Humic acids and fulvic acids extracted from leonardite, reaction product with formaldehyde, potassium hydrogen sulfite, sodium hydrogen sulfite and potassium hydroxide

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

from March 25, 2009 to August 28, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Humic acids, potassium salts

EC Number:

271-030-1

EC Name:

Humic acids, potassium salts

Cas Number:

68514-28-3

IUPAC Name:

68514-28-3

Details on test material:

- Name of test material (as cited in study report): Humic acids, potassium salts
- Molecular formula: not known - UVCB substance
- Molecular weight: not known - UVCB substance
- Substance type: technical product
- Physical state: solid
- Lot/batch No.: 16. 5. 2007/R
- Expiration date of the lot/batch: 05/2022
- Stability under test conditions: stable
- Storage condition of test material: dry conditions

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., Kolec u Kladna, Czech Republic
- Age at study initiation: (P) 9 wks
- Weight at study initiation: Males: cca 311-312 g; Females: cca 202-203 g
- Fasting period before study:
- Housing: Animals were housed in SPF animal room, 2 rats of the same sex in one plastic cage (40x25x20 cm) containing sterilised clean shavings of soft wood. During mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother.
- Diet (e.g. ad libitum): Complete peleted diet for rats and mice in SPF breeding - ST 1 BERGMAN, ad libitum. Diet was sterilised before using
- Water (e.g. ad libitum): Free access to drinking water (ad libitum).
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): Relative humidity 30-70%
- Photoperiod (hrs dark / hrs light): 12-hour light/12 hour dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was administered to the stomach by gavage as the solution in water for injection. The animals were treated 7 days per week at the same time.

VEHICLE
water for injection (Aqua pro injectione)
Manufacturer: Ardeapharma a.s., Ševětín
- Concentration in vehicle: 25, 50 or 100 g/L water
- Amount of vehicle (if gavage): 1 mL per 100 g of body weight.
- Batch No.: 0204190908, 0102220109, 0102050309, 0101030309

Details on mating procedure:

- M/F ratio per cage: 1/1
- Proof of pregnancy: sperm in vaginal smear
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and the homogeneity of application form were determined.
The stability of application form was monitored by the analyses of solution of test substance in water. The measurement was performed on two concentration levels (250 and 1000 mg/10mL). The solution was prepared by mixing of test substance and water for 5 minutes (cca 400 rpm) in a container for application form preparation. For the determination of stability the samples were taken from the middle of container content after required time intervals (0, 30, 60 and 120 minutes). Time interval 0 min. was the time after 5 minutes mixing. The solution was mixed all the time of monitoring.
The homogeneity of application form was monitored by the analyses of solution of test substance in water prepared by the same way as for the determination of the stability. The measurement was performed on two concentration levels (250 and 1000 mg/10mL). The samples were taken after 5 minute mixing from 3 given places - the bottom, the middle and the surface of container content.

The determination of test substance was performed on the basis of measurement of the absorbance of a water solution. Test substance stability and homogeneity were determined by measuring of an absorbance of water solution (application form) in visible range of spectrum.

Duration of treatment / exposure:

The treated groups were administered daily for the following period: males and females – 2 weeks prior to the mating period and then during the mating period.
Pregnant females were administered during pregnancy and till the 3rd day of lactation.
Males were then administered after mating period – totally for 54 days.
Nonpregnant females (mated females without parturition) were administered 26 days after the confirmed mating.

Frequency of treatment:

The animals were treated 7 days per week

Details on study schedule:

Mating Procedure
Animals were mated from the 15th day of study. Mating 1 : 1 (one male to one female) was used in this study. Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:

Basis:
actual ingested
250 mg/kg/day

Doses / concentrations 2

Remarks:

Doses / Concentrations:

Basis:
actual ingested
500 mg/kg/day

Doses / concentrations 3

Remarks:

Doses / Concentrations:

Basis:
actual ingested
1000 mg/kg/day

No. of animals per sex per dose:

Dose 250 mg/kg/day: 10 males + 10 females
Dose 500 mg/kg/day: 10 males + 10 females
Dose 1000 mg/kg/day: 10 males + 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels for study – 250, 500 and 1000 mg/kg/day were chosen on the basis of the results of the Study No. 26/07/7: Humic acids, potassium salts – Repeated Dose (28 days) Toxicity (Oral), VUOS-CETA Report No.0857, 2008.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

Health Condition Control:
All rats were observed pre-experimentally to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. During the administration period they were examined for behaviour changes each day before and during application.

CLINICAL OBSERVATIONS:
Clinical Observations of Males and Females
All rats were observed daily during the administration period.
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 – at the time of expectation of maximal effect of the test substance.
Animals were observed in natural conditions in their cages.

BODY WEIGHT:
The body weight of animals was recorded on automatic balances with group average computing module on specified days. All animals were weighed immediately before euthanasia too.
Weight increment was computed as an average per group per day (in grams). Non-pregnant females (females without parturition) were not included in calculation of averages in pregnancy and lactation period.

FOOD CONSUMPTION:
In a specified day the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed.
In males average values were calculated for each week of the study (except of mating period). Food consumption for animal/day was calculated from average values of each group.
The same way of calculation of average food consumption was used for females in premating period. In pregnancy and lactation period average individual values (grams/animal/day) were calculated for each week of the study. Average food consumption for each group was calculated from individual values. Nonpregnant females (females without parturition) were not included in calculation of average food of pregnant females.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
In all males of all groups surviving to scheduled necropsy the sperm parameters were examined: sperm motility and sperm morphology. Sperm specimens were prepared and examined according to the SOP No. M/45.

Sperm motility
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension.
The result of observation was evaluated subjectively according to following grades:
1 – fast progressive motility, 2 - slow progressive motility, 3 – no progressive motility, 4 – non-motile sperm.

Sperm morphology
Sperm samples were taken from one epididymis and sperm morphology were assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination.
All deviations – e.g. broken tail, abnormal form of tail, double head, amorphous head, no head, abnormal form of neck ¬– were recorded.

Biometry of Reproductive Organs
The absolute weights of testes, 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 recorded in females. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.

Litter observations:

PARAMETERS EXAMINED
Clinical Observation of Pups
All pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 0 or 1 post-partum) and the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.

Postmortem examinations (parental animals):

Pathological Examination
Parental males were killed at the end of the administration period – after 54 days of administration.
Parental females were killed on the 4th day of lactation. Mated females without delivery were killed 27th day after confirmed mating.
Then they were macroscopically examined for any pathological changes with special attention to the organs of the reproductive systems. All macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Biometry of Reproduction Organs
The absolute weights of testes, 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 recorded in females. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.

The following tissue and organs were collected from all killed males and females at necropsy and fixed in neutral 4% formaldehyde solution (v/v) for further histopathological evaluation: relevant gross lesions, pituitary gland, coagulation gland, prostate gland, seminal vesicles, epididymis and testes, cervix of uterus, ovaries, uterus and vagina. relevant gross lesions, pituitary gland, coagulation gland, prostate gland, seminal vesicles, epididymis and testes, cervix of uterus, ovaries, uterus and vagina.

Postmortem examinations (offspring):

Dead pups were sexed and externally examined; the stomach was examined for the presence of milk. Pups killed on the 4th day of lactation were sexed and subjected to external examination of the cranium, and to macroscopic examination of the thoracic and abdominal tissues and organs. All macroscopic changes were recorded.

Statistics:

The ANOVA test - Analysis of Variance (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, biometry of organs and number of pups. Control group with vehicle was compared with three treated groups.
The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the summary tables.

Reproductive indices:

For each of parental females the following parameters were calculated:
Loss of offspring - individual
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 alive at postnatal day 4

For each dose group the fertility parameters were calculated.
Fertility parameters group
Percentage 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 bearing live pups / number of pregnant females) x 100
Percentage of postnatal loss days 0-4 post partum: (number of dead pups on day 4 post partum*/ number of live pups at first litter check) x 100
Note: * without still born pups (dead pups with anaerial lungs)

Offspring viability indices:

Viability index: (number of live pups on day 4 post partum / number of pups born alive+) x 100
Note: + with dead pups with aerial lungs

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
The application of the test substance did not cause the death of any female or male.
In parental males negative influence of the test substance on clinical status was not found out.
In parental females clinical changes were observed only sporadically at the dose level 1000 mg/kg/day (dyspnoea and piloerrection in one female).

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
The test substance did not affect growth of parental males and females of the dose levels 250 and 500 mg/kg/day.
The body weight of parental animals of the dose level 1000 mg/kg /day was slightly negatively influenced while the food consumption was balanced with control. This body weight difference was not statistically significant and dose dependent.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Observation of sperm motility and sperm morphology detect impaired quality of sperm in males of the dose levels 500 and 1000 mg/kg/day. But these changes were not accompanied by microscopical affections in testes - detailed examination of testes did not revealed damage of spermatogenesis at treated males.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Examination of reproductive system of parental females showed increased occurrence of cysts in ovaries in treated females – this finding was not dose dependent. Hyperplasia of vaginal epithelial cells was detected only at the dose level 1000 mg/kg/day. These microscopical affections in ovaries and vagina did not affected fertility of parental females.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Biometry of organs also proved no statistically significant and no dose dependent differences in treated animals. Slight increasing of uterus weight (without changes of microscopical structure) was recorded in all treated groups – this finding without treatment-related distribution was not considered as finding of toxicological significance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological examination of reproductive system of parental males showed only increased incidence of lymphocyte infiltration in epididymides of males at the dose levels 500 and 1000 mg/kg/day (dependent on dose level). Microscopical changes diagnosed in pituitary gland were not considered as changes of toxicological significance – vacuolation of cell cytoplasm probably related with processing of organs for histology and this affection is commonly observed in Wistar rats.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Sex:

male

Basis for effect level:

other: clinical signs; mortality; body weight; food consumption and compound intake; water consumption and compound intake; gross pathology; organ weights; histopathology; mating index; fertility index; sperm characterization

2

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Sex:

female

Basis for effect level:

other: clinical signs; mortality; body weight; food consumption and compound intake; water consumption, gross pathology; organ weights; histopathology; mating index; fertility index; number of implantation sites; duration of pregnancy.

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:

not examined

Histopathological findings:

not examined

Details on results (F1)

Observation of pups – the number of pups, sex ratio, average weight of litter, average body weight of pups on the day of parturition/1st day after parturition and the 4th day of lactation and postnatal development of pups were unaffected by the test substance treatment. Vice versa the numbers of live pups at the dose levels 500 and 1000 mg/kg/day were higher than in control. Death of one pup in lactation period was recorded at the dose level 1000 mg/kg/day. Macroscopic abnormalities were detected sporadically: haemorrhages on abdominal skin, blood clots in abdominal cavity and flatulence of stomach (in one pup at the highest dose) was found out during necropsy of one pup of the dose level 1000 mg/kg/day.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: birth index; live birth index; pregnancy index; litter size; litter weight; pup weight; sex ratio; survival index; viability index; l

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Reproduction data

Reproduction data
Observed parameters	Dose level
	0	250	500	1000
Pairs started (N)	10	10	10	10
Females showing evidence of copulation (N)	10	10	10	10
Females achieving pregnancy (N)	9	10	9	9
Females with abortion (N)	0	0	0	1
Conceiving days (duration of mating) 1 – 5 (N)	10	10	10	10
Conceiving days (duration of mating) 6 – 13 (N)	0	0	0	0
Pregnancy ≤ 21 days (N)	2	3	2	1
Pregnancy = 22 days (N)	5	7	7	5
Pregnancy ≥ 23 days (N)	2	0	0	2
Females with live pups born (N)	9	10	9	8
Females with live pups at day 4 after parturition (N)	9	10	9	8
Corpora lutea/pregnant females (average)	16.78	15.20	17.10	15.89
Implantations/pregnant females (average)	13.78	13.70	14.44	12.67
Live pups/mother at birth (average)	12.00	12.30	13.67	13.13
Live pups/mother at day 4 after parturition (average)	12.00	12.30	13.67	13.10
Sex ratio (M/F) at birth (average)	6.70/5.30	6.40/5.90	7.90/5.80	6.90/6.30
Sex ratio (M/F) at day 4 after parturition (average)	6.70/5.30	6.40/5.90	7.90/5.80	6.80/6.30
Litter weight at birth (average)	84.20	82.80	89.90	85.60
Litter weight at day 4 after parturition (average)	123.10	121.20	129.70	123.90
Pup weight at birth (average)	7.10	6.70	6.60	6.50
Pup weight at day 4 after parturition (average)	10.40	9.90	9.60	9.60
ABNORMAL PUPS
Mothers with 0 (N)	9	10	9	7
Mothers with 1 (N)	0	0	0	1
Mothers with ≥ 2 (N)	0	0	0	0

Table 2: Fertility parameters

Fertility parameters
Calculated parameters	Dose level
	0	250	500	1000
Percentage of mating	100.00	100.00	100.00	100.00
Fertility index	90.00	100.00	90.00	90.00
Conception index	90.00	100.00	90.00	90.00
Gestation index	100.00	100.00	100.00	88.89
Percentage of postnatal loss	0.00	0.00	0.00	0.95
Viability index	100.00	100.00	100.00	99.05
LOSS OF OFFSPRING
Pre-implantation (corpora lutea minus implants)
Pregnant females with 0 – 5 (N)	7	9	8	6
Pregnant females with 6 - 10 (N)	2	1	1	3
Pregnant females with 11 - 15 (N)	0	0	0	0
Pregnant females with ≥ 16 (N)	0	0	0	0
Pre-natal/post-implantations (implants minus live births)
Pregnant females with 0 (N)	2	1	4	5
Pregnant females with 1 (N)	3	6	4	2
Pregnant females with 2 (N)	3	1	0	1
Pregnant females with ≥ 3 (N)	1	2	1	0
Post-natal (live births minus alive at post-natal day 4)
Pregnant females with 0 (N)	9	10	9	7
Pregnant females with 1 (N)	0	0	0	1
Pregnant females with 2 (N)	0	0	0	0
Pregnant females with ≥ 3 (N)	0	0	0	0

Applicant's summary and conclusion

Conclusions:

The negative influence of the test substance treatment expressed mainly at the highest dose level (limit dose): effect on body weight of parental females, sperm quality of parental males and misroscopical structure of reproduction organs (epididymides, vagina) in parental males and females were observed. Microscopical changes of sperms and structure of epididymis was recorded also at the middle dose level. The test substance administered at the highest dose level had probably slight negative influence on early prenatal development (abortion in one female).

Food consumption, clinical status and macroscopic structure of reproductive organs of parental males and females were not markedly affected by treatment of the test substance. Number of females achieving pregnancy, durations of mating and pregnancy, number of pups, sex ratio, average weight of litter, average body weight and postnatal development of pups were unaffected by the test substance treatment.
The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION of males was established as 250 mg/kg body weight/day.
The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION of females and DEVELOPMENT was established as 500 mg/kg body weight/day.

Executive summary:

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

Methods

Wistar rats of SPF quality were used for testing.The test substance was administered dissolved in water using a stomach tube; oral application of rats was made daily. The concentrations of solutions at all dose levels were adjusted to ensure the administered volume of 1 mL per of body weight. Four groups of animals were included in the study - 3 treated groups (doses 250, 500, 1000 mg/kg of body weight/day) and one control group (vehicle only). Each group consisted of 10 males and 10 females. The dose levels for study – 250, 500 and 1000 mg/kg/day were chosen on the basis of the results of the Study No. 26/07/7: Humic acids, potassium salts – Repeated Dose (28 days) Toxicity (Oral), VUOS-CETA Report No.0857, 2008.

The treated groups were administered daily for the following periods:

males and females – 2 weeks prior to the mating period and during the mating period,

pregnant females  – during pregnancy and till the 3^rdday of lactation,

males  –  after mating period – totally for 54 days,

nonpregnant females (mated females without parturition) – for 26 days after the confirmed mating.

   

During the study 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, weight of litters, sex or vitality) were also recorded.

The study was finished by gross necropsy of animals. In all males of all groups the sperm parameters: sperm motility and sperm morphology were examined. The selected organs from parental animals were removed for weighing and histopathological examination.

Results 

Body weight, food consumption, clinical status, weight and macroscopic structure of reproductive organs of treated parental males were unaffected by treatment of the test substance.

The test substance had negative effect on sperm quality of treated males. Sperm motility was affected in males of the dose level 1000 mg/kg/day and microscopical examination of sperms revealed increased percentage portion of morphologically changed sperms in males of the dose levels 500 and 1000 mg/kg/day. These findings were not accompanied by damage of spermiogenesis in testicular tubules. 

Histopathological examination of reproductive system of parental males showed increased incidence of lymphocyte infiltration in interstitium and epithelium of epididymidesin males of the dose levels 500 and 1000 mg/kg/day.

Food consumption, clinical status and macroscopic structure of reproductive organs of treated parental females were not markedly affected by treatment of the test substance. 

Growth of parental females was slightly negatively influenced by the test substance administration: the body weight of females of the dose level 1000 mg/kg/day was slightly decreased during premating period and pregnancy. During lactation period the body weight difference between control females and females of the dose level 1000 mg/kg/day was minimal.

Slight increase of absolute and relative weight of uterus of treated females independent on dose level was detected during biometry of reproductive organs.

Histopathological examination of reproductive system of parental females revealed increased incidence of microscopic affections in vagina (hyperplasia of epithelium) of females of the dose level 1000 mg/kg/day. In ovaries of females of the dose level 500 mg/kg/day increased occurrence of cysts was detected but this affection was recorded at the highest dose level only sporadically.  

Observation of pups – the number of pups, sex ratio, average weight of litter, average body weight and postnatal development of pups were unaffected and well-balanced with the control group. Macroscopic abnormality – haemorrhage on skin of abdomen and flatulence of stomach was recorded sporadically (only in 1 pup at the dose level 1000 mg/kg/day).

 

Reproduction parameters – number of females achieving pregnancy and accompanying conception index were well-balanced in control and treated females. Gestation index was slightly decreased only at the highest dose level (there was one aborted females). Durations of mating and pregnancy were similar in control and treated females.

Pre-implantation and post-implantation losses were relative well balanced at treated groups and control group. In control animals and animals treated by the middle and the lowest dose level no postnatal loss was recorded. At the highest dose group slightly increased postnatal loss and decreased viability index was detected in comparison with control.

    

Conclusion

The negative influence of the test substance treatment expressed mainly at the highest dose level (limit dose): effect on body weight of parental females, sperm quality of parental males and misroscopical structure of reproduction organs (epididymides, vagina) in parental males and females were observed. Microscopical changes of sperms and structure of epididymis was recorded also at the middle dose level. The test substance administered at the highest dose level had probably slight negative influence on early prenatal development (abortion in one female).

Food consumption, clinical status and macroscopic structure of reproductive organs of  parental males and females were not markedly affected by treatment of the test substance. Number of females achieving pregnancy, durations of mating and pregnancy, number of pups, sex ratio, average weight of litter, average body weight and postnatal development of pups were unaffected by the test substance treatment.

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTIONof males was established as 250 mg/kg body weight/day.        

The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION of females and DEVELOPMENT was established as 500 mg/kg body weight/day.