Cd, Cu and As, and radionuclides K40 and Cs137 in muscle, liver and kidney.

Postmortem examinations (offspring):

Reported separately (Juntes et al., 2007)

Statistics:

No data on methods

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slightly higher body weight gain in the treated group. No data for food consumption.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Slightly higher body weight gain in the treated group. No data for food consumption.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Body weight and food consumption (parental animals): Slightly higher body weight gain in the treated group due to slightly lower nutritional value of the control pastures; food consumption: no data. The nutritional value of grass was analysed four times per year and proved to be slightly but non-significantly better in the electrofilter ash treated pastures.

Test substance intake (parental animals): No differences between the treatment groups in concentrations of Pb, Cd, Cu and As, or radionuclides K40 and Cs137 in muscle, liver and kidney.

Dose descriptor:

other: No data

Based on:

other: analysis of Pb, Cd, Cu and As, and radionuclides K40 and Cs137 in muscle, liver and kidney

Sex:

female

Basis for effect level:

other: No data

Remarks on result:

not measured/tested

Remarks:

Effect level not specified

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Reproductive effects observed:

not specified

No significant differences in health status, the studied parameters or the reproductive performance between experimental and control animals were found.

Conclusions:

Maintenance of sheep on electofilter ash treated pastures had no effects on health or reproduction on sheep.

Executive summary:

In order to find out appropriate means to utilize electrofilter ash dumping areas, pastures and hay production areas for sheep were experimentally cultivated and effects on sheep health and breeding studied. Ash deposits were spread out in dumping areas in the depth of 2-4 meters followed by several months of watering and compression to stabilize the ground. The second phase included conveying, spreading, compression and preparation of earth deposit at the depth of 0.5 meters, seeding of grass and corresponding one year maintenance to make the pasture suitable for grazing and production of hay for winter nutrition. Experimental and control groups of 10 older ewes and 10 one year old ewes grazed on the pasture cultivated with electrofilter ash or ordinary pastures in the surrounding area, respectively. Health condition, body weight, hematology, clinical chemistry and reproductive performance were monitored during the study. Animals from each group were slaughtered after one or two years and hematological, clinical chemistry, gross pathological, histopathological and analytical chemistry analyses were carried out. No significant differences in health status, the studied parameters or the reproductive performance between experimental and control animals were found.

Reference 5

Endpoint:

fertility, other

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Study period 10 Dec 1950 - 14 Apr 1954 (main study 3 years for 6 cows followed by an off-dose period of 4 months for 3 cows)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Doctoral dissertation. Scientific publication with well-documented data.

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

Fertility study in grazing cows.

GLP compliance:

no

Limit test:

no

Species:

other: cattle

Strain:

other: Schwarzbunte Niederungsrasse

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Oldenburg, Germany (a region without no known fly ash residues from coal combustion)
- Age at study initiation: 2.75 - 6.75 years
- Weight at study initiation: 535 - 710 kg
- Housing: Cattle pen and grazinge
- Diet (e.g. ad libitum): fodder root vegetables, hay and straw (ad libitum) supplemented with mash and fortified fodder.

At arrival a careful clinical examination was carried out for all animals. All animals were in good nutritional state.

ENVIRONMENTAL CONDITIONS
Animals were kept on farm. They had no contact with other cattle.
The cattle pen was cleaned regularly, and it was dry and ventilated with fresh air.

IN-LIFE DATES:
Exposure period: From: 10 Dec 1950 To: 15 Dec 1953
Off-dose observation period: From: 16 Dec 1953 To: 14 Apr 1954

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks:

Test material mixed with feed.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed with the feed.
Animals were fed twice a day using individual boxes to assure the complete 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. On pasture the fly ash was mixed with grass cuts.

Details on mating procedure:

All animals were naturally mated. Eight out of 9 animals were pregnant at initiation of the experiment.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Exposure period: 3 years

Frequency of treatment:

Twice a day

Details on study schedule:

not applicable

Remarks:

Doses / Concentrations:
300 g / animal / day (about 0.43 g/kg bw/day [assuming an average body weight of 700 kg])
Basis:
nominal conc.
fed to the animals of group 1 and group 2

Remarks:

Doses / Concentrations:
1500 - 1800 g / animal / day (about 2.1-2.6 g/kg bw/day)
Basis:
nominal conc.
fed to the animals of group 3. Additionally approx. 300 g / animal / day.

Remarks:

Doses / Concentrations:

Basis:
nominal conc.
resulting from calculated amounts of coal fly ash precipitation to the grazing land in the experimental geographic area and the daily intake of the grazing test animals.

No. of animals per sex per dose:

9 cows were divided into 3 groups (3 animals/group):
Group 1 (animal # 1-3): served as concurrent no treatment control animals during the first two years and received 300 g fly ash/day/animal (about 0.43 g/kg bw/day) during the 3rd year of the experiment
Group 2 (animal # 4-6): 300 g fly ash/day/animal (about 0.43g/kg bw/day) during the first and second year of the experiment, during the 3rd year no treatment.
Group 3 (animal # 7-9): 1500 - 1800 g fly ash/day/animal (about 2.1-2.6 g/kg bw/day) during all 3 years of the experiment

Control animals:

other: yes, concurrent no treatment (for the first two years of the exposure period)

Details on study design:

To exclude possible individual differences in the sensitivity for coal fly ash, a feeding strategy conversion for groups 1 and 2 was performed: Animals of group 1 received fly ash only in the third year, whereas the animals of group 2 received the fly ash only in the first two years of the 3-year duration.

Positive control:

no

Parental animals: Observations and examinations:

REGULAR CLINICAL OBSERVATIONS: made:
First year: every 10 days
Second year: every 14 days
Third year: every 4 weeks

DETAILED CLINICAL OBSERVATIONS: Yes, including parasitological, bacteriological and serological analyses:
1) at arrival
2) at the end of the second year
3) at the end of the exposure period of 3 years
4) at the end of the off-dose period

BODY WEIGHT: measurements:
First year: every 10 days
Second year: every 14 days
Third year: every 4 weeks

MILK PRODUCTION:
Milk production of every cow was recorded daily.

Sperm parameters (parental animals):

not applicable

Litter observations:

The parturition of all calves was described, but the calves were not further mentioned in the report.

Postmortem examinations (parental animals):

SACRIFICE
- Maternal animals: 2 animals of each group were euthanised at the end of the exposure period, the remaining animals were euthanised at the end of the off-dose observation period of 4 months.

GROSS NECROPSY
All animals were macroscopically examined.

HISTOPATHOLOGY / ORGAN WEIGHTS
Samples from: Liver, kidneys, pyloric region of the stomach, mesenterial lymph nodes, small and large intestine.

Postmortem examinations (offspring):

not examined

Statistics:

not applicable (n=3)

Reproductive indices:

not examined

Offspring viability indices:

not examined

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

See the tables below.
Brucella abortus, Tuberculosis, liver Trematodes and other infectous diseases (no data about viral infections) and complications following accidental
injuries (following natural matings, for instance) confound the interpretation of the individual observations. However, no dose-dependent changes of the sexual behaviour, pregnancy rate or development of conceptus and parturition were noted. The general condition of the animals was not affected by the test substance administration, as the milk production was even slightly higher in the both treated groups compared to the controls.

Although treatment of group 1 and 2 animals was reversed after 2 years the pathological findings observed at the end of the 3-year treatment period did not show treatment-related effects. No major pathological alterations were observed in the gastro-intestinal mucosa or other potential target organs. Therefore, in conclusion, long-term oral administration of the fly ash at up to 2100mg/kg/day to female cows for 3 years was not associated with toxicologically relevant pathological alterations.

Dose descriptor:

NOAEL

Effect level:

2 100 mg/kg bw/day

Sex:

female

Basis for effect level:

other: overall effects clinical signs; mortality; body weight; macroscopic pathology; histopathology; pregnancy rate dose level was calculated based on average body weight of 700 kg

Mortality / viability:

no mortality observed

Description (incidence and severity):

2 calves in group 1 and one calf in group 2 were born with an infection. This was not considered to be related to the treatment. One calf in group 1 was born prematurely. No reason for this could be identified.

Reproductive effects observed:

not specified

Table 2: Chemical analysis on coal combustion fly ash (Scholven)

Component	Laboratory A (51 samples)		Laboratory B*	Laboratory C*
	Mean (%)	Range (%)	(%)	(%)
Ignition loss	10.01	3.89-20.60	11,76	8.93
SiO2	41.59	36.41-47.39	44.7	38.5
Al2O3	21.72	14.04-31.50	20.6	20.0
Fe2O3	14.25	6.33-21.96	9.47	14.9
MgO	2.67	0.93-4.12	2.49	2.3
CaO	4.84	2.47-10.03	6.72	5.3
SO3	1.19	0.46-2.82	0.64	1.0
P2O5	0.53	0.12-1.31	0.18	0.51
CuO	0.0075*	-	0.0069	0.0085
ZnO	-	-	0.0078	0.040
PbO	0.0011*	-	<0.0009	0.0014
MnO	-	-	-	0.15
CoO	-	-	-	0.0011
F	-	-	-	0.008
B2O3	-	-	-	0.0021
As2O3	0.0012*	-	0.0012	0.0013
Sulphite S	-	-	0.0012	-

*only 1 sample analysed

Table 3: Individual observations

Animal	Off-dose period	Age, body weight and pregnancy status at initiation of exposure	Clinical signs	Pregnancy rate^a	Sexual behaviour	Body weight (in kg at similar stages of pregnancy, measured in 1950, 1951, 1952 and 1953)	Milk production (kg /d; mean values)
1	no	5 years, 657 kg, 5^thmonth	No clinical signs of toxicological relevance were observed.	4/6	No disturbances of conception and parturition.	657, 697, 720, 744	10.9
2	yes	2.75 years, 535 kg, 9^thmonth	No clinical signs of toxicological relevance were observed.	4/4	No disturbances of conception and parturition up to the 3^rdbirth in 1953. During the 4^thpregnancy an infection with Brucella abortus occured, followed by a preterm delivery in the 8^thmonth of pregnancy.	535, n.d., 590, 682	10.7
3^b	no	5 years, 585 kg, not pregnant	No clinical signs of toxicological relevance were observed.	3/3	No disturbances of conception and parturition up to the 2^ndbirth in April, 1953. In November 1953 an abortion occured after a Brucella infection.	n.d., n.d., 554, 709	11.6
4	yes	4.75 years, 625 kg, 5^thmonth	Observed clinical signs (uterus catarrh, bronchial catarrh) couldn´t be associated with fly ash administration, as the milk production was highest during fly ash administration	3/6	Mastitis due to a Streptococcus infection occured early after experimental start. Infection with Brucella abortus during post exposure period.	625, n.d., 695, 691	11.3
5	no	4.5 years, 645 kg, 9^thmonth	No clinical signs of toxicological relevance were observed.	4/6	No disturbances of conception and parturition up to the 3^rdbirth in 1953. During the 4^thpregnancy an infection with Brucella abortus occured, followed by an abortion in the 8^thmonth of pregnancy.	645, 636, 686, n.d.	13.7
6	no	5 years, 710 kg, 9^thmonth	No clinical signs of toxicological relevance were observed. The occuring mastitis could not be associated with fly ash administration.	4/4	No disturbances of conception and parturition up to the 3^rdbirth. During the 4^thpregnancy the animal was euthanised after an accident with fracturing of the lumbar vertebrae.	710, 727, n.d., 812	12.9
7	no	5.5 years, 627 kg, 7^thmonth	No clinical signs of toxicological relevance were observed.	4/4	Abortion in February 1951 without evidence of bacterial infection. No disturbances of conception and parturition were observed for the following 3 pregnancies.	627, 683, 685, n.d.	11.9
8	no	5.75 years, 670 kg, 7^thmonth	No clinical signs of toxicological relevance were observed.	4/7	No disturbances of conception and parturition up to the 3^rdbirth.During the 4^thpregnancy the animal was euthanised.	670, 719, 732, n.d.	10.3
9	yes	6.75 years, 682 kg, 9^thmonth	No clinical signs of toxico logical relevance were observed.	4/4	No disturbances of conception and parturition during all 4 pregnancies and deliveries.	682, 765, 784, 809	12.3

n.d. = not determined

^anumber of pregnancies / number of natural matings

^bAnimal n:o 3 was incorporated to the experiment in March 1952 as a replacement for another animal, which was euthanised due to diseases of the lung and intestine. This was not considered treatment-related, as this animal belonged to the control group.

 

 

Table 4:Individual macroscopic and microscopic pathological findings

Ani- mal / Treat- ment	Necropsy findings	Histological findings^ain kidney, liver, stomach, lymph nodes and intestine samples
1	infectious diseases: Liver Trematodes, Tuberculosis; changes in claws, considered normal under the given circumstances	no histopathological changes
2	infectious diseases: Liver Trematodes, Tuberculosis, Brucella abortus; changes in claws, considered normal under the given circumstances	liver: high glycogen deposition and fatty changes; kidney: glomerulonephritis, brownish pigmentation in tubular epithelial cells, mild nephrosis; lymph nodes: pigmentation
3	infectious diseases: Liver Trematodes, Brucella abortus; changes in uterus and vagina considered to be a consequence of Brucella infection	no histopathological changes
4	infectious diseases: Tuberculosis, Streptococcus, Brucella abortus	kidney: glomerulonephritis, pigmentation of tubular epithelial cells; liver: fatty changes, centrilobular glycogen deposits
5	infectious diseases: Liver Trematodes, Tuberculosis, Brucella abortus; petechial changes of the gall bladder mucosa, notconsidered toxicologically relevant;	no histopathological changes
6	infectious diseases: Tuberculosis; fracture of the lumbar vertebra; necropsy findings of this animal were omitted, as the animal suffered for 6 weeks from the downer cow syndrome due to the lumbar vertebra fracture.	kidneys: lymphocytic interstitial infiltration; liver: mild perivascular lymphocytic infiltration;
7	infectious diseases: Liver Trematodes; mild erythema^b´in the pyloric mucosa of the abomasum, the amounts of sand in the gastro-oesophageal mucosa was considered normal and there were no defects, bleeding or necrosis in the mucosa of the gastro-oesophageal tract	no histopathological changes with the exception of a single focal fatty change in liver
8	infectious diseases: Liver Trematodes; the amounts of sand in the gastro-oesophageal mucosa was considered normal, and there were no irritation of gastro-oesophageal mucosae	no histopathological changes
9	infectious diseases: Liver Trematodes; the amounts of sand in the gastro-oesophageal mucosa was considered normal; mild erythema in the abomasal mucosa, and there were no defects, bleeding or necrosis in the mucosa of the gastro-oesophageal tract	kidney: chronic glomerulonephritis, focal chronic interstitial nephritis, protein deposition in tubular epithelial cells; liver: hydropic degeneration with cell lysis, mild centrilobular fatty change and glycogen deposits; lymph nodes: pigmentation

^aHistological findings detected in animals 2, 4 and 9 (groups 1, 2 and 3, respectively) were considered to be of no toxicological relevance, because no dose-response relationship was found.

^bThe mild erythemas in some gastrooesophageal tracts were considered to be of no toxicological relevance, as this finding were also observed in 25 out of 30 animals analysed at the butchery of Hannover, Germany.

 

 

Conclusions:

No treatment-related effects were observed. A NOAEL for female cattle is 2100 mg/kg bw/day. Chemical analyses suggest low oral bioavailability of the analysed constituents of coal fly ash.

Executive summary:

Groups of 3 milking cows were exposed daily to coal fly ash mixed with fodder at 0, 300 or 1500-1800 g/day (equivalent with about 0, 430 or 2100-2600 mg/kg bw/day) for 3 years in a non-GLP study. (After 2 years the control group and the low dose group were switched.) Observations included clinical signs, body weight, milk production, reproduction and parturition, chemical analyses of milk, excreta and tissues as well as macroscopic pathology and histopathology. Male reproduction parameters and litters were not examined. No treatment-related effects were observed. A NOAEL of about 2100 mg/kg bw/day was identified. Chemical analyses suggested low oral bioavailability of As, Mn, Pb, Fe, Cu, Zn and Co from coal fly ash.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 100 mg/kg bw/day

Study duration:

chronic

Species:

cattle

Quality of whole database:

Doctoral dissertation from 1955. Reproductive functions studied after chronic high-dose exposure.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A published reproductive study in sheep maintained on pastures treated with electrofilter ash did not indicate toxic effects on ewes, effects on fertility, or effects on body weight development or viability of the offspring.Similarly, no effects on fertility or birth were found in milking cows after oral exposure to coal fly ash at doses up to about 2100 mg/kg bw/day for 2 years (Herrmann, 1955).

Short description of key information:
No effects observed

Justification for selection of Effect on fertility via oral route:
The study were selected, because they addresses the reproductive effects after chronic high dose exposure to coal fly ash and provide additional information onreproductive toxicity of ash.

Effects on developmental toxicity

Description of key information

No effects observed.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Principles of method if other than guideline:

This non-GLP preliminary dose range finding study did not follow a specific OECD guideline, but it was designed to allow selection of appropriate dose levels for the upcoming OECD No. 414 study.

GLP compliance:

no

Species:

rat

Strain:

Wistar

Remarks:

Hannover Wistar

Route of administration:

oral: gavage

Vehicle:

other: 1% aqueous methylcellulose

Details on exposure:

Animals (one control and one test item treated group) were treated daily by oral gavage administration, from Gestation Day 6 (GD6) up to and including GD19 (sperm positive day = day 0 of pregnancy, GD0). Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD20.

Analytical verification of doses or concentrations:

yes

Remarks:

Analysis of test item formulations for concentration and/or homogeneity was performed at the Test Site using an XRF (X-ray fluorescent spectroscopy) method. Top, middle and bottom samples were taken from the test item formulations once during the stud

Details on mating procedure:

Mating Procedure

The oestrus cycle of female animals was examined shortly before start of pairing. After acclimation, the females were paired according to their oestrus cycle with males in the morning for approximately 2-3 hours (1 male: 1 female) until at least 6 sperm positive females/group were attained. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (gestation day 0, GD 0). Sperm positive females were separated and caged individually.

Duration of treatment / exposure:

Animals (one control and one test item treated group) were treated daily by oral gavage administration, from Gestation Day 6 (GD6) up to and including GD19 (sperm positive day = day 0 of pregnancy, GD0). Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD20.

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

14 female animals, 7 mated, pregnant, and evaluated female animals/group, 2 groups (one control and one test item-treated group); 14 male animals for mating; no study-procedures were carried out on the male animals; untreated, proven breeders from Charles River Laboratories Hungary Kft. spare colony were used.

Control animals:

yes, concurrent vehicle

Details on study design:

The objective of the preliminary study was to determine the Maximum Tolerated Dose (MTD) and to assess the effects of the test item on pregnant females and on the developing conceptuses in order to set the dose levels for the main developmental toxicity study.

Animals (one control and one test item treated group) were treated daily by oral gavage administration, from Gestation Day 6 (GD6) up to and including GD19 (sperm positive day = day 0 of pregnancy, GD0). Caesarean sections, necropsy of dams and examination of uterine contents were performed on GD20.

The dose levels were set by the Sponsor in agreement with the Study Director based on the available data, including the results of a 28-day oral toxicity (OECD 407) study, with the aim of inducing toxic effects but no death or suffering at the highest dose (1000 mg/kg bw/day). Control dams were treated with vehicle, 1% aqueous methylcellulose solution, only.

Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Gross macroscopic examination was performed at necropsy. Maternal reproductive parameters associated with uterine examination were evaluated, and the foetuses were weighed and examined for external abnormalities. Placentae were examined macroscopically.

Fourteen sperm positive females were included in the study, seven in each group. The number of confirmed pregnant dams was 7 in each group.

Maternal examinations:

Parameters monitored during the study included mortality and clinical observations, body weight, body weight gain and individual food consumption. Gross macroscopic examination was performed at necropsy. Maternal reproductive parameters associated with uterine examination were evaluated. Placentae were examined macroscopically.

Ovaries and uterine content:

The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses.

The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted, the number and percentage of pre- and post-implantation losses were calculated. The degree of resorption was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.

Fetal examinations:

Each foetus was weighed individually (accuracy ±0.01 g) and subjected to external examination. The gender of foetuses was determined according to the appearance of the anogenital distance.

All external abnormalities found during the foetal examinations were recorded; photographic records were made additionally.

Statistics:

The statistical evaluation was performed with SAS v9.2 in case of Provantis v.9, or SPSS PC+4.0 (SPSS Hungary Kft, Budapest) in the case of data tabulated in Excel, by an appropriate statistical method. In case of the SAS v9.2 software package (within the validated Provantis system) the following decision tree was applied automatically for statistical evaluation of numeric data. The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where the tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. In case of a positive result, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of P<0.05 or P<0.01 as appropriate. If the Shapiro-Wilk or Levene tests showed significance on the data, then a non-parametric analysis was used. A Kruskal-Wallis analysis of variance was used after Rank Transformation. In case of a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of P<0.05 or P<0.01 as appropriate. In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. For positive results, the inter-group comparisons were performed using Mann-Whitney U-test. The Chi squared test was used for comparing group incidences against the controls.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was significant difference in the number of foetuses with retarded body weight for males in the Top dose group compared to the Control group. Taking into account the historical control data, the finding was considered biologically non- significant.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Conclusions:

In conclusion, Mixed ash, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 1000 mg/kg/day, was not associated with maternal toxicity, embryotoxicity, foetotoxicity or any teratogenicity effect.

In selecting dose levels for the following OECD 414 study, 1000 mg/kg body weight/day is suitable as a Top dose. Using a factor of 3, the dose levels suggested are 1000, 300 and 100 mg/kg bw/day for the main OECD 414 study.

Executive summary:

No animal was found dead during the study.

No clinical signs, test item-related effects on body weight or food consumption parameters, were observed in the study.

No test item related macroscopic findings were present at necropsy in any of the animals in the study.

There was no statistically significant difference in foetal death in any test item treated groups compared to the control. The mean number of corpora lutea was comparable with the control in all test item treated groups. No significant differences were noted in preimplantation loss or number of implantations of the test item treated groups when compared to the control.

The early and the late embryonic loss values of the test item treated groups, and the number of dead foetuses were comparable with control. There was no statistically significant difference in the postimplantation loss or total intrauterine mortality between the test item treated and control groups.

No toxicologically relevant adverse effect of the test item was observed on the foetal parameters, including mean foetal weight and in the sex distribution in the Top dose group. There was significant difference in the number of foetuses with retarded body weight for males in the Top dose group compared to the Control group.

Placentas were normal for all observed animals.

There was no test item related effect on the external development of foetuses in the study.

Reference 2

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Species:

other: Study waived

Reference 3

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Species:

rabbit

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 January 2020 - 24 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes

Remarks:

GLP compliant, according to the Hungarian Good Laboratory Practice Regulations of 42/2014. (VIII. 19.) EMMI decree of the Ministry of Human Capacities

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:
Batch number 11122018
- Purity, including information on contaminants, isomers, etc.: 100%

RADIOLABELLING INFORMATION (if applicable): Not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C), protected from air (under argon or nitrogen)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage:
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis:
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium:
- Reactivity of the test material with the incubation material used (e.g. plastic ware):

Species:

rat

Strain:

Wistar

Remarks:

HanWistar, Crl:WI (Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633 Sulzfeld, Germany) from SPF colony
- Age at study initiation: Young adult female rats, nulliparous and non-pregnant, 11-12 weeks old at mating.
- Weight at study initiation: 191 - 254 g (the variation did not exceed ± 20% of the mean weight)
- Fasting period before study: None
- Housing: Standard laboratory conditions; individual housing
- Diet (e.g. ad libitum): The animals were provided with ssniff® SM R/M-Z+H Autoclavable Complete Diet for Rats/Mice – Breeding and Maintenance (Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) and tap water (in water bottles) as for human consumption, ad libitum.
- Water (e.g. ad libitum): The animals were provided with ssniff® SM R/M-Z+H Autoclavable Complete Diet for Rats/Mice – Breeding and Maintenance (Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany) and tap water (in water bottles) as for human consumption, ad libitum.
- Acclimation period: at least 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8-24.0°C (target: 22 ± 3°C)
- Humidity (%): 26-66% (target: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: To: From 02 Jan 2020 (start of acclimation) to 13 Feb 2020

Route of administration:

oral: gavage

Vehicle:

other: 1% aqueous methylcellulose solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle (1% aqueous methylcellulose solution) at the appropriate concentrations according to the dose level and volume selected, in the Pharmacy of Charles River Laboratories Hungary Kft.
Formulations were be prepared fresh prior to administration to animals.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the available information provided by the Sponsor as well as results of the trial formulation, 1% aqueous methylcellulose solution was selected as vehicle for this study in agreement with the Sponsor.
- Concentration in vehicle: 1%
- Lot/batch no. (if required): Dispense codes: S11172 / S11173

Details on analytical verification of doses or concentrations:

Analysis of the formulations for homogeneity and concentration of test item were performed using a validated gravimetric analysis method (Charles River Laboratories Hungary Kft. Study code: 18/288-316AN) in the Analytical Department of the Test Facility under GLP two times during the study.

Top, middle and bottom duplicate samples were taken from test item formulations for formulation analysis for homogeneity and concentration, one set (Set1G) to analyse (which was collected in replicates as practical) and two sets (Set2G and Set3G) as back-up, if required for any confirmatory analyses. Duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

This gravimetric formulation analysis was conducted under the control of the Contributing Scientist (at the Analytical Department of Charles River Laboratories
Hungary Kft.) in compliance with the relevant SOPs of the Test Facility. The results of the formulation analysis is included in Appendix 5.

Acceptance criteria for homogeneity was that the relative standard deviation (RSD) of the replicates must be less than 10%.

Details on mating procedure:

The oestrus cycle of female animals was examined shortly before start of pairing, and then daily, up to the day of mating. On the first week of the mating, only half of the animals were examined daily for their oestrus cycles. After acclimation, the females were paired according to their oestrus cycle with males in the morning for approximately 2 hours (1 male:1-3 female) until 24 sperm positive females/group were attained. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (gestation day 0, GD 0). Sperm positive females were separated and caged individually.

Randomisation

The sperm-positive, assumed pregnant females were allocated to the experimental groups (on each mating day) in such a way that the group averages of the body weight were as similar as possible. Females paired with the same male were allocated to different groups on the same mating day. A computer software (Provantis) was used to verify homogeneity/variation among/within groups.

Duration of treatment / exposure:

Start of treatment Gestation Day GD6
End of treatment Gestation Day GD19

Frequency of treatment:

daily by oral (gavage) administration

Duration of test:

07 January 2020 - 13 February 2020

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

100 female animals, 25 mated female animals/group, 4 groups (one control and 3 test item-treated groups);
22, 24, 20, 24 pregnant and evaluated female animals (with implantation sites at necropsy) per Control, Low, Mid and High dose groups, respectively;
56 male animals for mating;
no study-procedures were carried out on the male animals

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were set by the Sponsor in consultation with the Study Director, based on the available data and information from previous experimental work, including the results of a limit test in a dose range finding (DRF) study.
In the DRF study, no signs of systemic toxicity were observed at the limit dose of 1000 mg/kg bw/day. A statistically significant increase of the number of runts in the DRF study was considered to be incidental, and the top dose of 1000 mg/kg for the main study is considered appropriate. Based on this information, the doses of 1000, 300 and 100 mg/kg bw/day are deemed suitable for the purpose of the study. The oral route was selected as it is the most relevant route of human exposure.
- Rationale for animal assignment (if not random):
The sperm-positive, assumed pregnant females were allocated to the experimental groups (on each mating day) in such a way that the group averages of the body weight were as similar as possible. Females paired with the same male were allocated to different groups on the same mating day. A computer software (Provantis) was used to verify homogeneity/variation among/within groups.
- Fasting period before blood sampling for (rat) dam thyroid hormones:
- Time of day for (rat) dam blood sampling: At termination

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made usually twice daily (at the beginning and end of each working day, or before treatment and when the peak of the clinical observations,
if any, is observed after treatment). Only one clinical observation was made in the afternoon on those days when detailed clinical observation is made in the morning.
When signs of toxicity were noted, animals might be observed more frequently.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals at the onset of treatment (GD 6) then weekly.
The animals were monitored for any changes including pertinent behavioural changes
and signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes,
occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation,
piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and
response to handling as well as the presence of clonic or tonic movements, stereotypies
(e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation,
walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and
coma.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded with precision of ±1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Body weight gain were calculated for each interval, including GD0-6, GD6-20 and GD0-20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was measured with precision of ± 1 g on GD 0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption was also calculated for each interval, including GD 0-6, GD6-10, GD 6-20 and GD 0-20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: dam's viscera, weight of the thyroid gland with parathyroid glands, ovaries, uterus, cervix, placentas, anogenital distance of each foetus, foetuse's thymus, great arteries and skeleton.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Plasma: Yes
- Serum: No
- Volume collected: volume target of at least 125 µL for the first aliquot and at least 75 µL

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [ half per litter ]
- Skeletal examinations: Yes: [ half per litter ]
- Head examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
- Anogenital distance of all live rodent pups: The anogenital distance of each foetus was measured, and the sex of each assigned based on the distance. The sex of the foetuses was reconfirmed by examining the internal sex organs.

Statistics:

SPSS PC+4.0: The heterogeneity of variance between groups: Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then Duncan's Multiple Range test (for inter-group differences). Where significant heterogeneity was found, Kolmogorow-Smirnow test was used. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the were performed using Mann-Whitney U-test (inter-group comparisons). The Chi-squared test was used for non-continuous data.
SAS v9.2 (within Provantis): The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests. If no significant heterogeneity, an Anova/Ancova test was carried out. If the result was positive, Dunnett (Multiple Range) test was used for inter-group differences (<0.05 or <0.01). If either of the Shapiro-Wilk or Levene tests showed significance on the data, then a non-parametric analysis was used. A Kruskal-Wallis analysis of variance was used after Rank Transformation. In case of a positive result, the inter-group comparisons were performed using Dunn test (<0.05 or <0.01). For non-continuous data, the Cochran-Amitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control. Non-pregnant females, females with no implantation or ≤ 5 implantation sites, and females showing signs of abnormal pathology and/or misdosing were excluded from selected statistical analysis; in-life individual data was reported as applicable.
The limit for growth-retarded foetuses (runt) was calculated from the average and standard deviation of the body weights of the vehicle control foetuses. A foetus was considered as growth retarded (runt) when their body weight is less than the control average – 2 standard deviations), by using the SAS v9.2.

Historical control data:

Historical control data is presented in the Appendix 9 HISTORICAL CONTROL DATA OF HANNOVER
WISTAR RATS of the attached study report.

Clinical signs:

no effects observed

Description (incidence and severity):

Noisy respiration was observed in 1 out of 20 and 1 out of 24 animals in the Mid and High dose group. The finding appeared from Day 12 until Day 19; the symptom occurred for a maximum of eight days.

Piloerection was present in 1 out of 24 animals in the High group. The finding appeared from Day 12 until Day 19; the symptom occurred maximum eight days.

Red discharge around the right eye was observed in 1 out of 24 animals in the High dose group.

The clinical signs were attributed to local respiratory effect of test item (aspiration of a small amount of formulation), there was no evidence for systemic toxicity.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item related body weight decrease was observed in any of the dose groups.

No significantly lower or higher corrected body weight, corrected body weight gain, and net body weight gain was observed in animals in any of the dose groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No changes in the food consumption was observed in any of the dose groups during the treatment period.

Food efficiency:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

During the macroscopic examination of dams, the thyroid gland with parathyroid glands were retained from all animals and the organ weights recorded. There were no statistical differences in the organ weights between the control and the Dose groups.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. The result of the examination showed that there was a significant difference between the control and the low dose group in the number of multinucleated giant cell (parathyroid, unilateral, minimal), but this finding was considered to be a normal background finding and not related to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: Parameters according to OECD Test Guideline 414

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was statistically significant higher number of foetuses with retarded body weight
in the High dose group (p<0.05). However, the number of affected litters was identical
to the concurrent controls (and it is in line with Historic Data).

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, non-treatment-related

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There were no external variations in the test item treated groups. There was an incidental external malformation only in the High dose group. Absent of lower jaw (mandible) was observed.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Most of the skeletal findings correspond to the current historical control or the concurrent study control data, or were considered to be incidental findings without dose response. Based on the skeletal findings the number of malformed / intact foetuses were comparable with the control in the Low, Mid and High dose groups. The number of variations were higher in all test item treated groups. The number of affected foetuses with variation was statistically significantly higher in the Mid dose group compared to the control group (p<0.05). However, all data was considered to be in the normal range and there was no dose related trend.

In the case of most of the skeletal variations, the foetal or litter based incidence in the test item treated groups was comparable with the current study control or historical control values. Therefore, they were considered as biologically not relevant findings and not related to the test item treatment.

Most of the findings correspond to the study control data or have isolated occurrence that were considered incidental ascribed to individual variability.

Based on these results the test item did not affect adversely the intrauterine development by higher incidence of malformations, there were no adverse effects on external, visceral or skeletal evaluated data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The findings correspond with the current historical control or the study control data or have isolated occurrence that was considered incidental, ascribed to individual variability and not related to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Parameters according to OECD Test Guideline 414

Key result

Developmental effects observed:

no

Conclusions:

In conclusion, Mixed Ash, when administered daily by oral gavage to pregnant
Hannover Wistar rats from gestation days GD6 to GD19 at 1000 mg/kg bw/day
induced no maternal systemic toxicity and no effect on body weight or growth. No
effect on the embryotoxicity and foetotoxicity was observed (number of foetuses
with retarded body weight and malformations) in the study based on overall
development.
The following no-observed-adverse-effect (NOAEL) levels were derived:
NOAELmaternal toxicity: 1000 mg/kg bw/day
Based on no maternal systemic effects at 1000 mg/kg bw/day.
NOAELembryotoxicity: 1000 mg/kg bw/day
Based on the lack of any test-item related intra-uterine effect in any treatment group.
NOAELfoetotoxicity: 1000 mg/kg bw/day
Based on no effect on runts at 1000 mg/kg bw/day.
NOAELteratogenecity: 1000 mg/kg bw/day
Based on the lack of treatment related malformations in any dose group.

Executive summary:

Mixed Ash, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 1000 mg/kg bw/day induced no maternal systemic toxicity and no effect on body weight or growth in a GLP-compliant OECD 414 Prenatal Developmental Toxicity test. No effect on the embryotoxicity and foetotoxicity was observed (number of foetuses with retarded body weight and malformations) in the study based on overall development. No mortality occurred during the study.

All test item formulations were within the range of 98-108% of nominal concentration and were found to be homogenous. No test item was detected in the vehicle control samples.

In the higher dose group, some symptoms were present. Noisy respiration was observed in 1 out of 20 and 1 out of 24 animals in the Mid and High dose group. The finding appeared from Day 12 until Day 19, the symptom occurred for a maximum of eight days.

Piloerection was present in 1 out of 24 animals in the High group. The finding appeared from Day 12 until Day 19, the symptom occurred for a maximum of eight days. Red discharge around right eye was observed in 1 out of 24 animals.

No test item related body weight effects were observed in any of the dose groups. No changes in the food consumption in any of the dose groups was observed during the treatment period. No test item related macroscopic and microscopic findings were present at necropsy in the surviving animals.

There were no statistically significant changes in the concentration of the T3, T4 and TSH level between the Control and the Dose groups. There were no statistical differences in the organ weights between the control and the Dose groups. There were no statistically significant differences in the intra-uterine parameters in the test item treated animals when compared to the controls. There was no toxicologically significant difference in the foetal weights of either sex of foetuses, sex distribution or on anogenital distance between the control and treatment groups. There were no adverse test item related effects on external, visceral or skeletal development of foetuses in the study.

Foetal malformations observed in the study were all considered to be incidental. They showed no dose dependency and thus were not regarded as a test item related effect.

The following no-observed-adverse-effect (NOAEL) levels were derived:

NOAELmaternal toxicity: 1000 mg/kg bw/day

Based on no maternal systemic effects at 1000 mg/kg bw/day.

NOAELembryotoxicity: 1000 mg/kg bw/day

Based on the lack of any test-item related intra-uterine effect in any treatment group.

NOAELfoetotoxicity: 1000 mg/kg bw/day

Based on no effect on runts at 1000 mg/kg bw/day.

NOAELteratogenecity: 1000 mg/kg bw/day

Based on the lack of treatment related malformations in any dose group.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Prenatal Developmental toxicity study (OECD 414):

In conclusion, Mixed Ash, when administered daily by oral gavage to pregnant Hannover Wistar rats from gestation days GD6 to GD19 at 1000 mg/kg bw/day
induced no maternal systemic toxicity and no effect on body weight or growth. No
effect on the embryotoxicity and foetotoxicity was observed (number of foetuses
with retarded body weight and malformations) in the study based on overall
development.
The following no-observed-adverse-effect (NOAEL) levels were derived:
NOAELmaternal toxicity: 1000 mg/kg bw/day
Based on no maternal systemic effects at 1000 mg/kg bw/day.
NOAELembryotoxicity: 1000 mg/kg bw/day
Based on the lack of any test-item related intra-uterine effect in any treatment group.
NOAELfoetotoxicity: 1000 mg/kg bw/day
Based on no effect on runts at 1000 mg/kg bw/day.
NOAELteratogenecity: 1000 mg/kg bw/day
Based on the lack of treatment related malformations in any dose group.

Justification for classification or non-classification

The information available is conclusive for classification. No classification is required based on.

(1) No reproductive or developmental effects observed in OECD 414 Prenatal developmental toxicity Test at doses up to 1000 mg/kg bw/day (2) Low systemic toxicological activity and no effects on reproductive organs reported in repeated dose toxicity studies or in the literature.