Ashes (residues), nonhazardous municipal solid waste

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12.03.2008-12.08.2008

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

Test material information

Constituent 1

Reference substance name:

Ashes (residues)

EC Number:

268-627-4

EC Name:

Ashes (residues)

Cas Number:

68131-74-8

Test material form:

solid: bulk

Details on test material:

- Name of test material (as cited in study report): Ashes (residues)
- Substance type: technical product
- Physical state: solid
- Main components: SiO2 (42.12%), Al2O3 (32.16%), Fe2O3 (10.88%), TiO2, MnO, MgO, CaO, Na2O, K2O, SO3 - constituents with content less than 10%
- Expiration date of the lot/batch: unlimited
- Appearance: greyish black powder

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: 6-7 weeks
- Fasting period before study: none
- Housing: Animals were housed in plastic cages (40x25x20cm) containing sterilised clean shavings of soft wood: 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: ad libitum, 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. Diet was sterilised before using. Composition of food: Wheat, Oats, Fish meal powder, Dried snail-clover, Soya extracted groats, Wheat sprouts, Dehydrated yeast, Calcium carbonate, Vitamin and Mineral complex. Nutrient content of the diet: Crude protein – min. 21%, Drip – max. 14%, Fat – min. 3%, Fiber – max. 4.1%, Ash – max. 7%, Calcium – min. 1%, Phosphorus – min. 0.8%, Magnesium – min. 0.2%, Sodium – max. 0.25%;
- Water: ad libitum, Free access to drinking water. Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law. Water was sterilised before using.
- Acclimation period: at least, 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3oC
- Humidity (%): 30-70%
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12 hour dark cycle


STUDY TIME SCHEDULE
Animal arrival: 12. 3. 2008
Acclimatisation: 6 days
Administration:
1st - 14th day of study (before mating) – males and females 18.03. - 04.04.2008
15th - 21st day of study – males and females (mating period) 01.04. - 08.04.2008
22nd - 41st day of study – males 09.04. - 01.05.2008
22nd - 50th day of study – pregnant females (1st – 21st day of pregnancy) 14.04.-09.05.2008
22nd - 53rd day of study – non-pregnant females 14.04.-12.05.2008
51st - 53rd day of study – females in lactation period (1st – 3rd day of lactation) 10.05. – 13.05.2008
Necropsies: males – till 42nd day of study 29.04. - 02.05.2008
mothers and pups – 4th day of lactation 26.04. - 13.05.2008
non-pregnant females – 54th – 56th day of study 12.05. - 15.05.2008
End of histopathological examination: 12.08.2008
Final report elaboration: 14.11.2008

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% methylcellulose (in water for injection)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The concentrations of suspension at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The vehicle control group was administered by the 0.5% methyl cellulose in water in the same volume. The application form – suspensions of test substance in 0.5% methyl cellulose in water for injections was prepared daily before administration. These suspensions were mixed for 10 minutes by magnetic stirrer.

The following dose level groups: 1000 mg/kg/day, 400 mg/kg/day, 160 mg/kg/day and one control group (vehicle only) were chosen for the study.

VEHICLE
The test substance was administered in 0.5% methyl cellulose in water for injection (Aqua pro injectione).
Aqua pro injectione
Manufacturer: ArdeaPharma, Třeboňská 229, 373 63 Ševětín, Česká republika
Batch number: 0408031207
Attest No.: 2174 (pharmaceutical - ČL 2005)

Methylcellulosum
Manufacturer: Dr. Kulich Pharma sro., Piletická 178/61, 500 03 Hradec Králové - Slatina
Batch number: DT157078
Attest No.: 0330/1106/538 (pharmaceutical - ČL 2005)

Details on mating procedure:

Animals were mated in 15th – 21st day of study. Mating 1:1 (one male to one female) was used in this study.
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.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

ADMINISTRATION
1st - 14th day of study (before mating) – males and females
15th - 21st day of study – males and females (mating period)
22nd - 41st day of study – males
22nd - 50th day of study – pregnant females (1st – 21st day of pregnancy)
22nd - 53rd day of study – non-pregnant females
51st - 53rd day of study – females in lactation period (1st – 3rd day of lactation)

Frequency of treatment:

The animals were treated for 7 days per week at the same time (8.00 – 10.00 am).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

160 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)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

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.

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
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
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 pituitary gland 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 - 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.

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)

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

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.

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).

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

Overall reproductive toxicity

Reproductive effects observed:

not specified

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: decrease of males body weight (1000 mg/kg/day), decrease weight of male pituitary gland (1000 mg/kg/day), and increased weight of mothers uterus (400 mg/kg/day).
Statistically non-significant effects manifested in: decrease 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.

At the dose level of 160 mg/kg/day the examinations did not detect adverse effects of the test substance on reproduction parameters and on the health of parental animals. At the dose levels 160 and 400 mg/kg/day did not detect negative effects on pups.
Based on the results summarized above it could be done the following conclusion about NOAEL of the test substance:

The dose level of 160 mg/kg body weight/day could be regarded as the NOAEL (No Observed Adverse Effect Level) for parents health and reproduction.

The NOAEL for pups health effects was established as 400 mg/kg body weight/day.

Executive summary:

Introduction

The test substance, Ashes (residues), was tested for reproduction toxicity using the method - OECD Test Guideline No. 421 Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on July 27th 1995.    

Study performance

Wistar rats of SPF quality were used for testing. The test substance was administered orally, dissolved in 0.5% methylcellulose in water for injection (Aqua pro injectione), by stomach tube.

The test substance consists of various oxides, the most of which are water insoluble (see study VUOS: Ashes (residues) Water Solubility – No. 46/07/46, Study No. 0827, 2008). Therefore a suitable analytical method cannot be found for homogeneity and stability testing. Since undissolved particles of the test substance (were mixed for 10 minutes by magnetic stirrer) are easily visible in the application form, homogeneity was checked by eye. Stability of the test substance in the application form could not be verified but there is no indication that a mixture of rigid oxides would be unstable in its 0.5% methyl cellulose in water suspension for that short time period (1 hour).

The treated groups were administered daily for the following periods:

males and females – prior to the mating period and during the mating period,

pregnant females – during pregnancy and till 3rd day of lactation,

males – after mating period till 41st day of study,

nonpregnant females – till 54th-56th day of study.

The animals were treated for 7 days per week at the same time. The concentrations of suspensions in all three dose levels were adjusted to ensure the administered volume of 1 mL per 100 g of body weight. 

The following dose level groups: 1000 mg/kg/day, 400 mg/kg/day, 160 mg/kg/day and one control group (vehicle only) were chosen for the study.

During the study the clinical observation and health status control were performed daily. The body weight and food consumption were measured weekly or in specified time intervals. Vaginal smears were prepared daily during mating period (until the presence of spermatozoa). Reproduction parameters relevant to pups (number of pups, body weight, sex or vitality) were also recorded.

The study was finished by gross necropsy of animals. The selected organs from parental animals were removed for weighing and histopathological examination.

Results  

The administration of the test substance Ashes (residues) did not affect the 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: decrease of male body weight (1000 mg/kg/day), decrease of the weight of male pituitary gland (1000 mg/kg/day), and increased weight of mother uterus (400 mg/kg/day). 

Statistically non-significant effects manifested in: decrease 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 level of 400 mg/kg/day and decrease of number of pups at the dose level of 1000 mg/kg/day.

At the dose level of 160 mg/kg/day the examinations did not detect adverse effects of the test substance on reproduction. At the dose levels 160 and 400 mg/kg/day no negative effects on pups were detected.   

Conclusion

Based on the results summarized above it could be done the following conclusion about NOAEL of the test substance:

The dose level of 160 mg/kg body weight/day could be regarded as the NOAEL (No Observed Adverse Effect Level) for parents.        

The NOAEL for pups was established as 400 mg/kg body weight/day.