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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 December 2008 to 13 July 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD test guideline in compliance with GLP and reported with a valid GLP certificate.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
Identification: DUSANTOX 86lot number: 005/07Purity: Quality of the test article was declared with Certificate of Analysis No 040707/A9Storage conditions: Long term storage: at room temperature, protected against sunlight Stable: at room temperature for 24 months.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Test Animals: 48 adult males and 48 females of Wistar rats from Velaz Praha, Czech Republic were used. The animals were acclimatised for 20 days before the study in the condition identical to the condition during the experiment. The acclimatisation was made according to SOPB-00188-AH, Animals quarantine. The mean bodyweight of animals at the start of experiment was in males 269.8g, in females 185g. Twelve males and twelve females were used in the control and in all dose groups. Husbandry: All animals were kept in cages with bedding in the experimental animal house with standard conditions. The sanitation was made according to standard operation procedure. The temperature was 22± 2°C, relative humidity 45 to 65%. They were followed by a room thermometer and hydrometer placed in the room. The room is equipped with central air-conditioner. The light regime was artificial 12-hour light / 12-hour dark cycle. The keeping of animals was organised to timetable:1. 4 animals in the cage (males and female separately)2. 1 male and 1 female in the cage (mating)3. 1 pregnant female individually4. 1 female and offspring individuallyFood and water: For feeding conventional laboratory diet (Top Dovo, PD Horné Dubové – Naháč) was used with an unlimited supply of drinking water.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
Control article/VehicleIdentification: Olive oil (Olivae oleum raffinatum), product by G. HeessLot number: L 809182Purity: Quality of control article was declared with Certificate.Storage condition: The control article was stored under laboratory conditions in dark container. Justification for choice of vehicle: Dusantox 86 is not soluble in water, but olive oil was used as vehicle. Method of application: The test article was administered per os with the metal stomachic tube everyday at 8 a.m. Doses were calculated on the current bodyweight. The animals were weighed weekly. The fixed application volume of 0.5mL/100g bodyweight was used. Dusantox 86 is not soluble in water; the olive oil was used as a vehicle. The test substance was soluble in olive oil as vehicle every day. The vehicle was applied to control animals in volume 0.5mL/100g bodyweight.
Details on mating procedure:
Normally, 1:1 (1 male to 1 female) mating was used in this study. The female was place with the same male until pregnancy occurs or 14 days have elapsed. Every morning during the mating period the females were examined for the presence if sperm or vaginal plugs. Day 0 of pregnancy was defined as the day a vaginal plug or sperm are found. The pregnant female was kept individually up to 4th day post-partum.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not specified.
Duration of treatment / exposure:
Males - 28 daysFemales - up to four days after birth (54 days)
Frequency of treatment:
Daily
Details on study schedule:
Daily dosing of both sexes began 14 days before mating. Dosing was continued in both sexes during mating period (max. 14 days – 2 complete oestrus cycles). Males were dosed after the mating period at least until the minimum total dosing period of 28 days. They were sacrificed by the overdose of anaesthetic (dietylether). Daily dosing of females were continued throughout pregnancy and up to the 4th day after birth. Female showing no evidence of copulation were dosed 29 days after the last day of mating period. They were sacrificed by overdose of anaesthetic. Females with offpring were sacrificed on day 4 post-partum.
Doses / concentrations
Remarks:
Doses / Concentrations:0, 5, 25 & 50 mg/kg bw/dayBasis:actual ingested
No. of animals per sex per dose:
12 animals per sex, per dose. (Total of 48 rats)
Control animals:
yes, concurrent vehicle
Details on study design:
Groups Designation and Dose Levels: Three test groups and control group (vehicle) were used. Twelve animals of both sexes were used in every level group and in the control group.The animals were marked by serial number 1 – 48. All cages were marked with the number of the group, the dose and the serial number of the animals. The animals in the cage were marked by line I – IIII on the tail. Males were coloured with black and females with red. Selection of doses was estimated from available information about the test article. The low dose was estimated for 5mg.kg-1. The high dose level – 50mg.kg-1 represents 10-fold of the low dose level, medium dose level – 25mg.kg-1 represents 5-fold of the low dose level. Males (black)Females (red)ControlOlive oil(12) 1 -12(12) 1 – 12Low dose group5mg.kg-1(12) 13 – 24(12) 13 – 24Medium dose group25mg.kg-1(12) 25 – 36(12) 25 – 36High dose group50mg.kg-1(12) 37 – 48(12) 37 – 48
Positive control:
Not utilised in this study.

Examinations

Parental animals: Observations and examinations:
General clinical observations were made once a day 1 hour after the test application. The health condition of the animals, reaction of the animals to the applied substance, their condition were monitored every day and recorded. The number of pregnant females was recorded.Bodyweight and Food Consumption: Individual weighing of the adult animals was performed once a week. The bodyweights of animals were recorded.
Oestrous cyclicity (parental animals):
Not measured
Sperm parameters (parental animals):
Not measured
Litter observations:
The number of live pups, weight of litters and sex of pups were observed.
Postmortem examinations (parental animals):
The animals were anesthetized with diethylether and necropsied. Males: at the end of the mating period (after 28 days of treatment period) and pregnant females after 4 days post-partum and non-pregnant females 28 days after mating period. Kidneys, testes, epididymides, uterus and ovaries were weighed during the necropsy. The samples from these organs with prostate and glands vesicles were processed for microscopic examination. Those tissue samples were fixed in bouin solution and prepared in the paraffin technical. The tissue segments were sectioned at 10μm by a Leica micrptome and stained with hematoxylin and eosin. The histological section was examined under a light microscope. Macroscopic Evaluation: At the time of death the animals were examined macroscopically for any abnormalities or pathological changes. Special attention was given to the organs of the reproductive system and the kidneys as target organ. The number of corpora lutea and count of implantation sites was monitored. Histopathological Examination: Histopathological evaluations have a prominent role in reproductive toxicity assessment. Detailed histological examination was performed on the uterus, ovaries, testes, epididydimes and kidneys (the target organ) of the animals of control and highest (50mg.kg-1) dose groups. The histopathological examination was made by standard operation procedures.
Postmortem examinations (offspring):
Postmorten exmainations not performed on offspring.
Statistics:
The results obtained during the study were statistically evaluated by statistical programme Statgraphics. Statistical evaluated was made separately for males, females and litters. For identification homogeneity groups were used the Bartllet’s test. In the case of homogeneity One-Way Analysis of variance with consecutive Multiple Rangers tests was accomplished. In case non homogeneity Kruskal-Wallis One Way Analysis by Ranks was applied.
Reproductive indices:
After sacrificed of all females were calculated reproduction indices Number of females with implantsFertility Index = -------------------------------- x 100 Number of females mating Number of females delivering live youngGestation (Pregnancy) Index = --------------------------------------------- x 100 Number of females with evidence of pregnancy Number of live offspringLive Birth Index = --------------------------------- x 100 Number of offspring delivered
Offspring viability indices:
Number of live offspring at lactation day 4Viability (Survival) Index = --------------------------------------------- x 100Number of live offspring delivered

Results and discussion

Results: P0 (first parental animals)

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
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Mating Procedure: Twelve animals both sexes were used in every level groups. Every female were placed with a single male from the same dose level (1:1 mating) until copulation (1 to 14 days). Females were examined for presence of sperm or vaginal plug daily at 6:30 a.m. The day 0 of the pregnancy was defined as the day presence of vaginal plug or sperm. The copulation of the majority of females in control and all dose groups were established in the first five days. Pregnancy: Twelve pairs in every dose group and in the control group were started. The evidence of copulation was demonstrated in control and in dose group 50mg.kg-1 in twelve females. The sperm of vaginal plugs were found in nine females in dose group 5mg.kg-1 and in eleven females in dose group 25 mg.kg-1. In three females (No 14, 15 & 24) in dose group 5mg.kg-1 and one female (No 30) in dose group of 25 mg.kg-1 were not observed the sperm or vaginal plugs. These females were not pregnant. The majority of females length of pregnancy were 23 days – seven female in the control and the dose group 50mg.kg-1, four animals in the dose group 5mg.kg-1 and six in the dose group of 25mg.kg-1. The length of pregnancy 22 days was established in three females in the control group, in five females in the dose group 5 and 25mg.kg-1 and in four females in the dose group 50mg.kg-1. 21 days of pregnancy were established in one female in the control and the dose group of 50 mg.kg-1. Pathology: Histopathological evaluation have a prominent role in reproductive toxicity assessment. After finishing the test all animals were sacrificed by overdose of anaesthetic and moved to histological evaluation. Special attention was paid to the reproductive system and to the kidneys as target organ. Pathology Results and DiscussionStatistical Analysis of Organ weight Data: Relative weights of organs in the treated animals were not significantly different from those in the control group.Relative weight of observed organs was calculated from formula: A (g)R (%) = ------------ x 100 Weight (g) Gross Pathology (Macroscopical Evaluation): All animals survived until the necropsy. One macerate dead foetus (prenatal age about 18-19 days) was found in the female No 9 from control group in cornis left of uterus. An individual cyst of 2mm was observed in the ovary left of female No 39 from 50mg.kg-1 dose group. Strinking structura of liver tubuli in female No 45 and a change of liver colouration (light brown) in female No 48 were observed, both females from 50mg.kg-1 dose group. Histopathological ExaminationLiver: Several small mononuclear nodules in parenchyma of liver were observed in female No 45 from 50mg.kg-1 dose group. Moderate diffuse small-droplet vacuolization of hepatocytes and individual mononuclear nodules were found in female No 48 950 mg.kg-1 dose group). Kidneys: Focal occurrence of caryopycnosis was seen in 3 females (No 37, 43 & 46) of 50 mg.kg-1 dose group and in 1 female (No 6) from control group. Small focal interstitial lymphocytic infiltrations were observed in 1 (No 4) female and in 1 male (No 5) from the control group. Sporadic glomerular atrophy was found in 1 female (No 44) and in 3 males (No 38, 41 & 44) of 50 mg.kg-1 dose group. Uterus: Focal accumulation of macrophages with yellowish pigment was observed in 1 female (No 1) in the control group. Inflammatory cells infiltration of mucosa and focal inflammatory lesions in submucosa were seen in 1 female (No 9) in cornis left. This lesion was associated with finding of dead foetus. Testes: Hypoplasia of existing cells in the several seinoferous tubles and marked loss of spermatogenic cells were observed in male No 9 in the control group. Epididymis: Moderate perivascular interstitial lymphocytic infiltration was seen in male No 45 in the 50 mg.kg-1 dose group. Prostate: Focal small interstitial lymphocytic lesions were found in 1 male of the control group (No 3) and in 3 males in the 50 mg.kg-1 dose group (No 41, 46 & 48).In the remaining organs and tissues no histopathologic changes were observed. Discussion of Pathological Results: The pathological examination has shown no extensive changes in organs and tissues of rats. The nature and frequency of lesions in the animals in the high dose groups were similar to the control group. We have not seen microscopical change related to the effect of Dusantox 86.

Effect levels (P0)

Dose descriptor:
NOEC
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical observation, bodyweight of animals, food consumption, length of pregnancy, number of pups and weight of litter, number of corpora lutea and implantation sites, relative weight of organs or macroscopical and histological changes.

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)

Alive and dead pups were counted on days 1 and 4. In the control group in female No 3 after birth two pups dead and in female No 9 one death macerate foetus was established. 102 alive and seven dead pups were born in this group. In the dose group 5mg.kg-1 in female No 18 after birth two dead pups were observed. 79 alive and 4 dead pups were in this group. Three pups dead were seen in female No 32 after birth on the dose group 25mg.kg-1. In this group 79 alive and 11 dead pups were born. In the dose group 50mg.kg-1 112 alive and one dead pups were born. In the control and also dose group of 25mg.kg-1 and 50mg.kg-1 were observed two dead pups after 4th day. There was observed in the control group in one female mean count of live pups 9 and in dose group 5 mg.kg-1 and 50 mg.kg-1 and 7 in the dose group 25mg.kg-1 after 1st day. There was seen at one female count of live pups 8 in the control group, 9 in dose group 5mg.kg-1 and 50 mg.kg-1 and 7 in the dose group 25 mg.kg-1 after 4th day. The litters were weighed on day 1 and 4. Mean weight of litter after birth was 53.33g in the control group, 51.67g in the dose group 5mg.kg-1, 43.64g in the dose group 25mg.kg-1 and 58.75 in the dose group 50mg.kg-1. Mean weight of litter at 4th day was 70.83g in the control group, 75.56g in the dose group 5mg.kg-1, 61.356g in the dose group 25mg.kg-1 and 77.92g in the dose group 50mg.kg-1. Offspring were sexed on the 4th day post-partum. Mean sex ratio was 4 males/4 females in the control and in the dose group 5mg.kg-1, 3 males/4females in the dose group 25mg.kg-1 and 4 males/5 females in the dose group 50mg.kg-1.

Effect levels (F1)

Dose descriptor:
NOEC
Generation:
F1
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical observation, bodyweight of animals, food consumption, length of pregnancy, number of pups and weight of litter, number of corpora lutea and implantation sites, relative weight of organs or macroscopical and histological changes.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Bodyweight, Males

Dose level

N

Bodyweight (g) / Day

1

7

14

21

28

Control

 

12/12

272.50± 12.88

286.17± 15.05

301.67± 20.38

321.67± 21.67

330.00± 26.63

Low

5mg.kg-1

12/12

265.83± 16.21

280.83± 16.21

295.00± 15.08

319.17± 18.32

325.83± 18.81

Medium

25mg.kg-1

12/12

270.00± 19.54

287.50± 23.79

299.17± 25.75

326.67± 29.65

331.67± 30.40

High

50mg.kg-1

12/12

270.83± 15.64

281.67± 19.92

299.17± 27.12

322.50± 31.66

329.17± 31.18

 

Table 2. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Bodyweight, Pre-Mating Period, Females

Dose level

N

Bodyweight (g) / Day

1

7

14

 

Control

12/12

185.83± 9.96

186.67± 10.73

190.83± 9.00

Low

5mg.kg-1

12/12

186.67± 9.85

185.83± 10.84

190.00± 11.28

Medium

25mg.kg-1

12/12

183.33± 11.55

185.00± 7.98

188.33± 10.30

High

50mg.kg-1

12/12

184.17± 7.93

186.67± 12.31

188.33± 10.30

 

Table 3. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Bodyweight, Mating Period, Females

Dose level

N

Bodyweight (g) / Day

1

7

14

 

Control

12/12

198.33± 9.37

215.83± 12.40

235.00± 11.68

Low

5mg.kg-1

12/12

199.17± 10.84

216.67± 16.14

232.50± 23.79

Medium

25mg.kg-1

12/12

198.33± 11.93

209.17± 13.11

230.83± 14.43

High

50mg.kg-1

12/12

200.83± 11.65

210.83± 12.40

230.83± 11.65

 

Table 4. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Bodyweight, Pregnancy, Females

Dose level

N

Bodyweight (g) / Day

1

14

21

 

Control

12/12

200.00± 9.54

235.00± 11.68

281.67± 27.1

Low

5mg.kg-1

12/12

202.22± 9.72

240.00± 18.71

288.89± 28.92

Medium

25mg.kg-1

12/12

208.18± 13.28

232.73± 14.89

280.00± 24.08

High

50mg.kg-1

12/12

210.83± 12.40

232.50± 14.22

283.33± 25.70

 

N-Number of animals, the results are presented as mean± SD

 

Table 5. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Bodyweight, F1 generation (Offspring)

Dose level

Bodyweight (g) / Day

N

1

N

4

 

Control

102

53.33± 30.25

101

70.83± 38.49

Low

5mg.kg-1

78

51.67± 23.98

78

75.56± 34.68

Medium

25mg.kg-1

79

43.64± 23.99

77

61.36± 35.08

High

50mg.kg-1

112

58.75± 17.34

110

77.92± 25.89

N-Number of pups, the results are presented as mean± SD

 

Table 6. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Mean Number of Corpora Lutea and Implantation Sites

Dose level

N

Corpora Lutea

Implantation Sites

 

Control

12/12

13.00± 2.13

11.08± 3.50

Low

5mg.kg-1

12/9

10.67± 1.41

10.33± 1.00

Medium

25mg.kg-1

12/11

11.73± 1.56

11.36± 1.21

High

50mg.kg-1

12/12

12.58± 2.39

11.83± 1.95

N-Number of females, the results are presented as mean± SD

 

Table 11. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Relative Weight of Organs, Males

Dose level

N

Kidneys (g)

Testis (g)

Epididymis (g)

Right

Left

Right

Left

Right

Left

 

Control

12/12

0.36± 0.03

0.36± 0.02

0.46± 0.05

0.48± 0.07

0.089± 0.013

0.090± 0.010

Low

5mg.kg-1

12/12

0.36± 0.03

0.35± 0.03

0.48± 0.04

0.47± 0.04

0.086± 0.011

0.089± 0.012

Medium 25mg.kg-1

12/12

0.36± 0.03

0.35± 0.03

0.46± 0.06

0.47± 0.06

0.088± 0.011

0.085± 0.010

High

50mg.kg-1

12/12

0.38± 0.04

0.37± 0.04

0.49± 0.05

0.51± 0.07

0.094± 0.011

0.097± 0.011

 

Table 12. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Relative Weight of Organs, Females

Dose level

N

Kidneys (g)

Ovaries (g)

Uterus (g)

Right

Left

Right

Left

 

Control

12/12

0.037± 0.03

0.37± 0.02

0.38± 0.06

0.36± 0.07

0.29± 0.09

Low

5mg.kg-1

12/12

0.37± 0.04

0.35± 0.04

0.40± 0.08

0.41± 0.10

0.27± 0.04

Medium

25mg.kg-1

12/12

0.37± 0.03

0.36± 0.03

0.38± 0.11

0.38± 0.09

0.29± 0.07

High

50mg.kg-1

12/12

0.38± 0.04

0.36± 0.04

0.34± 0.03

0.37± 0.05

0.27± 0.05

 

N-Number of animals, the results are presented as mean± SD

 

Table 13. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Percentage Evaluation of Results in Females

Dose level

N

Sperm-positive (%)

Pregnant (%)

With Live Pups (%)

 

Control

12/12

100

100

83.33

Low 5

mg.kg-1

12/9

75

75

66.67

Medium

25mg.kg-1

12/11

91.67

91.67

83.33

High

50mg.kg-1

12/12

100

100

100

N-Number of females

 

Table 14. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – F1 Generation (Offspring)

Dose level

Number of Pups

Live Pups (%)

Death Pups (%)

Total

Mean/1 Female

Live

Death

 

Control

109

9

102

7

93.58

6.42

Low

5mg.kg-1

82

9

78

4

95.12

4.88

Medium

25mg.kg-1

90

8

79

11

87.78

12.22

High

50mg.kg-1

113

9

112

1

99.12

0.89

 

Table 15. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Indices of Reproductive Parameters (%)

Dose level

Fertility

Gestation

Live Birth

Viability

 

Control

100

83.33

93.58

98.80

Low

5mg.kg-1

75

88.89

95.12

100

Medium

25mg.kg-1

91.67

90.91

87.78

97.47

High

50mg.kg-1

100

100

99.12

98.21

 

Table 16. Dusantox 86. Reproduction/Developmental Toxicity Screening Test (OECD 421) – Sex Distribution of Pups

Dose level

Total Number

Males

%

Females

%

 

Control

100

53

53.00

47

47.00

Low

5mg.kg-1

78

37

47.44

41

52.56

Medium

25mg.kg-1

77

36

46.75

41

53.25

High

50 mg.kg-1

110

49

49.00

61

61.00

Sex distribution – 4thday post-partum

Applicant's summary and conclusion

Conclusions:
The results of this study indicated that per os application of the test article Dusnatox 86 didn’t show any significant changes in all used doses in comparison with the control animals:in clinical observation, bodyweight of animals, food consumption, length of pregnancy, number of pups and weight of litter, number of corpora lutea and implantation sites, relative weight of organs or macroscopical and histological changes.
Executive summary:

This study was used to provide initial information on possible effects on reproduction and/or development, which may be evoked by Dusantox 86. The Wistar rats, 48 adult males and 48 adult females were divided into four groups. Three groups received the test article Dusantox 86 in graduated doses. The control group received the vehicle. Dusantox 86 is not soluble in water; olive oil was used as a vehicle. The dose of 5mg.kg-1was estimated as the low dose, the dose of 25mg.kg-1as the medium dose level and as the high dose level was selected 50mg.kg-1. Doses were calculated on the current bodyweight. The test article and vehicle were administered per os by metal stomachic tube every day. The males were applied 28 days – 14 days per-mating, 14 days mating. This dosing period (28 days) is considered sufficient to enable detection of the majority of effects on male fertility and spermatogenesis. The females were applicated 54 days – 14 days pre-mating, 14 days mating (with the objective of covering at least two complete oestrus cycles), 22 days gestation, and 4 days lactation. The application volume 0.5mL/100g weight was administered. All animals were monitored for signs of toxicity during application of the test article. All animals were weighed weekly. The bodyweight, food consumption and clinical observation were observed during the study. After finishing the test all animals were sacrificed by overdosing of anaesthetic and moved to macroscopical and histological evaluation. The organs of the reproductive system (the uterus, ovaries, testes, epididydimes, and accessory sex organs) and the kidneys as target organ were monitored. The number of implantation sites and corpora lutea was recorded. The results were elaborated in for of tables. The statistical programme Statgraphics was used for statistically evaluation of the results.

There were no test article-related deaths of animal during the study. There were not any visible signs of intoxication in all animals during clinical observation. The incidence of smooth stool in one animal of dose group 50mg.kg-1males and diarrhoea in one male of dose group 25mg.kg-1were observed. The incidence of alopecia in one female dose group of 50mg.kg-1was observed. These incidences were temporary and may not be indicate relation to the test application. The bodyweight of all animals moderately increased during the study. There were no significant differences between all dose groups and the control groups.

The food consumption was adequate. In control (olive oil) and highest (50mg.kg-1) dose groups 12 females were pregnant. In dose group 5mg.kg-19 females were pregnant and in dose group 25mg.kg-111 females were pregnant. In female dose group 5mg.kg-1three animals and in dose group 25 mg.kg-1one animal were not pregnant. The number of alive pups in all females of the control and all dose groups was stable. The relative weight of kidneys and reproduction organs in both sexes were without statistical differences. Number of corpora lutea and count of implantation sites in females of all dose groups and in the control group were without statistical differences. Detailed histological examination was performed on reproduction organs and kidneys (target organs) of the males and female of the control and the highest dose group. Macroscopical and histopathological findings showed that Dusantox 86 did not cause pathological lesions in organs of treated animals. The sporadic presence of lesions in the animals of the high dose groups was similar to control groups.

CONCLUSION

The results of this study indicated that per os application of the test article Dusnatox 86 didn’t show any significant changes in all used doses in comparison with the control animals:

in clinical observation, bodyweight of animals, food consumption, length of pregnancy, number of pups and weight of litter, number of corpora lutea and implantation sites, relative weight of organs or macroscopical and histological changes.