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Toxicological information

Toxicity to reproduction

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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:
guideline study

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

Constituent 1
Reference substance name:
Ashes (residues), coal
EC Number:
931-322-8
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 / concentrationsopen allclose all
Dose / conc.:
160 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
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)

Clinical signs:
no effects observed
Description (incidence and severity):
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).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
Males
There were no unscheduled deaths during all the study.

Females/Mothers
There were no unscheduled deaths during all the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
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.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
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.
Reproductive performance:
no effects observed
Description (incidence and severity):
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)

open allclose all
Dose descriptor:
NOEL
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no effects were noted in the parental animals at 160 mg/kg bw/day
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
1 000 mg/kg bw/day (actual dose received)
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.

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
- Only one pup death after birth at the dose level 400 mg/kg/day was observed.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- 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 than control weight of pups.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
- 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.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- No differences in development of pups were observed in treated groups compared with the control group.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

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)

open allclose all
Dose descriptor:
NOEL
Generation:
F1
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects were observed in pups up to and including the dose level of 400 mg/kg bw/day.
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
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

Target system / organ toxicity (F1)

Critical effects observed:
no

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 1stday

64.54

57.98

60.44

58.21

Weight of litter 4thday

106.8

96.42

97.73

99.90

Weight of pups 1stday

5.61

5.86

5.72

5.99

Weight of pups 4thday

9.23

9.75

9.37

10.28

Number of pups 1stday

11.50

9.89

10.57

9.71

Sex of live pups 1stday

 5.6M + 5.9F

5.6M + 4.3F

5.4M +5.2F

5.4M + 5.0F

Number of pups 4thday

11.50

9.89

10.40

9.71

Sex of live pups 4thday

 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.