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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:
2010-10-13 to 2011-04-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Remarks:
OECD 421

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
, the highest dose level exceeded the acceptability criteria of OECD 421
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Diethyl oxalate
EC Number:
202-464-1
EC Name:
Diethyl oxalate
Cas Number:
95-92-1
Molecular formula:
C6H10O4
IUPAC Name:
diethyl oxalate
Test material form:
other: colourless oily liquid
Details on test material:
- Name of test material: Diethyloxalate

Test animals

Species:
rat
Strain:
other: Wistar Han
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Koleč u Kladna, Czech Republic
- sex: males and virgin females
- Age at study initiation: 10 weeks
- Fasting period before study: no

- 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: Complete peleted diet for rats and mice in SPF breeding - ST 1 BERGMAN. Diet was sterilised before using.

- Water: Free access to drinking water (ad libitum). Water was sterilised before using.

- Acclimation period: 5 days

- Identification: Identification of animals was made by colour marks on fur (system 1 – 10), each cage was marked with the number of study, number of animals, sex, number of cage, name and dose of the test substance and mark of group.

- Additional Information: The standard pelleted laboratory animal diet was analysed for nutrients (once a year) and bacteriologically examined (every two months) on a regular basis. Results were retained in the CETA archives. Reports of analysis of water (twice a year) were retained in the CETA archives. Results of sterilizer effectivity control (performed once a year) were retained in the CETA archives.
Analysis of diet and water and steriliser control, did not reveal any findings that could affect study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature: 22+/-3°C
- Humidity: a relative humidity of 30-70%
- Air changes: Animals were housed in controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour
- Photoperiod: 12-hour light/12 hour dark cycle.

Study Time Schedule
Date of animal arrival: 2010-10-13
Start of administration: 2010-10-19
End of administration: males 2010-11-15, females 2010-12-10
Clinical examination: 2010-10-13 to 2010-12-11
Necropsy and biometry of organs: males 2010-11-16, females 2010-11-23 to 2012-12-11
Histopathological examination: 2011-01-19 to 2011-03-15

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The application form (test substance suspension in olive oil) was prepared daily just before administration.The concentrations of suspensions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.

VEHICLE
- Justification for use and choice of vehicle: olive oil (pharmaceutical quality), manufacturer: Dr.Kulich Pharma s.r.o., standard vehicle
- Lot/batch no.: 4874601
Details on mating procedure:
- M/F ratio per cage: Mating 1 : 1 (one male to one female) was used in this study.
- Length of cohabitation: until the presence of spermatozoa
- Proof of pregnancy: 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.
- 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 determination of Diethyloxalate was performed by gas chromatography based on a method developed at the test facility. Test substance stability and homogeneity were determined by means of peak areas of main components of Diethyloxalate. The analyses were performed by GC method.
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 - during pregnancy and till the 3rd day of lactation
males were then administered after mating period – totally for 28 days
non-pregnant females (mated females without parturition) were administered 25 days after the confirmed mating.
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
90 mg/kg bw/day (actual dose received)
Dose / conc.:
270 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Each group consisted of 12 males and 12 females.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses for the study were chosen with respect to the results of the dose-range finding experiment.

Examinations

Parental animals: Observations and examinations:
MORTALITY CONTROL: Yes
Daily during the treatment periods for vitality or mortality changes.

HEALTH CONDITION CONTROL: Yes
Daily during the acclimatization and the experimental part.

CLINICAL OBSERVATIONS: Yes
Clinical Observation of males and females daily during the administration period (in their cages) in order to record possible clinical effects after application and all changes in behaviour of animals.

BODY WEIGHT: Yes
- Time schedule for examinations: on specified days, all animals were weighed immediately before euthanasia too.
Weight increment was computed as an average per group per time interval (in grams). Nonpregnant females were not included in the calculation of averages in the pregnancy and lactation period.
males - weekly
females - weekly in premating and mating period
during pregnancy: 0., 7th, 14th, 20th day
during lactation: 0. or 1st and 4th day

FOOD CONSUMPTION: Yes
On 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). The food consumption animal/day was calculated from the average values of each group.
The same way of calculation of average food consumption was used for females in the 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 and aborted females (females without parturition) were not included in calculation of average food of pregnant females.
males - weekly
females - weekly during premating period and after mating period
during pregnancy: 0., 7th, 14th, 20th day
during lactation: 0. or 1st and 4th day

EXAMINATION OF VAGINAL SMEARS: Yes
The pregnancy was determined by the presence of spermatozoa in vaginal smear. The vaginal smears were carried out daily in the morning during mating period. The smears were stained and the presence of sperm was evaluated. Day 0 of pregnancy was defined as the day when sperms were found.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
In all males of all groups surviving to scheduled necropsy the following sperm parameters were examined: sperm motility and sperm morphology.

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.
Litter observations:
PARAMETERS EXAMINED
BODY WEIGHT: Yes
pups (litters) – 0, or 1st and 4th day

CLINICAL OBSERVATION: Yes
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):
SACRIFICE and PATHOLOGICAL EXAMINATION:
Parental males - at the end of the administration period – after 28 days of administration.
Parental females were killed on the 4th day of lactation. Mated females without delivery were killed on day 26th 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.

BIOMETRIC OF REPRODUCTIVE ORGANS: Yes
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 indices - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.

HISTOLOGICAL TECHNIQUE
The following tissue and organs were collected from all killed males and females at necropsy and fixed in neutral 4% formaldehyde suspension (v/v) for further histopathological evaluation:
relevant gross lesions, pituitary gland, coagulation gland, prostate gland, seminal vesicles, epididymis and testes (fixed in Davidson´s suspension), cervix of uterus, ovaries, uterus and vagina.
For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.

Detailed histological examination was performed on testes (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure). Spermatogenesis and spermatogenic cycle were evaluated according to criteria mentioned in the publication: Hess, R.A.; Quantitative and qualitative Characteristics of the Stages and Transitions in the Cycle of the Rat Seminiferous Epithelium: Light Microscopic Observation of Perfusion-Fixed and Plastic-Embedded Testes (Biology of Reproduction 43, 525-542, 1990). Pathological changes were evaluated according to criteria mentioned in the publication: Creasy, D.M.; Evaluation of Testicular Toxicity in Safety Evaluation Studies: The Appropriate Use of Spermatogenic Staging (Toxicologic Pathology 25, 119-131, 1997).
Postmortem examinations (offspring):
SACRIFICE AND GROSS EXAMINATION OF DEAD PUPS
- on the 4th day of lactation
- dead pups were sexed and externally examined; the stomach was examined for the presence of milk.
- these animals were 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 and 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 counted: Pre-implantation loss, Post implantation loss, Post-natal loss
For each dose group reproduction parameters were counted: Mating index, Fertility index, Gestation index
Offspring viability indices:
Survival index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
males:
- At the mid and highest dose levels treatment-related effects were detected during the health condition control and clinical observation since the 2nd week of application.
- At the mid dose level of 90 mg/kg bw/day, sporadic piloerection, secretion around nostrils, changes in behaviour or dyspnoea were recorded.
- At the highest dose level of 270 mg/kg bw/day, piloerection was observed in most of the males. Some males showed red secretion around nostrils or eyes and changes in behaviour (nervousness, scramble, apathy or droopiness). Dyspnoea and diarrhoea were recorded only sporadically. In the last week of the study cachexy was observed in three males.

females:
- At the highest dose level the treatment-related effects were detected during the health condition control and clinical observation since 2nd week of application: Piloerection (observed in all females); red secretion around nostrils or eyes and changes in behaviour (nervousness, scramble, apathy or droopiness) in some females
- At the end of pregnancy and during the lactation period almost all females of the high dose group showed cachexy, anaemia and light stool. Alopecia and lurch walking were recorded only sporadically.
One moribund female (cachexy, apathy and complicated abort) was found.
Mortality:
mortality observed, treatment-related
Description (incidence):
- At the dose level of of 270 mg/kg bw/day one female died at the 21st day after the mating period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
males:
- Decreased body weights and body weight increments were recorded at the mid and highest dose level during the whole study period. Statistically significant differences were detected at the highest dose level of 270 mg/kg bw/day.

females:
- Body weights of treatment females was dose-dependent decreased during the whole application period. Statistically significant differences were detected at the highest dose level of 270 mg/kg bw/day.
Before mating period: In the 1st week, the average body weight increments of females at the dose level of 270 mg/kg bw/day were slightly decreased as compared to the control group. In the 2nd week the body weight increments of the treated females at the dose level of 270 mg/kg bw/day were statistically significantly decreased as compared to the control group.
Pregnancy: The average body weight increments of mothers at all dose levels were dose-dependently decreased against control mothers for the whole time of pregnancy with statistical significance at the dose level 270 mg/kg bw/day.
Lactation: The average body weight increments of treated mothers at all dose levels were dose-dependently decreased with statistical significance at the dose level 270 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
males:
- The average food consumption of males at all dose levels was dose-dependently decreased against the control males during the whole time of the application period.

females:
- The average food consumption of treated females was dose-dependently decreased as compared to the control females.
Pre-mating period: The average food consumption of females at all dose levels was dose-dependently decreased against control females during the whole time of the pre-mating period.
Pregnancy: The average food consumption of the mothers at all dose levels was dose-dependently decreased against control mothers during the whole time of pregnancy.
Lactation: The average food consumption of treated females was dose-dependently decreased as compared to the control females.
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, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
males: (The incidence of affected males is expressed in numeric form and ranged in sequence of the dose levels 0-30-90-270 mg/kg bw/day further in the text.)
- testes: slight degeneration and atrophy of germ epithelium in testicular tubules of 5-4-3-1 males (it was recorded only in sporadic - less than 10% of the tubules); marked degeneration and atrophy of germ epithelium in testicular tubules or alteration of tubules or retention of spermaides of 0-0-0-3 males (marked damage - more than 20% of the tubules); vacuolation of cytoplasm of sporadic spermiogonium or Sertoli-like cells in 0-0-1-3 males, atrophy of Leydig cells in 0-0-0-3 males, degeneration of spermatides in different stages in 0-0-2-1 males and phagocytosis of spermatides by Sertoli cells in 0-0-0-1 male. The presence of oxalate crystals in artery was recorded in testes of 0-0-0-12 males.
- epididymis: tubules with degeneration and necrosis of epithelium with accumulation of cells dentrites and sperm in the lumen in one male in the mid dose group were observed. The presence of tubules without spermiocytes in the lumen were observed in one treated male in the high dose group
- prostate gland: lymphocyte infiltration of the interstitium or oedema in 2-0-1-1 males. Functional hyperplasia was detected in 0-1-0-0 male and atrophy of alveolar epithelium occurred in the prostate gland in 0-1-0-6 males.
- Atrophy of epithelium of seminal vesicle and coagulation glands were recorded in 0-0-0-4 males and a decreased quantity of spermiocytes as well as development stages of spermiogenesis in the vas deferens were found in 0-0-0-1 male.
- Histopathological changes of the pituitary gland were recorded only sporadically. The presence of oxalate crystals in parenchyma was recorded in 0-0-0-2 males and hyperfunction of the pituitary gland were observed in 0-1-0-0 male.
- kidneys: marked oxalate nephrosis (fare gone oxalate nephrosis with protein casts in the tubules) in 0-0-0-12 males, mild oxalate nephrosis (presence of oxalate crystals, atrophy or necrosis of epithelium) in 0-0-12-0 males and glomerulonephrosis (I.degree) in 0-0-4-0 males.
- The changes in the lymphatic organs were recorded only at the highest dose level of 270 mg/kg bw/day. Atrophic changes in the thymus and atrophy of PALS (periarteriolar lymphoid sheaths) in the spleen or other atrophic changes were observed in 2 males. In the lymph nodes, the following microscopical changes were found: erythrocytophages in dilated sinuses in 4 males and atrophic changes in 2 males.

females:
- Hypoplasia of uterus was recorded in 0-0-0-2 females. In the vagina of 0-0-1-2 females, hypertrophy and hyperplasia of mucined epithelium or atrophy were found. Other findings in the uterus or ovaries were recorded only sporadically. No significant pathological changes were recorded in the pituitary glands of all control and treated females. Only two petty cysts were recorded in one female at the dose level of 90 mg/kg bw/day.
- Treatment related significant changes were recorded in the kidneys of 0-1-12-12 females. Mild oxalate nephropathy with pronounced deposition of birefringent oxalate crystals in dilated cortical tubules lined by flattened epithelium, marked foci of basophilic cortical tubuli, focal peritubular fibrosis and mild focal mononuclear cells infiltrates were recorded in 0-1-12-0 females. In all females at the highest dose level of 270 mg/kg bw/day following changes were more important: marked oxalate nephropathy characterized by severe deposition of birefringent oxalate crystals in dilated cortical tubules lined by flattened epithelium, marked peritubular fibrosis and focal mononuclear cells infiltrates.
- Hemosiderosis and anaemia of the spleen were recorded in 0-0-1-5 females and atrophy of the thymus was found in 0-0-0-5 females.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
- Worse sperm motility was recorded at the mid and high dose level. Fast progressive motion of sperms was observed in 9-10-6-6 males (sequence of the dose levels: 0-30-90-270 mg/kg bw/day). The presence of sperms with changed motility was also increased in the mid and high dose levels. Non-motile sperms were recorded in one male at the highest dose level of 270 mg/kg bw/day.
The percentile proportion of morphologically changed sperms was similar in control males and males at the lowest dose level. Increased percentages of affected sperms were recorded at the mid (9.33%) and highest (8.25%) dose level as compared to the control group (2.75%).
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
- The presence of sperm was not found in one female at the dose levels of 30 and 270 mg/kg bw/day and in two females at the dose level of 90 mg/kg bw/day.
- Aborted females were recorded only in the mid and high dose level.
- The number of females bearing live pups and females with live pups at day 4 after parturition was decreased with dose dependence.
- The number of corpora lutea was slightly decreased at the dose level of 270 mg/kg bw/day. The number of implantations of treated females was lower as compared to the control group (markedly at the dose level of 270 mg/kg bw/day).
- Pre-implantation losses were slightly increased at the dose level of 270 mg/kg bw/day. Markedly increased post-implantation losses were recorded at the dose level of 90 and 270 mg/kg bw/day. No significant differences in post-natal losses were detected between the control and treated mothers.
- The number of pups per litter and litter weights were lower in all treated groups without clear dose relationship.
- The average weight of the litters and average pup weights were decreased at birth and also at day 4 after parturition in the mid and the highest dose level. Abnormal pups were found only at the dose level of 270 mg/kg bw/day.

- In treated males the mating index was slightly decreased as compared to the control males without dose relationship. The fertility index was relatively well-balanced as compared to the control males.
- In treated females the mating index was slightly decreased as compared to the control females without dose relationship. Fertility index and survival index were relatively well-balanced as compared to the control group.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on nephrotoxicity and secondary effects at 90 and 270 mg/kg bw/d.

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
90 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- One pup at the dose level of 30 mg/kg bw/day died during the lactation period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At the dose level of 90 mg/kg bw/day, the average weight of the litters and average weight of the pups were lower when compared to the control group, but without a statistical significance.
- At the dose level of 270 mg/kg bw/day the average body weights of the pups were statistically significantly lower as compared to the control group on the first examination of the litters after parturition and persisted after four days of lactation. The average weight of the litter was decreased in both intervals of weighing.
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
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- At the dose level of 270 mg/kg bw/day pups with empty stomach or with a little milk were recorded in 4 litters out of 7. Cachexy of pups and pale or dry skin were recorded in two litters.
Histopathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
- One pup at the dose levels of 30 and 90 mg/kg bw/day showed pale skin or stomach without milk. At the dose level of 270 mg/kg bw/day stomach without milk or with a little milk were often recorded in all litters.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on adverse effects in pups secondary to parental toxicity at 90 and 270 mg/kg bw/d.

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
90 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes

Applicant's summary and conclusion

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 27th 1995.

Methods

Wistar rats of SPF quality were used for testing. The test substance was administered dissolved in olive oil using a stomach tube; oral application of rats was made daily. The concentrations of suspensions at all dose levels were adjusted to ensure the administered volume of 1 mL per 100 g of body weight. Four groups of animals were included in the study - 3 treated groups (doses 30, 90, 270 mg/kg of body weight/day) and one contro1 group (vehicle only). Each group consisted of 12 males and 12 females. The dose levels for the study were chosen on the basis of the results of the Dose-range finding experiment with 14-day application period. 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 3rd day of lactation,

males - after mating period- totally for 28 days,

nonpregnant females (mated females without parturition) - for 25 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

Administration of the test substance affected clinical status, growth of parental animals, weight of organs and microscopic structure of reproductive male organs and development of pups (increased pre-implantation and post-implantation losses).

The NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION was established at 30 mg/kg body weight/day.

The NOAEL (No Observed Adverse Effect Level) for the DEVELOPMENT of pups was established at 30 mg/kg body weight/day.