Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Apr - 20 Jun 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
screening study (OECD 422)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Apr - 20 Jun 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary
Limit test:
no
Species:
rat
Strain:
other: Hsd.Han: of Wistar origin
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt.Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 83 - 85 days (males), 83 - 85 days (females)
- Weight at study initiation: 330 - 384 g (males), 208 - 246 g (females); The weight variation did not exceed ± 20 per cent of the mean weight.
- Housing: 2 animals of the same sex per cage (before mating), 1 male and 1 female per cage (mating), individually (pregnant females), 2 animals per cage (males after mating), 2 or 3 animals of the same sex per cage (recovery animals) in Type III polypropylene/polycarbonate (Size: 22 x 32 x 19 cm (width x length x height)); certified laboratory wood bedding (Lignocel® Hygienic Animal Bedding, J. Rettenmaier & Söhne GmbH+Co.KG, Rosenberg, Germany) suitable as nesting material
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice – breeding and maintenance (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY: The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Budapest,Hungary).


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Details on route of administration:
The route of application was selected in compliance with international guidelines. The oral route is the anticipated route of human exposure to the test item.
Vehicle:
polyethylene glycol
Remarks:
polyethylene glycol 400 (PEG 400)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was formulated in the vehicle at concentrations of 20, 60 and 200 mg/mL in the formulation laboratory of the Test Facility beforehand not longer than for three days before the administration and was stored in a refrigerator until use.

VEHICLE
- Justification for use and choice of vehicle: The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front.
- Concentration in vehicle: 20, 60 and 20 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: 15I040501 (batch number 1), 16I284004 (batch number 2)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations (control of concentration) was performed twice during the study. Five aliquots of 1 mL of each formulation) (20, 60 and 200 mg/mL) and five aliquots of 1 mL control substance (vehicle) were taken and analyzed. The samples were stored at 5 ± 3 °C before the analysis. Concentration of the test item in the dosing formulations varied in the range of 101% to 105% in comparison to the nominal values. The formulation samples were homogenous at both analytical occasions. Recovery of the test material from formulations in the vehicle was 102 and 99% at ~1 and ~300 mg/mL concentrations, respectively. A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation in a separate analytical report. The test material proved to be stable at room temperature for 1 day (recovery was 106% of starting concentrations at 1 mg/mL and 105% at 300 mg/mL) and at 5 ± 3°C for 3 days (recovery was 102% of starting concentrations at 1 mg/mL and 105% at 300 mg/mL).
Duration of treatment / exposure:
56 days (males, control and test groups)
56, 57 or 64 days (females, control and test groups, depending on the effectiveness of mating)
55 days and 14 day post-exposure observation period (recovery groups)
Frequency of treatment:
once daily at similar time (± 2 h), 7 days/week
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Treatment period: 12/sex (control and test groups)
Treatment and recovery period: 5/sex (control group and 1000 mg/kg bw/day)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen on the basis of the results of a preliminary toxicity screening test with the tets material in rats. The high and mid-high doses were chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.
- Post-exposure recovery period in satellite groups: 14 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily and once daily
- Cage side observations included: morbidity and mortality (twice daily), general clinical observations (once daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the times of weekly weighing, prior to and during the mating until necropsy; weekly during the recovery period

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of dosing and weekly thereafter and on the day of necropsy (parental males); on the first day of dosing and weekly thereafter and on gestation days 0, 7, 14 and 21 and on days 0 (within 24 h after parturition), 4 and 13 post-partum (parental females); on day of necropsy (females subjected to organ weighing); weekly during treatment and post-treatment observation period (recovery groups)

FOOD CONSUMPTION:
The food consumption was determined weekly by reweighing the non-consumed diet during the treatment period except mating phase (pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13). Food consumption of male animals was also determined by weekly interval during post-mating period. The food consumption of animals assigned to the recovery groups were weighed by weekly interval during the treatment and post-treatment observation period.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of necropsy (control and test groups), at the end of the 14 days post-treatment period (recovery groups)
- Anaesthetic used for blood collection: Yes (Isofluran)
- Animals fasted: Yes
- How many animals: 5 males and 5 females randomly selected from each group (control and test groups), all animals (recovery groups)
- Parameters examined: white blood cell (leukocyte) count, red blood cell (erythrocyte) count, hemoglobin concentration, hematocrit (relative volume of erythrocytes), mean corpuscular (erythrocyte) volume, mean corpuscular (erythrocyte) hemoglobin, mean corpuscular (erythrocyte) hemoglobin concentration, platelet (thrombocyte) count, reticulocytes, differential white blood cell count (neutrophil, monocyte, lymphocyte, basophil, eosinophil), blood coagulation (activated partial thromboplastin time, prothrombin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy (control and test groups), at the end of the 14 days post-treatment period (recovery groups)
- Animals fasted: Yes
- How many animals: 5 males and 5 females randomly selected from each group (control and test groups), all animals (recovery groups)
- Parameters examined: alanine aminotransferase activity, aspartate aminotransferase activity, total bilirubin concentration, creatinine concentration, urea concentration, glucose concentration, cholesterol concentration, bile acids, sodium concentration, potassium concentration, albumin concentration, total protein concentration

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week (Day 56) but before the blood sampling
- Dose groups that were examined: 5 male and 5 female animals randomly selected from each group
- Battery of functions tested: sensory activity / grip strength / motor activity / other: modified Irwin test

IMMUNOLOGY: No

OTHER: Determination of serum levels of thyroid hormones (T4) from all parental male animals at termination on Day 56.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Terminally (one day after the last treatment), animals were euthanized by exsanguination after verification of deep narcosis by Isofluran and were subjected to gross necropsy (parental male animals: after the optionally extended post-mating period on Day 56; dams not selected for toxicology examinations: on post-partum days 14 - 22 or shortly thereafter (Days 57 or 64); dams selected for toxicology examinations: shortly after post-partum days 15 - 21 (Day 56); recovery animals after 14-day post-treatment observation period (Day 69). Examination of the external appearance and the appearance of the tissues and organs. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded. The uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, ovaries of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution. Thyroid gland was preserved from all adult males and females for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with larynx.
All organs showing macroscopic lesions and the following organs were preserved in 4% buffered formaldehyde solution (except testes and epididymides; see above) for five male and five female animals randomly selected from each group: adrenal glands, aorta, bone with bone marrow and joint (femur), brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), eyes (lachrymal gland with Harderian glands), female mammary gland, gonads (testes with epididymides, ovaries, uterus with fallopian tube and vagina), gross lesions, heart, kidneys, large intestines (caecum, colon, rectum, including Peyer’s patches), liver, lungs (with main stem bronchi; inflation with fixative and then immersion), lymph nodes (submandibular, mesenteric), muscle (quadriceps), esophagus, pancreas, pituitary, prostate, salivary glands (submandibular), sciatic nerve, seminal vesicle with coagulating gland, skin, small intestines (representative regions: duodenum, ileum, jejunum), spinal cord (at three levels: cervical, mid-thoracic and lumbar), spleen, sternum, stomach, thymus, thyroid + parathyroid, trachea, urinary bladder.
The following organs of male adults were weighed: brain, testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole. In addition, for 5 males and females randomly selected from each group, and for recovery animals, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.


HISTOPATHOLOGY: Yes
Detailed histological examination was performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. Additionally, these organs were processed and examined histologically in not mated male animals (1/17 at 100 mg/kg bw/day and 1/17 at 300 mg/kg bw/day), in non-pregnant female and its male pair at 100 mg/kg bw/day and in one dam at 300 mg/kg bw/day based on macroscopic observation. Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose including recovery groups. Histological examinations were performed on the liver in all animals and on organs with macroscopic findings (thymus, uterus) in the low and mid dose groups.
Statistics:
The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible. For evaluation of data obtained during the recovery period, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed. Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
Control, 100, 300 and 1000 mg/kg bw/day: soft stool caused by the vehicle from Day 3 up to the termination of treatment

FEMALES
Control, 100, 300 and 1000 mg/kg bw/day: soft stool caused by the vehicle from Day 3 up to the termination of treatment
Control: alopecia on the forelimbs and on the abdomen in 1/12 animal from gestation Day 9 and gestation Day 21, respectively, up to the termination of the treatment
300 mg/kg bw/day: alopecia on the forelimbs in 1/12 animal between lactation days 4 and 14
1000 mg/kg bw/day: alopecia on the abdomen in 1/11 dams between lactation day 0 and 17
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
1000 mg/kg bw/day: statistically significant slightly increased mean body weight gain comparing to their control during the first week of the recovery period; slightly increased summarized body weight gain during the post-treatment observation period (not statistically significant)
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
MALES
1000 mg/kg bw/day: statistically significant slightly decreased mean daily food consumption during the second week of the premating period
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
100 mg/kg bw/day: statistically significant slightly decreased mean percentage of basophil granulocytes, mean concentration of hemoglobin and mean hematocrit value; statistically significant shorter mean activated thromboplastin time
300 mg/kg bw/day: statistically significant slightly decreased mean percentage of basophil granulocytes, mean concentration of hemoglobin and mean hematocrit value
1000 mg/kg bw/day: statistically significant slightly decreased mean percentage of basophil granulocytes; statistically significant slightly decreased mean percentage of lymphocytes at the end of the recovery period

FEMALES
1000 mg/kg bw/day: statistically significant decreased mean percentage of neutrophil granulocytes, statistically significant increased percentage of lymphocytes and mean platelet count
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
100 mg/kg bw/day: statistically significant slightly decreased mean concentration of albumin
300 mg/kg bw/day: statistically significant slightly decreased mean concentration of albumin
1000 mg/kg bw/day: statistically significant slightly decreased mean activity of alanine aminotransferase; statistically significant slightly decreased mean concentration of potassium in the recovery period

FEMALES
300 mg/kg bw/day: statistically significant slightly increased mean urea concentration
1000 mg/kg bw/day: statistically significant slightly increased mean urea and creatinine concentration
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
1000 mg/kg bw/day: statistically significant slightly increased absolute kidney weight at the end of the recovery period

FEMALES
300 mg/kg bw/day: statistically significant slightly increased absolute liver weight and liver weight relative to brain weight
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
MALES
Control: thymic hemorrhage in 1/12 animal; 3 mm cavity at the renal medulla in 1/5 animal at the end of the recovery period
300 mg/kg bw/day: pale liver in 1/12 animals, thymic hemorrhage in 1/12 animal (non-treatment-related)
1000 mg/kg bw/day: pale liver in 4/12 animals, thymic hemorrhage in 1/12 animal (non-treatment-related)

FEMALES
Control: thymic hemorrhage in 1/12 animal, hydrometra in 2/12 animals, alopecia on the skin of forelimbs and abdomen in 1/12 animal
300 mg/kg bw/day: hydrometra in 1/12 animal (non-treatment-related), dilatation of cecum in 1/12 animal (non-treatment-related), alopecia on the skin of forelimbs in 1/12 animal (non-treatment-related)
1000 mg/kg bw/day: pale liver in 3/11 dams, thymic hemorrhage in 1/11 dam (non-treatment-related), alopecia on the abdomen in 1/11 dam (non-treatment-related), slight hydrometra in 1/11 dam (non-treatment-related); slight hydrometra in 1/1 non-pregnant female (non-treatment-related)
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
MALES
Control: acute thymic hemorrhage in 1/5 animal; hyperplasia of bronchus associated lymphoid tissue in 1/5 animal; alveolar emphysema of minimal degree in 1/5 animal at the end of the recovery period; hyperplasia of bronchus associated lymphoid tissue in 1/5 animal at the end of the recovery period; renal cyst in 1/5 animal
300 mg/kg bw/day: centrilobular microvesicular vacuolation in hepatocytes/ hepatic lipidosis in 3/12 animals; acute thymic hemorrhage in 1/5 animal (non-treatment-related)
1000 mg/kg bw/day: centrilobular microvesicular vacuolation in hepatocytes/ hepatic lipidosis in 7/12 animals; alveolar emphysema in 1/5 animal (non-treatment-related); acute thymic hemorrhage in 1/5 animal (non-treatment-related); alveolar emphysema of minimal degree in 1/5 animal at the end of the recovery period (non-treatment-related)

FEMALES
Control: dilatation of uterine horns in 2/12 animals; alveolar emphysema in 1/5 animal; acute thymic hemorrhage in 1/5 animal; hyperplasia of bronchus associated lymphoid tissue in 1/5 animal at the end of the recovery period
300 mg/kg bw/day: dilatation of uterine horns in 1/12 animal (non-treatment-related)
1000 mg/kg bw/day: dilatation of uterine horns in 1/12 animal at the end of the treatment period and in 2/5 animals at the end of the recovery period (non-treatment-related); centrilobular microvesicular vacuolation in hepatocytes/ hepatic lipidosis in 3/12 animals; alveolar emphysema in 1/5 animal (non-treatment-related); acute thymic hemorrhage in 1/5 animal (non-treatment-related); hyperplasia of bronchus associated lymphoid tissue in 1/5 animal (non-treatment-related)
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the thyroid hormone (free T4) level in parental male animals compared to the control.
Details on results:
CLINICAL SIGNS
Soft stool was observed in all animals at all dose groups and was attributed to the vehicle. Alopecia is a common spontaneous finding in this strain of experimental rats and is seen also in untreated rats. The effect occurred in single animals of control, mid and high dose groups in this study. Therefore, it was considered to be an incidental finding and not related to the treatment with the test item.

BODY WEIGHT AND BODY WEIGHT GAIN
Statistical significant slightly increased mean body weight gain was observed in males during the first week of the recovery period at the highest dose group. This resulted in a slightly higher summarized body weight gain during the post-treatment observation period and caused no significant difference with respect to the control in the mean body weight. This difference was not considered treatment-related.

FOOD CONSUMPTION
The slightly lower mean daily food consumption in males of the high dose group (1000 mg/kg bw/day) compared to controls was with minor degree and was transient and therefore was judged to be toxicologically not relevant.

HAEMATOLOGICAL FINDINGS
The differences in several haemotological parameters with respect to control were judged to be indicative of biological variation and toxicologically not relevant. Changes in basophil granulocytes, hemoglobin, hematocrit and activated thromboplastin time in treated male animals were of minor degree and not related to the doses at the end of the treatment period. Moreover, all values were well within the historical ranges and statistical significances were mainly originated from the relative high value of the control group (hemoglobin, hematocrit and activated thromboplastin time). Slight changes in the neutrophil granulocytes, lymphocytes and platelet count were only observed in female animals at the end of the post-treatment observation period but not at the end of the treatment period.

CLINICAL BIOCHEMISTRY FINDINGS
Higher concentrations of urea and creatinine were indicative of altered renal function in in female animals at 300 or 1000 mg/kg bw/day. Changes in concentrations of creatinine and urea in serum were considered to be signs of adaptation of the organ to the altered demands. Without supporting histopathological changes, these effects were judged to be toxicologically not relevant. The slight, but statistically significant differences compared to the controls with respect to albumin and potassium concentration, were considered to have little or no toxicological relevance. Decrease in enzyme activity of alanine aminotransferase has no toxicological relevance without tissue damage. Albumin concentrations changed only in the lower dose groups but not at the high dose.

GROSS PATHOLOGICAL FINDINGS
Hemorrhage in the thymus was due to circulatory disturbance developed during the exsanguination procedure. Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats. Cecum dilatation was an individual change probably due to an increased water accumulation as there were no accompanying histological findings. Alopecia on the skin is a common observation in this strain of experimental rats, which occurred with similar incidence in the control and test item treated female animals (300 and 1000 mg/kg bw/day) in this study. Therefore, these macroscopic changes were considered to be independent from the treatment with the test item.

ORGAN WEIGHTS
The slight increases in liver weights in females at 300 mg/kg bw/day were not considered to be toxicologically relevant due to the small degree and in the lack of associated histopathological alterations as well as in the absence of similar changes in the high dose group.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC
The dilatation of uterine horns observed in females is not associated with inflammatory or other pathological lesions and is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
The treatment related observed hepatic lipidosis was of minimal or mild degree and and is considered to be a slight reversible liver injury and it might be in connection with a disturbance of energy metabolism of affected hepatocytes.
The acute changes in the lungs (alveolar emphysema) and thymus (hemorrhage) in control or high dose treated animals were considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguinations.
Hyperplasia of bronchus associated lymphoid tissue as observed in males and females of the control as well as of the highest dose group is a physiological immune-morphological phenomenon, without toxicological significance.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1: Summary of clinical signs in males (pre-mating, mating and post-mating periods)

Observations

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Treatment period*

Recovery period

Treatment period*

Recovery period

Normal

17/17

5/5

12/12

12/12

17/17

5/5

Soft stool

17/17

5/5

12/12

12/12

17/17

5/5

Frequency of observations: number of animals (cage) with observation/number of animals (cage) examined

* Including animals of recovery group

 

Table 2: Summary of clinical signs in females

Observations

Control

100 mg/kg bw/day

300 mg/kg bw/day

600 mg/kg bw/day

Pre-mating and mating periods

Normal

12/12

12/12

12/12

12/12

Soft stool

12/12

12/12

12/12

12/12

Post-mating periods

Normal

-

1/1

-

1/1

Soft stool

-

1/1

-

1/1

Gestation period

Normal

11/12

11/11

12/12

11/11

Soft stool

12/12

11/11

12/12

11/11

Skin: Alopecia

1/12

0/11

0/12

0/11

Lactation period

Normal

11/12

11/11

11/12

10/11

Soft stool

12/12

11/11

12/12

11/11

Skin: Alopecia

1/12

0/11

1/12

1/11

Recovery animals

Normal

5/5

5/5

5/5

5/5

Soft stool

5/5

5/5

5/5

5/5

 

Table 3: Summary of body weight gain in males

Group

 

Body weight gain (g) between

Pre-mating days

Mating and post-mating days

Total

0 – 7

7 – 13

0 – 13

13 – 20

20 – 27

27 – 34

34 – 41

41 – 48

48 - 55

0 – 55

Control

Mean

17.9

17.2

35.1

14.5

11.3

10.2

7.9

10.9

11.5

101.4

SD

4.5

4.1

6.1

4.7

4.2

6.7

5.1

3.5

4.5

17.2

n

17

17

17

17

17

17

17

17

17

17

100 mg/kg bw/day

Mean

18.3

15.6

33.9

15.7

10.8

11.0

8.4

10.9

12.1

102.8

SD

7.7

5.6

10.1

4.6

5.2

4.0

4.8

8.4

7.1

18.9

n

12

12

12

12

12

12

12

12

12

12

300 mg/kg bw/day

Mean

15.8

16.6

32.4

10.8

12.7

10.2

10.0

13.6

10.8

100.4

SD

6.2

4.1

9.1

8.5

6.8

4.3

4.0

4.7

3.7

22.2

n

12

12

12

12

12

12

12

12

12

12

1000 mg/kg bw/day

Mean

16.3

15.0

31.3

12.2

11.5

13.1

7.4

11.2

8.0

94.6

SD

5.6

4.3

8.1

7.5

4.6

5.4

3.4

4.9

4.3

20.0

n

17

17

17

17

17

17

17

17

17

17

 

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS = Not Significant

 

Table 4: Summary of body weight gain in males of the recovery group

Group

 

Body weight gain (g) between

Recovery days

0 – 6

6 – 13

0 – 13

Control

Mean

4.8

0.8

5.6

SD

3.8

3.1

6.5

n

5

5

5

1000 mg/kg bw/day

Mean

11.0*

3.6

14.6

SD

4.4

4.6

7.8

n

5

5

5

 

*

NS

NS

* p < 0.05

 

Table 5: Summary of body weight gain in females

Group

 

Body weight gain (g) between

Pre-mating days

Treatment days

Total

0 – 7

7 – 13

0 – 13

13 – 20

20 – 27

27 – 34

34 – 41

41 – 48

0 – 55

Control

Mean

8.2

7.2

15.4

6.0

2.6

7.0

4.0

1.8

39.6

SD

6.4

7.1

6.8

4.0

7.6

4.5

2.6

4.9

10.0

n

17

17

17

5

5

5

5

5

5

100 mg/kg bw/day

Mean

11.3

6.4

17.8

-

-

-

-

-

-

SD

9.1

7.7

4.5

-

-

-

-

-

-

n

12

12

12

-

-

-

-

-

-

300 mg/kg bw/day

Mean

13.3

5.1

18.3

-

-

-

-

-

-

SD

7.1

5.0

9.0

-

-

-

-

-

-

n

12

12

12

-

-

-

-

-

-

1000 mg/kg bw/day

Mean

10.1

8.4

18.5

8.8

-2.2

9.6

0.6

5.0

39.4

SD

9.6

6.4

9.5

2.3

6.9

4.2

4.5

1.2

9.4

n

17

17

17

5

5

5

5

5

5

 

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS = Not Significant

 

Table 6: Summary of body weight gain in females of the recovery group

Group

 

Body weight gain (g) between

Recovery days

0 – 6

6 – 13

0 – 13

Control

Mean

0.8

0.0

0.8

SD

7.7

1.9

7.3

n

5

5

5

1000 mg/kg bw/day

Mean

6.6

-1.0

5.6

SD

6.66

3.9

3.0

n

5

5

5

 

NS

NS

NS

NS = Not Significant

 

 

Table 7: Summary of food consumption in males

Group

 

Daily mean food consumption (g/animal/day)

Pre-mating days

Post-mating days

Recovery period

0 – 7

7 – 13

20 – 27

27 – 34

34 – 41

41 – 48

48 – 55

Day 0 – 6

Day 6 – 13

Control

Mean

25.9

27.4

24.9

24.9

24.5

25.0

25.1

24.4

24.6

SD

1.93

1.46

1.59

1.86

1.25

1.26

1.54

0.55

0.35

n

8.00

8.00

8.00

8.00

8.00

8.00

8.00

2.00

2.00

100 mg/kg bw/day

Mean

26.0

27.1

24.8

24.9

24.9

25.1

24.9

-

-

SD

1.52

2.03

1.09

0.85

0.73

1.66

1.66

-

-

n

6

6

5

5

6

6

6

-

-

±%

0

-1

0

0

1

0

-1

-

-

300 mg/kg bw/day

Mean

25.8

26.2

24.4

24.6

24.6

25.1

25.1

-

-

SD

2.22

1.31

1.38

1.16

0.94

1.27

1.16

-

-

n

6

6

6

6

6

6

6

-

-

±%

-1

-4

-2

-1

0

1

0

-

-

1000 mg/kg bw/day

Mean

24.7

25.2*

24.0

24.2

24.0

24.3

23.6

25.1

25.6

SD

2.30

1.80

2.16

1.91

1.65

1.54

1.3

1.73

2.54

n

8

8

8

8

8

8

8

2

2

±%

-5

-8

-4

-3

-2

-3

-6

3

4

 

NS

DN

NS

NS

NS

NS

NS

NS

NS

* p < 0.05

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 8: Summary of food consumption in females

Group

 

 

Daily mean food consumption (g/animal/day)

Pre-mating days

Post-mating days

Recovery period

0 – 7

7 – 13

13 – 20

20 – 27

27 – 34

34 – 41

41 – 48

48 – 55

Day 0 – 6

Day 6 – 13

Control

Mean

19.7

19.4

17.7

19.3

18.3

17.7

17.3

17.0

16.9

17.3

SD

2.36

1.06

1.94

0.12

0.99

0.62

0.84

0.00

1.77

0.61

n

8

8

2

2

2

2

2

2

2

2

100 mg/kg bw/day

Mean

19.8

19.0

-

-

-

-

-

-

-

-

SD

1.53

1.26

-

-

-

-

-

-

-

-

n

6

6

-

-

-

-

-

-

-

-

±%

0

-2

-

-

-

-

-

-

-

-

300 mg/kg bw/day

Mean

18.9

18.8

-

-

-

-

-

-

-

-

SD

1.18

1.69

 

 

 

 

 

 

 

 

n

6

6

-

-

-

-

-

-

-

-

±%

-4

-3

-

-

-

-

-

-

-

-

1000 mg/kg bw/day

Mean

18.6

19.0

17.4

16.5

17.8

16.6

17.5

16.8

18.6

18.9

SD

0.96

2.00

0.62

1.92

1.03

1.16

0.45

0.13

0.12

0.02

n

8

8

2

2

2

2

2

2

2

2

±%

-6

-2

-2

-14

-3

-6

1

-1

10

10

 

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS = Not Significant

 

 

Table 9: Summary of hematology

Group

 

Basophil [%]

Hemoglobin [g/L]

Hematocrit [L/L]

Neutrophil [%]

Lymphocyte [%]

Mean platelet count [×10E9/L]

Activated partial thromboplastin time [sec]

MALES – TREATMENT PERIOD

Control

Mean

0.16

175.40

0.47

23.80

72.40

891.60

23.60

SD

0.05

9.34

0.03

5.06

5.74

118.00

2.60

n

5

5

5

5

5

5

5

100 mg/kg bw/day

Mean

0.04*

161.60**

0.43**

25.08

70.62

901.40

20.48*

SD

0.09

4.72

0.01

5.03

5.26

88.05

0.96

n

5

5

5

5

5

5

5

±%

-75

-8

-9

5

-2

1

-13

300 mg/kg bw/day

Mean

0.02**

163.80**

0.44*

20.96

74.20

897.40

21.72

SD

0.04

2.95

0.00

3.74

4.21

137.05

1.91

n

5

5

5

5

5

5

5

±%

-88

-7

-6

-12

2

1

-8

1000 mg/kg bw/day

Mean

0.04**

169.00

0.45

17.64

77.98

796.60

22.22

SD

0.05

3.67

0.01

6.23

7.44

54.62

1.43

n

5

5

5

5

5

5

5

±%

-75

-4

-5

-26

8

-11

-6

 

DN

DN

U

NS

NS

NS

DN

MALES – RECOVERY PERIOD

Control

Mean

0.08

176.20

0.47

16.44

78.94

823.80

19.48

SD

0.04

3.11

0.01

3.44

3.44

83.96

3.49

n

5

5

5

5

5

5

5

1000 mg/kg bw/day

Mean

0.02

107.40

0.46

22.28

72.40*

833.40

20.24

SD

0.04

5.81

0.01

5.06

4.94

42.55

1.85

n

5

5

5

5

5

5

5

±%

-75

-3

-2

36

-8

1

4

 

NS

NS

NS

NS

*

NS

NS

FEMALES – TREATMENT PERIOD

Control

Mean

0.10

159.20

0.43

25.68

69.78

1016.20

23.44

SD

0.22

8.32

0.01

7.76

7.54

151.26

1.11

n

5

5

5

5

5

5

5

100 mg/kg bw/day

Mean

0.04

169.00

0.45

16.70

79.02

1079.80

23.94

SD

0.09

9.67

0.02

3.57

2.94

181.70

2.09

n

5

5

5

5

5

5

5

±%

-60

6

4

-35

13

6

2

300 mg/kg bw/day

Mean

0.10

152.00

0.41

21.18

75.50

1064.20

22.12

SD

0.10

15.02

0.03

9.78

10.17

195.41

1.62

n

5

5

5

5

5

5

5

±%

0

-5

-4

-18

8

5

-6

1000 mg/kg bw/day

Mean

0.10

162.80

0.43

21.90

73.34

996.20

21.80

SD

0.10

5.67

0.01

4.39

5.74

79.97

0.70

n

5

5

5

5

5

5

4

±%

0

2

1

-15

5

-2

-7

 

NS

NS

NS

NS

NS

NS

NS

FEMALES – RECOVERY PERIOD

Control

Mean

0.14

160.20

0.44

18.94

74.84

744.40

21.76

SD

0.13

9.86

0.02

2.23

3.40

75.68

1.33

n

5

5

5

5

5

5

5

1000 mg/kg bw/day

Mean

0.04

164.20

0.45

15.18*

80.86*

852.00*

20.30

SD

0.09

5.45

0.01

1.92

2.28

67.62

1.18

n

5

5

5

5

5

5

5

±%

-71

2

3

-20

8

14

-7

 

NS

NS

NS

*

*

*

NS

* p < 0.05, **p < 0.01

NS = Not Significant

U = Mann-Whitney U-test vs. Control

DN = Duncan´s multiple range test

 

Table 10: Summary of clinical biochemistry findings

Group

 

Albumin [g/L]

Alanine aminotransferase activity [U/L]

Urea [mmol/L]

Creatinine [µmol/L]

Potassium [mmol/L]

MALES – TREATMENT PERIOD

Control

Mean

37.16

70.98

8.10

28.04

4.71

SD

1.08

18.19

0.99

3.22

0.54

n

5

5

5

5

5

100 mg/kg bw/day

Mean

35.04**

56.78

8.22

31.24

4.67

SD

1.33

13.38

0.90

2.22

0.15

n

5

5

5

5

5

±%

-6

-20

1

11

-1

300 mg/kg bw/day

Mean

35.74*

56.76

8.20

29.90

4.74

SD

0.69

8.28

1.12

3.21

0.22

n

5

5

5

5

5

±%

-4

-20

1

7

1

1000 mg/kg bw/day

Mean

36.70

49.66*

8.68

30.50

4.57

SD

0.76

6.45

1.23

1.47

0.45

n

5

5

5

5

5

±%

-1

-30

7

9

-3

 

DN

DN

NS

NS

NS

MALES – RECOVERY PERIOD

Control

Mean

36.36

60.22

8.04

27.28

4.79

SD

0.92

7.23

0.63

1.19

0.19

n

5

5

5

5

5

1000 mg/kg bw/day

Mean

35.60

69.60

1.72

26.40

4.42*

SD

0.69

11.83

0.31

3.50

0.19

n

5

5

5

5

5

±%

-2

16

11

-3

-8

 

NS

NS

NS

NS

*

FEMALES – TREATMENT PERIOD

Control

Mean

34.24

53.30

7.87

27.56

4.39

SD

0.46

12.42

1.32

1.44

0.47

n

5

5

5

5

5

100 mg/kg bw/day

Mean

35.02

50.46

8.90

30.34

4.13

SD

1.04

4.34

0.90

2.34

0.18

n

5

5

5

5

5

±%

2

-5

13

10

-6

300 mg/kg bw/day

Mean

34.26

64.36

10.07*

30.42

4.65

SD

1.23

5.54

1.12

2.01

0.38

n

5

5

5

5

5

±%

0

21

28

10

6

1000 mg/kg bw/day

Mean

33.48

54.94

10.35*

32.30*

4.39

SD

0.37

19.24

1.97

3.78

0.20

n

5

5

5

5

5

±%

-2

3

32

17

0

 

NS

NS

DN

DN

NS

FEMALES – RECOVERY PERIOD

Control

Mean

39.22

53.32

7.66

31.86

4.19

SD

1.70

14.32

0.81

4.40

0.31

n

5

5

5

5

5

1000 mg/kg bw/day

Mean

39.04

62.18

8.24

32.98

3.95

SD

1.69

20.47

0.66

4.23

0.15

n

5

5

5

5

5

±%

0

17

8

4

-6

 

NS

NS

NS

NS

NS

* p < 0.05, **p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 11: Summary of necropsy findings

Organs

Observations

Frequency of observations per group

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Main group

Recovery group

 

 

Main group

Recovery group

MALES

 

No macroscopic finding

11/12

4/5

12/12

10/12

8/12

5/5

Thymus

Hemorrhages

1/12

0/5

0/12

1/12

1/12

0/5

Kidney

3 mm cavity at the renal medulla

0/12

1/5

0/12

0/12

0/12

0/5

Liver

Pale

0/12

0/5

0/12

1/12

4/12

0/5

FEMALES - DAMS

 

No macroscopic finding

8/12

5/5

11/11

9/12

6/11

3/5

Thymus

Hemorrhages

1/12

0/5

0/11

0/12

1/11

0/5

Uterus

Hydrometra

2/12

0/5

0/11

1/12

1/11

2/5

Cecum

Dilatation

0/12

0/5

0/11

0/12

3/11

0/5

Liver

Pale

0/12

0/5

0/11

0/12

3/11

0/5

Skin

Alopecia

1/12

0/5

0/11

1/12

1/11

0/5

FEMALES – NON-PREGNANT

 

No macroscopic finding

NA

NA

1/1

NA

0/1

NA

Uterus

Hydrometra

NA

NA

0/1

NA

1/1

NA

Frequency of observation =number of animals with observations / number of animals examined

NA = not applicable

 

Table 12: Summary of organ weights

 

 

Body weight [g]

Organ weight [g]

Organ weight relative to body weight [%]

Organ weight relative to brain weight [%]

Group

 

 

Liver

Kidney

Liver

Kidney

Liver

Kidney

MALES – TREATMENT PERIOD

Control

Mean

448.4

11.31

2.70

2.604

0.622

518.31

123.81

SD

24.45

1.05

0.17

0.179

0.037

40.16

7.90

n

12

5

5

5

5

5

5

100 mg/kg bw/day

Mean

443.5

11.65

2.62

2.632

0.595

533.25

120.14

SD

29.02

1.86

0.22

0.235

0.039

70.18

7.46

n

12

5

5

5

5

5

5

±%

-1

3

-3

1

-4

3

-3

300 mg/kg bw/day

Mean

444.8

12.43

2.77

2.740

0.610

578.38

129.37

SD

34.75

0.84

0.32

0.201

0.057

43.08

19.33

n

12

5

5

5

5

5

5

±%

-1

10

3

5

-2

12

4

1000 mg/kg bw/day

Mean

430.7

12.07

12.82

2.768

0.647

555.54

129.84  

SD

34.57

1.25

0.30

0.263

0.082

43.19

14.08

n

12

5

5

5

5

5

5

±%

-4

7

4

6

4

7

5

 

NS

NS

NS

NS

NS

NS

NS

MALES – RECOVERY PERIOD

Control

Mean

448.8

11.68

2.54

2.599

0.566

436.09

116.69

SD

23.21

1.19

0.19

0.193

0.018

45.94

7.51

n

5

5

5

5

5

5

5

1000 mg/kg bw/day

Mean

458.2

11.93

2.82*

2.607

0.618

544.92

129.01

SD

27.07

0.94

0.13

0.187

0.049

41.69

9.65

n

5

5

5

5

5

5

5

±%

2

2

11

0

9

2

11

 

NS

NS

*

NS

NS

NS

NS

FEMALES – TREATMENT PERIOD

Control

Mean

248.8

7.92

1.78

3.183

0.716

392.68

88.44

SD

7.29

0.45

0.12

0.137

0.052

15.47

7.80

n

5

5

5

5

5

5

5

100 mg/kg bw/day

Mean

263.6

8.29

1.83

3.142

0.695

406.12

89.58

SD

8.88

0.63

0.09

0.152

0.026

40.55

4.22

n

5

5

5

5

5

5

5

±%

6

5

3

-1

-3

3

1

300 mg/kg bw/day

Mean

270.8*

9.45*

1.88

3.481

0.695

469.07*

93.36

SD

14.20

1.23

0.11

0.295

0.049

61.20

6.89

n

5

5

5

5

5

5

5

±%

9

19

6

9

-3

19

6

1000 mg/kg bw/day

Mean

264.0

9.00

1.94

3.404

0.737

447.20

96.67

SD

13.11

1.05

0.13

0.288

0.063

40.25

5.75

n

5

5

5

5

5

5

5

±%

6

14

9

7

3

14

9

 

DN

DN

NS

NS

NS

DN

NS

FEMALES – RECOVERY PERIOD

Control

Mean

255.6

6.99

1.56

2.738

0.614

350.45

78.40

SD

18.20

0.86

0.12

0.309

0.057

47.29

6.87

n

5

5

5

5

5

5

5

1000 mg/kg bw/day

Mean

264.6

7.17

1.67

2.705

0.630

371.57

86.39

SD

18.70

0.81

0.12

0.118

0.021

33.63

4.98

n

5

5

5

5

5

5

5

±%

4

3

7

-1

3

6

10

 

NS

NS

NS

NS

NS

NS

NS

* p < 0.05, **p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 13: Summary of histopathological findings

Organs

Observations

Incidence of observations per group

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Main group

Recovery group

 

 

Main group

 

Recovery group

MALES

Kidneys

Cyst

0/5

1/5

-

-

0/5

0/5

Liver

Centrilobular vacuolation in hepatocytes

0/12

0/5

0/12

3/12

7/12

0/5

Lungs

Alveolar emphysema

0/5

1/5

-

-

1/5

1/5

Hyperplasia of BALT

1/5

1/5

-

-

0/5

0/5

Thymus

Acute hemorrhage

1/5

0/5

-

1/1

1/5

0/5

FEMALES

Kidneys

Cyst

0/5

0/5

-

-

0/5

0/5

Liver

Centrilobular vacuolation in hepatocytes

0/12

0/5

0/11

0/11

3/12

0/5

Lungs

Alveolar emphysema

1/5

0/5

-

-

1/5

0/5

Hyperplasia of BALT

0/5

1/5

-

-

1/5

0/5

Thymus

Acute hemorrhage

1/5

0/5

-

-

1/5

0/5

Uterus with cervix

Dilatation

2/12

0/5

0/1

1/1

1/12

2/5

Incidence of observation =number of animals with observations / number of animals examined

BALT = bronchus associated lymphoid tissue

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for systemic toxicity after oral administration via gavage of the test substance to male Hsd.Han: Wistar rats was 100 mg/kg bw/day. The NOAEL for systemic toxicity after oral administration via gavage of the test substance to female Hsd.Han: Wistar rats was 300 mg/kg bw/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted in 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test)
Version / remarks:
adopted in 2000
GLP compliance:
yes (incl. QA statement)
Remarks:
Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate
EC Number:
276-957-5
EC Name:
7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate
Cas Number:
72869-86-4
Molecular formula:
C23H38N2O8
IUPAC Name:
7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate

Test animals

Species:
rat
Strain:
other: Hsd.Han: of Wistar origin
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt.Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 83 - 85 days (males), 83 - 85 days (females)
- Weight at study initiation: 330 - 384 g (males), 208 - 246 g (females); The weight variation did not exceed ± 20 per cent of the mean weight.
- Housing: 2 animals of the same sex per cage (before mating), 1 male and 1 female per cage (mating), individually (pregnant females), 2 animals per cage (males after mating), 2 or 3 animals of the same sex per cage (recovery animals) in Type III polypropylene/polycarbonate (Size: 22 x 32 x 19 cm (width x length x height)); certified laboratory wood bedding (Lignocel® Hygienic Animal Bedding, J. Rettenmaier & Söhne GmbH+Co.KG, Rosenberg, Germany) suitable as nesting material
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice – breeding and maintenance (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY: The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Budapest,Hungary).


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
polyethylene glycol 400 (PEG 400)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was formulated in the vehicle at concentrations of 20, 60 and 200 mg/mL in the formulation laboratory of the Test Facility beforehand not longer than for three days before the administration and was stored in a refrigerator until use.

VEHICLE
- Justification for use and choice of vehicle: The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front.
- Concentration in vehicle: 20, 60 and 20 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: 15I040501 (batch number 1), 16I284004 (batch number 2)
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until copulation occurred
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations (control of concentration) was performed twice during the study. Five aliquots of 1 mL of each formulation (20, 60 and 200 mg/mL) and five aliquots of 1 mL control substance (vehicle) were taken and analyzed. The samples were stored at 5 ± 3 °C before the analysis. Concentration of the test item in the dosing formulations varied in the range of 101% to 105% in comparison to the nominal values. The formulation samples were homogenous at both analytical occasions. Recovery of the test material from formulations in the vehicle was 102 and 99% at ~1 and ~300 mg/mL concentrations, respectively. A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation in a separate analytical report. The test material proved to be stable at room temperature for 1 day (recovery was 106% of starting concentrations at 1 mg/mL and 105% at 300 mg/mL) and at 5 ± 3°C for 3 days (recovery was 102% of starting concentrations at 1 mg/mL and 105% at 300 mg/mL).
Duration of treatment / exposure:
56 days (males, control and test groups)
56, 57 or 64 days (females, control and test groups, depending on the effectiveness of mating)
55 days and 14 day post-exposure observation period (recovery groups)
Frequency of treatment:
once daily at similar time (± 2 h), 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Treatment period: 12/sex (control and test groups)
Treatment and recovery period: 5/sex (control group and 1000 mg/kg bw/day)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen on the basis of the results of a preliminary toxicity screening test with the tets material in rats. The high and mid-high doses were chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily and once daily
- Cage side observations included: morbidity and mortality (twice daily), general clinical observations (once daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the times of weekly weighing, prior to and during the mating until necropsy; weekly during the recovery period

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of dosing and weekly thereafter and on the day of necropsy (parental males); on the first day of dosing and weekly thereafter and on gestation days 0, 7, 14 and 21 and on days 0 (within 24 h after parturition), 4 and 13 post-partum (parental females); on day of necropsy (females subjected to organ weighing); weekly during treatment and post-treatment observation period (recovery groups)

FOOD CONSUMPTION:
The food consumption was determined weekly by reweighing the non-consumed diet during the treatment period except mating phase (pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13). Food consumption of male animals was also determined by weekly interval during post-mating period. The food consumption of animals assigned to the recovery groups were weighed by weekly interval during the treatment and post-treatment observation period.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of necropsy (control and test groups), at the end of the 14 days post-treatment period (recovery groups)
- Anaesthetic used for blood collection: Yes (Isofluran)
- Animals fasted: Yes
- How many animals: 5 males and 5 females randomly selected from each group (control and test groups), all animals (recovery groups)
- Parameters examined: white blood cell (leukocyte) count, red blood cell (erythrocyte) count, hemoglobin concentration, hematocrit (relative volume of erythrocytes), mean corpuscular (erythrocyte) volume, mean corpuscular (erythrocyte) hemoglobin, mean corpuscular (erythrocyte) hemoglobin concentration, platelet (thrombocyte) count, reticulocytes, differential white blood cell count (neutrophil, monocyte, lymphocyte, basophil, eosinophil), blood coagulation (activated partial thromboplastin time, prothrombin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy (control and test groups), at the end of the 14 days post-treatment period (recovery groups)
- Animals fasted: Yes
- How many animals: 5 males and 5 females randomly selected from each group (control and test groups), all animals (recovery groups)
- Parameters examined: alanine aminotransferase activity, aspartate aminotransferase activity, total bilirubin concentration, creatinine concentration, urea concentration, glucose concentration, cholesterol concentration, bile acids, sodium concentration, potassium concentration, albumin concentration, total protein concentration

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week (Day 56) but before the blood sampling
- Dose groups that were examined: 5 male and 5 female animals randomly selected from each group
- Battery of functions tested: sensory activity / grip strength / motor activity / other: modified Irwin test

IMMUNOLOGY: No

OTHER: Determination of serum levels of thyroid hormones (T4) from all parental male animals at termination on Day 54.
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears each day before the treatment started from each animal being considered for study for two weeks. Estrous cycle was evaluated and considered at randomization. Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating. Vaginal smears were also prepared on the day of the necropsy. Vaginal smears were stained with 1% aqueous methylene blue solution and were examined with a light microscope.
Sperm parameters (parental animals):
Parameters examined in all parental males of the control and high dose group:
testis weight, epididymis weight, other: quantity and morphology of spermatogenic cells (spermatogonia, spermatocytes, spermatids and spermatozoa) representing different stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4 pups/sex/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other: runts (pups that are significantly smaller than normal pups)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead; on the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn) from pups died after the birth (dead pups)
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after the optionally extended post-mating period on Day 56.
- Maternal animals: All surviving animals not selected for toxicology examinations on post-partum days 14 - 22 or shortly thereafter (Days 57 or 64). All surviving animals selected for toxicology examinations shortly after post-partum days 15 - 21 (Day 56).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs of male adults were weighed: brain, testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole. In addition, for 5 males and females randomly selected from each group, and for recovery animals, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed. Detailed histological examination was performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. Additionally, these organs were processed and examined histologically in not mated male animals (1/17 at 100 mg/kg bw/day and 1/17 at 300 mg/kg bw/day), in non-pregnant female and its male pair at 100 mg/kg bw/day and in one dam at 300 mg/kg bw/day based on macroscopic observation. Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose including recovery groups. Histological examinations were performed on the liver in all animals and on organs with macroscopic findings (thymus, uterus) in the low and mid dose groups.
Postmortem examinations (offspring):
SACRIFICE
- All offspring were sacrificed on postnatal day 13 or shortly thereafter.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Dead pups and pups euthanized at day 13 post-partum, or shortly thereafter, were carefully examined for gross abnormalities.

GROSS NECROPSY
- Gross necropsy consisted of examinations for gross abnormalities.

OTHER: The serum level of T4 (thyroid hormone) was determined in pups sacrificed on Day 13.
Statistics:
The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible. For evaluation of data obtained during the recovery period, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed. Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.
Reproductive indices:
Copulatory index males [%] = Number of males with confirmed mating/Total number of males cohabited x 100
Copulatory index females [%] = Number of sperm positive females/Total number of females cohabited x 100
Fertility index males [%] = Number of males impregnating a female/Total number of males with confirmed mating x 100
Fertility index females [%] = Number of pregnant females/Number of sperm positive females x 100
Gestation index [%] = Number of females with live born pups/Number of pregnant females x 100
Offspring viability indices:
Post-implantation mortality (intrauterine mortality) [%] = (Number of implantations - Number of liveborns)/Number of implantations x 100
Post-natal mortality [%] = (Number of liveborns - Number of live pups on postnatal day 13)/Number of liveborns x 100
Survival index [%] = Number of live pups on postnatal day 13/Number of pups born x 100
Sex ratio [%] = (Number of pups examined - Number of males (females))/Number of pups examined x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
Control, 100, 300 and 1000 mg/kg bw/day: soft stool caused by the vehicle from Day 3 up to the termination of treatment

FEMALES
Control, 100, 300 and 1000 mg/kg bw/day: soft stool caused by the vehicle from Day 3 up to the termination of treatment
Control: alopecia on the forelimbs and on the abdomen in 1/12 animal from gestation Day 9 and gestation Day 21, respectively, up to the termination of the treatment
300 mg/kg bw/day: alopecia on the forelimbs in 1/12 animal between lactation days 4 and 14
1000 mg/kg bw/day: alopecia on the abdomen in 1/11 dams between lactation day 0 and 17
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
1000 mg/kg bw/day: statistically significant slightly increased mean body weight gain comparing to their control during the first week of the recovery period; slightly increased summarized body weight gain during the post-treatment observation period (not statistically significant)
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
MALES
1000 mg/kg bw/day: statistically significant slightly decreased mean daily food consumption during the second week of the premating period
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
100 mg/kg bw/day: statistically significant slightly decreased mean percentage of basophil granulocytes, mean concentration of hemoglobin and mean hematocrit value; statistically significant shorter mean activated thromboplastin time
300 mg/kg bw/day: statistically significant slightly decreased mean percentage of basophil granulocytes, mean concentration of hemoglobin and mean hematocrit value
1000 mg/kg bw/day: statistically significant slightly decreased mean percentage of basophil granulocytes; statistically significant slightly decreased mean percentage of lymphocytes at the end of the recovery period

FEMALES
1000 mg/kg bw/day: statistically significant decreased mean percentage of neutrophil granulocytes, statistically significant increased percentage of lymphocytes and mean platelet count
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
100 mg/kg bw/day: statistically significant slightly decreased mean concentration of albumin
300 mg/kg bw/day: statistically significant slightly decreased mean concentration of albumin
1000 mg/kg bw/day: statistically significant slightly decreased mean activity of alanine aminotransferase; statistically significant slightly decreased mean concentration of potassium in the recovery period

FEMALES
300 mg/kg bw/day: statistically significant slightly increased mean urea concentration
1000 mg/kg bw/day: statistically significant slightly increased mean urea and creatinine concentration
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
MALES
Control: acute thymic hemorrhage in 1/5 animal; hyperplasia of bronchus associated lymphoid tissue in 1/5 animal; alveolar emphysema of minimal degree in 1/5 animal at the end of the recovery period; hyperplasia of bronchus associated lymphoid tissue in 1/5 animal at the end of the recovery period; renal cyst in 1/5 animal
300 mg/kg bw/day: centrilobular microvesicular vacuolation in hepatocytes/ hepatic lipidosis in 3/12 animals; acute thymic hemorrhage in 1/5 animal (non-treatment-related)
1000 mg/kg bw/day: centrilobular microvesicular vacuolation in hepatocytes/ hepatic lipidosis in 7/12 animals; alveolar emphysema in 1/5 animal (non-treatment-related); acute thymic hemorrhage in 1/5 animal (non-treatment-related); alveolar emphysema of minimal degree in 1/5 animal at the end of the recovery period (non-treatment-related)

FEMALES
Control: dilatation of uterine horns in 2/12 animals; alveolar emphysema in 1/5 animal; acute thymic hemorrhage in 1/5 animal; hyperplasia of bronchus associated lymphoid tissue in 1/5 animal at the end of the recovery period
300 mg/kg bw/day: dilatation of uterine horns in 1/12 animal (non-treatment-related)
1000 mg/kg bw/day: dilatation of uterine horns in 1/12 animal at the end of the treatment period and in 2/5 animals at the end of the recovery period (non-treatment-related); centrilobular microvesicular vacuolation in hepatocytes/ hepatic lipidosis in 3/12 animals; alveolar emphysema in 1/5 animal (non-treatment-related); acute thymic hemorrhage in 1/5 animal (non-treatment-related); hyperplasia of bronchus associated lymphoid tissue in 1/5 animal (non-treatment-related)
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Description (incidence and severity):
There were no statistically significant differences in the thyroid hormone (free T4) level in parental male animals compared to the control.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
MALES
100 mg/kg bw/day: statistically significant slightly decreased copulatory index
300 mg/kg bw/day: statistically significant slightly decreased copulatory index

FEMALES
100 mg/kg bw/day: statistically significant slightly decreased fertility index
300 mg/kg bw/day: statistically significant increased mean number of pre-coital days and mean number of conceiving days
1000 mg/kg bw/day: statistically significant slightly decreased fertility index

Details on results (P0)

CLINICAL SIGNS
Soft stool was observed in all animals at all dose groups and was attributed to the vehicle. Alopecia is a common spontaneous finding in this strain of experimental rats and is seen also in untreated rats. The effect occurred in single animals of control, mid and high dose groups in this study. Therefore, it was considered to be an incidental finding and not related to the treatment with the test item.

BODY WEIGHT AND BODY WEIGHT GAIN
Statistical significant slightly increased mean body weight gain was observed in males during the first week of the recovery period at the highest dose group. This resulted in a slightly higher summarized body weight gain during the post-treatment observation period and caused no significant difference with respect to the control in the mean body weight. This difference was not considered treatment-related.

FOOD CONSUMPTION
The slightly lower mean daily food consumption in males of the high dose group (1000 mg/kg bw/day) compared to controls was with minor degree and was transient and therefore was judged to be toxicologically not relevant.

HAEMATOLOGICAL FINDINGS
The differences in several haemotological parameters with respect to control were judged to be indicative of biological variation and toxicologically not relevant. Changes in basophil granulocytes, hemoglobin, hematocrit and activated thromboplastin time in treated male animals were of minor degree and not related to the doses at the end of the treatment period. Moreover, all values were well within the historical ranges and statistical significances were mainly originated from the relative high value of the control group (hemoglobin, hematocrit and activated thromboplastin time). Slight changes in the neutrophil granulocytes, lymphocytes and platelet count were only observed in female animals at the end of the post-treatment observation period but not at the end of the treatment period.

CLINICAL BIOCHEMISTRY FINDINGS
Higher concentrations of urea and creatinine were indicative of altered renal function in in female animals at 300 or 1000 mg/kg bw/day. Changes in concentrations of creatinine and urea in serum were considered to be signs of adaptation of the organ to the altered demands. Without supporting histopathological changes, these effects were judged to be toxicologically not relevant. The slight, but statistically significant differences compared to the controls with respect to albumin and potassium concentration, were considered to have little or no toxicological relevance. Decrease in enzyme activity of alanine aminotransferase has no toxicological relevance without tissue damage. Albumin concentrations changed only in the lower dose groups but not at the high dose.

GROSS PATHOLOGICAL FINDINGS
Hemorrhage in the thymus was due to circulatory disturbance developed during the exsanguination procedure. Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats. Cecum dilatation was an individual change probably due to an increased water accumulation as there were no accompanying histological findings. Alopecia on the skin is a common observation in this strain of experimental rats, which occurred with similar incidence in the control and test item treated female animals (300 and 1000 mg/kg bw/day) in this study. Therefore, these macroscopic changes were considered to be independent from the treatment with the test item.

ORGAN WEIGHTS
The slight increases in liver weights in females at 300 mg/kg bw/day were not considered to be toxicologically relevant due to the small degree and in the lack of associated histopathological alterations as well as in the absence of similar changes in the high dose group.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC
The dilatation of uterine horns observed in females is not associated with inflammatory or other pathological lesions and is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
The treatment related observed hepatic lipidosis was of minimal or mild degree and and is considered to be a slight reversible liver injury and it might be in connection with a disturbance of energy metabolism of affected hepatocytes.
The acute changes in the lungs (alveolar emphysema) and thymus (hemorrhage) in control or high dose treated animals were considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguinations.
Hyperplasia of bronchus associated lymphoid tissue as observed in males and females of the control as well as of the highest dose group is a physiological immune-morphological phenomenon, without toxicological significance.

REPRODUCTIVE PERFORMANCE
The slight, but statistically significant differences in the copulatory index observed in males at 100 and 300 mg/kg bw/day as well as in the fertility index at 100 and 1000 mg/kg bw/day and in the mean number of pre-coital and conceiving days at 300 mg/kg bw/day in females were considered to be indicative of biological variation due to the lack of a clear and consistent dose-response relationsship. All values were within or marginal to historical control mean.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects at highest dose tested

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
100 mg/kg bw/day: swollen abdomen, smaller than normal size and dyspnea
300 mg/kg bw/day: swollen abdomen, smaller than normal size and dyspnea
1000 mg/kg bw/day: decreased percentage of of not suckled pups
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Control: one dead offspring on postnatal day 13
100 mg/kg bw/day: one stillborn offspring, two dead pups on postnatal day 0, two dead pups on postnatal day 13
300 mg/kg bw/day: two stillborn pups, two dead pups
1000 mg/kg bw/day: one stillborn pup, one dead offspring
Body weight and weight changes:
no effects observed
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:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the thyroid hormone (free T4) level in Day 13 pups compared to the control.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Control: no milk in the stomach of 1/1 dead offspring
100 mg/kg bw/day: hydrops fetalis in 1/1 stillborn; hole on the cranium, paleness and black colored content in the intestines in 1/2 dead pup on postnatal day 0; swollen abdomen and stomach as well as gas filled intestines in 1/2 dead pup on postnatal day 13
300 mg/kg bw/day: gas filled stomach and intestines in 1/2 dead pup; autolyzed visceral organs in 2/2 pups
1000 mg/kg bw/day: misshapen head (lack of eyes, mouth and nostrils) in 1/1 stillborn pup
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
SEX DISTRIBUTION
1000 mg/kg bw/day: statistically significant slightly decreased mean number of male pups per litter

ANOGENITAL DISTANCE
100 and 300 mg/kg bw/day: statistically significant slightly shorter anogenital distance in male pups (non-treatment-related)
1000 mg/kg bw/day: statistically significant slightly shorter anogenital distance in female pups (non-treatment-related)

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

GROSS PATHOLOGICAL FINDINGS
The observed alterations (hydrops fetalis, hole on the cranium, misshapen head) are spontaneous variation or malformation in experimental rats of this strain.

SEX DISTRIBUTION
The difference in the mean number of male pups per litter compared to the control was with minor degree and was due to the relative high value of control (6.4 ± 1.7 vs. historical control mean: 5.4 ± 2). Therefore, it was considered to be toxicologically not relevant.

ANOGENITAL DISTANCE
Due to the minor degree, the slight differences regarding the anogenital distance in males and females compared to the control were not considered to be toxicologically relevant.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects at highest dose tested

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Table 1: Summary of clinical signs in males (pre-mating, mating and post-mating periods)

Observations

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Treatment period*

Recovery period

Treatment period*

Recovery period

Normal

17/17

5/5

12/12

12/12

17/17

5/5

Soft stool

17/17

5/5

12/12

12/12

17/17

5/5

Frequency of observations: number of animals (cage) with observation/number of animals (cage) examined

* Including animals of recovery group

 

Table 2: Summary of clinical signs in females

Observations

Control

100 mg/kg bw/day

300 mg/kg bw/day

600 mg/kg bw/day

Pre-mating and mating periods

Normal

12/12

12/12

12/12

12/12

Soft stool

12/12

12/12

12/12

12/12

Post-mating periods

Normal

-

1/1

-

1/1

Soft stool

-

1/1

-

1/1

Gestation period

Normal

11/12

11/11

12/12

11/11

Soft stool

12/12

11/11

12/12

11/11

Skin: Alopecia

1/12

0/11

0/12

0/11

Lactation period

Normal

11/12

11/11

11/12

10/11

Soft stool

12/12

11/11

12/12

11/11

Skin: Alopecia

1/12

0/11

1/12

1/11

Recovery animals

Normal

5/5

5/5

5/5

5/5

Soft stool

5/5

5/5

5/5

5/5

 

Table 3: Summary of body weight gain in males

Group

 

Body weight gain (g) between

Pre-mating days

Mating and post-mating days

Total

0 – 7

7 – 13

0 – 13

13 – 20

20 – 27

27 – 34

34 – 41

41 – 48

48 - 55

0 – 55

Control

Mean

17.9

17.2

35.1

14.5

11.3

10.2

7.9

10.9

11.5

101.4

SD

4.5

4.1

6.1

4.7

4.2

6.7

5.1

3.5

4.5

17.2

n

17

17

17

17

17

17

17

17

17

17

100 mg/kg bw/day

Mean

18.3

15.6

33.9

15.7

10.8

11.0

8.4

10.9

12.1

102.8

SD

7.7

5.6

10.1

4.6

5.2

4.0

4.8

8.4

7.1

18.9

n

12

12

12

12

12

12

12

12

12

12

300 mg/kg bw/day

Mean

15.8

16.6

32.4

10.8

12.7

10.2

10.0

13.6

10.8

100.4

SD

6.2

4.1

9.1

8.5

6.8

4.3

4.0

4.7

3.7

22.2

n

12

12

12

12

12

12

12

12

12

12

1000 mg/kg bw/day

Mean

16.3

15.0

31.3

12.2

11.5

13.1

7.4

11.2

8.0

94.6

SD

5.6

4.3

8.1

7.5

4.6

5.4

3.4

4.9

4.3

20.0

n

17

17

17

17

17

17

17

17

17

17

 

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS = Not Significant

 

Table 4: Summary of body weight gain in males of the recovery group

Group

 

Body weight gain (g) between

Recovery days

0 – 6

6 – 13

0 – 13

Control

Mean

4.8

0.8

5.6

SD

3.8

3.1

6.5

n

5

5

5

1000 mg/kg bw/day

Mean

11.0*

3.6

14.6

SD

4.4

4.6

7.8

n

5

5

5

 

*

NS

NS

* p < 0.05

 

Table 5: Summary of body weight gain in females

Group

 

Body weight gain (g) between

Pre-mating days

Treatment days

Total

0 – 7

7 – 13

0 – 13

13 – 20

20 – 27

27 – 34

34 – 41

41 – 48

0 – 55

Control

Mean

8.2

7.2

15.4

6.0

2.6

7.0

4.0

1.8

39.6

SD

6.4

7.1

6.8

4.0

7.6

4.5

2.6

4.9

10.0

n

17

17

17

5

5

5

5

5

5

100 mg/kg bw/day

Mean

11.3

6.4

17.8

-

-

-

-

-

-

SD

9.1

7.7

4.5

-

-

-

-

-

-

n

12

12

12

-

-

-

-

-

-

300 mg/kg bw/day

Mean

13.3

5.1

18.3

-

-

-

-

-

-

SD

7.1

5.0

9.0

-

-

-

-

-

-

n

12

12

12

-

-

-

-

-

-

1000 mg/kg bw/day

Mean

10.1

8.4

18.5

8.8

-2.2

9.6

0.6

5.0

39.4

SD

9.6

6.4

9.5

2.3

6.9

4.2

4.5

1.2

9.4

n

17

17

17

5

5

5

5

5

5

 

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS = Not Significant

 

Table 6: Summary of body weight gain in females of the recovery group

Group

 

Body weight gain (g) between

Recovery days

0 – 6

6 – 13

0 – 13

Control

Mean

0.8

0.0

0.8

SD

7.7

1.9

7.3

n

5

5

5

1000 mg/kg bw/day

Mean

6.6

-1.0

5.6

SD

6.66

3.9

3.0

n

5

5

5

 

NS

NS

NS

NS = Not Significant

 

 

Table 7: Summary of food consumption in males

Group

 

Daily mean food consumption (g/animal/day)

Pre-mating days

Post-mating days

Recovery period

0 – 7

7 – 13

20 – 27

27 – 34

34 – 41

41 – 48

48 – 55

Day 0 – 6

Day 6 – 13

Control

Mean

25.9

27.4

24.9

24.9

24.5

25.0

25.1

24.4

24.6

SD

1.93

1.46

1.59

1.86

1.25

1.26

1.54

0.55

0.35

n

8.00

8.00

8.00

8.00

8.00

8.00

8.00

2.00

2.00

100 mg/kg bw/day

Mean

26.0

27.1

24.8

24.9

24.9

25.1

24.9

-

-

SD

1.52

2.03

1.09

0.85

0.73

1.66

1.66

-

-

n

6

6

5

5

6

6

6

-

-

±%

0

-1

0

0

1

0

-1

-

-

300 mg/kg bw/day

Mean

25.8

26.2

24.4

24.6

24.6

25.1

25.1

-

-

SD

2.22

1.31

1.38

1.16

0.94

1.27

1.16

-

-

n

6

6

6

6

6

6

6

-

-

±%

-1

-4

-2

-1

0

1

0

-

-

1000 mg/kg bw/day

Mean

24.7

25.2*

24.0

24.2

24.0

24.3

23.6

25.1

25.6

SD

2.30

1.80

2.16

1.91

1.65

1.54

1.3

1.73

2.54

n

8

8

8

8

8

8

8

2

2

±%

-5

-8

-4

-3

-2

-3

-6

3

4

 

NS

DN

NS

NS

NS

NS

NS

NS

NS

* p < 0.05

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 8: Summary of food consumption in females

Group

 

 

Daily mean food consumption (g/animal/day)

Pre-mating days

Post-mating days

Recovery period

0 – 7

7 – 13

13 – 20

20 – 27

27 – 34

34 – 41

41 – 48

48 – 55

Day 0 – 6

Day 6 – 13

Control

Mean

19.7

19.4

17.7

19.3

18.3

17.7

17.3

17.0

16.9

17.3

SD

2.36

1.06

1.94

0.12

0.99

0.62

0.84

0.00

1.77

0.61

n

8

8

2

2

2

2

2

2

2

2

100 mg/kg bw/day

Mean

19.8

19.0

-

-

-

-

-

-

-

-

SD

1.53

1.26

-

-

-

-

-

-

-

-

n

6

6

-

-

-

-

-

-

-

-

±%

0

-2

-

-

-

-

-

-

-

-

300 mg/kg bw/day

Mean

18.9

18.8

-

-

-

-

-

-

-

-

SD

1.18

1.69

 

 

 

 

 

 

 

 

n

6

6

-

-

-

-

-

-

-

-

±%

-4

-3

-

-

-

-

-

-

-

-

1000 mg/kg bw/day

Mean

18.6

19.0

17.4

16.5

17.8

16.6

17.5

16.8

18.6

18.9

SD

0.96

2.00

0.62

1.92

1.03

1.16

0.45

0.13

0.12

0.02

n

8

8

2

2

2

2

2

2

2

2

±%

-6

-2

-2

-14

-3

-6

1

-1

10

10

 

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS = Not Significant

 

 

Table 9: Summary of hematology

Group

 

Basophil [%]

Hemoglobin [g/L]

Hematocrit [L/L]

Neutrophil [%]

Lymphocyte [%]

Mean platelet count [×10E9/L]

Activated partial thromboplastin time [sec]

MALES – TREATMENT PERIOD

Control

Mean

0.16

175.40

0.47

23.80

72.40

891.60

23.60

SD

0.05

9.34

0.03

5.06

5.74

118.00

2.60

n

5

5

5

5

5

5

5

100 mg/kg bw/day

Mean

0.04*

161.60**

0.43**

25.08

70.62

901.40

20.48*

SD

0.09

4.72

0.01

5.03

5.26

88.05

0.96

n

5

5

5

5

5

5

5

±%

-75

-8

-9

5

-2

1

-13

300 mg/kg bw/day

Mean

0.02**

163.80**

0.44*

20.96

74.20

897.40

21.72

SD

0.04

2.95

0.00

3.74

4.21

137.05

1.91

n

5

5

5

5

5

5

5

±%

-88

-7

-6

-12

2

1

-8

1000 mg/kg bw/day

Mean

0.04**

169.00

0.45

17.64

77.98

796.60

22.22

SD

0.05

3.67

0.01

6.23

7.44

54.62

1.43

n

5

5

5

5

5

5

5

±%

-75

-4

-5

-26

8

-11

-6

 

DN

DN

U

NS

NS

NS

DN

MALES – RECOVERY PERIOD

Control

Mean

0.08

176.20

0.47

16.44

78.94

823.80

19.48

SD

0.04

3.11

0.01

3.44

3.44

83.96

3.49

n

5

5

5

5

5

5

5

1000 mg/kg bw/day

Mean

0.02

107.40

0.46

22.28

72.40*

833.40

20.24

SD

0.04

5.81

0.01

5.06

4.94

42.55

1.85

n

5

5

5

5

5

5

5

±%

-75

-3

-2

36

-8

1

4

 

NS

NS

NS

NS

*

NS

NS

FEMALES – TREATMENT PERIOD

Control

Mean

0.10

159.20

0.43

25.68

69.78

1016.20

23.44

SD

0.22

8.32

0.01

7.76

7.54

151.26

1.11

n

5

5

5

5

5

5

5

100 mg/kg bw/day

Mean

0.04

169.00

0.45

16.70

79.02

1079.80

23.94

SD

0.09

9.67

0.02

3.57

2.94

181.70

2.09

n

5

5

5

5

5

5

5

±%

-60

6

4

-35

13

6

2

300 mg/kg bw/day

Mean

0.10

152.00

0.41

21.18

75.50

1064.20

22.12

SD

0.10

15.02

0.03

9.78

10.17

195.41

1.62

n

5

5

5

5

5

5

5

±%

0

-5

-4

-18

8

5

-6

1000 mg/kg bw/day

Mean

0.10

162.80

0.43

21.90

73.34

996.20

21.80

SD

0.10

5.67

0.01

4.39

5.74

79.97

0.70

n

5

5

5

5

5

5

4

±%

0

2

1

-15

5

-2

-7

 

NS

NS

NS

NS

NS

NS

NS

FEMALES – RECOVERY PERIOD

Control

Mean

0.14

160.20

0.44

18.94

74.84

744.40

21.76

SD

0.13

9.86

0.02

2.23

3.40

75.68

1.33

n

5

5

5

5

5

5

5

1000 mg/kg bw/day

Mean

0.04

164.20

0.45

15.18*

80.86*

852.00*

20.30

SD

0.09

5.45

0.01

1.92

2.28

67.62

1.18

n

5

5

5

5

5

5

5

±%

-71

2

3

-20

8

14

-7

 

NS

NS

NS

*

*

*

NS

* p < 0.05, **p < 0.01

NS = Not Significant

U = Mann-Whitney U-test vs. Control

DN = Duncan´s multiple range test

 

Table 10: Summary of clinical biochemistry findings

Group

 

Albumin [g/L]

Alanine aminotransferase activity [U/L]

Urea [mmol/L]

Creatinine [µmol/L]

Potassium [mmol/L]

MALES – TREATMENT PERIOD

Control

Mean

37.16

70.98

8.10

28.04

4.71

SD

1.08

18.19

0.99

3.22

0.54

n

5

5

5

5

5

100 mg/kg bw/day

Mean

35.04**

56.78

8.22

31.24

4.67

SD

1.33

13.38

0.90

2.22

0.15

n

5

5

5

5

5

±%

-6

-20

1

11

-1

300 mg/kg bw/day

Mean

35.74*

56.76

8.20

29.90

4.74

SD

0.69

8.28

1.12

3.21

0.22

n

5

5

5

5

5

±%

-4

-20

1

7

1

1000 mg/kg bw/day

Mean

36.70

49.66*

8.68

30.50

4.57

SD

0.76

6.45

1.23

1.47

0.45

n

5

5

5

5

5

±%

-1

-30

7

9

-3

 

DN

DN

NS

NS

NS

MALES – RECOVERY PERIOD

Control

Mean

36.36

60.22

8.04

27.28

4.79

SD

0.92

7.23

0.63

1.19

0.19

n

5

5

5

5

5

1000 mg/kg bw/day

Mean

35.60

69.60

1.72

26.40

4.42*

SD

0.69

11.83

0.31

3.50

0.19

n

5

5

5

5

5

±%

-2

16

11

-3

-8

 

NS

NS

NS

NS

*

FEMALES – TREATMENT PERIOD

Control

Mean

34.24

53.30

7.87

27.56

4.39

SD

0.46

12.42

1.32

1.44

0.47

n

5

5

5

5

5

100 mg/kg bw/day

Mean

35.02

50.46

8.90

30.34

4.13

SD

1.04

4.34

0.90

2.34

0.18

n

5

5

5

5

5

±%

2

-5

13

10

-6

300 mg/kg bw/day

Mean

34.26

64.36

10.07*

30.42

4.65

SD

1.23

5.54

1.12

2.01

0.38

n

5

5

5

5

5

±%

0

21

28

10

6

1000 mg/kg bw/day

Mean

33.48

54.94

10.35*

32.30*

4.39

SD

0.37

19.24

1.97

3.78

0.20

n

5

5

5

5

5

±%

-2

3

32

17

0

 

NS

NS

DN

DN

NS

FEMALES – RECOVERY PERIOD

Control

Mean

39.22

53.32

7.66

31.86

4.19

SD

1.70

14.32

0.81

4.40

0.31

n

5

5

5

5

5

1000 mg/kg bw/day

Mean

39.04

62.18

8.24

32.98

3.95

SD

1.69

20.47

0.66

4.23

0.15

n

5

5

5

5

5

±%

0

17

8

4

-6

 

NS

NS

NS

NS

NS

* p < 0.05, **p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 11: Summary of necropsy findings

Organs

Observations

Frequency of observations per group

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Main group

Recovery group

 

 

Main group

Recovery group

MALES

 

No macroscopic finding

11/12

4/5

12/12

10/12

8/12

5/5

Thymus

Hemorrhages

1/12

0/5

0/12

1/12

1/12

0/5

Kidney

3 mm cavity at the renal medulla

0/12

1/5

0/12

0/12

0/12

0/5

Liver

Pale

0/12

0/5

0/12

1/12

4/12

0/5

FEMALES - DAMS

 

No macroscopic finding

8/12

5/5

11/11

9/12

6/11

3/5

Thymus

Hemorrhages

1/12

0/5

0/11

0/12

1/11

0/5

Uterus

Hydrometra

2/12

0/5

0/11

1/12

1/11

2/5

Cecum

Dilatation

0/12

0/5

0/11

0/12

3/11

0/5

Liver

Pale

0/12

0/5

0/11

0/12

3/11

0/5

Skin

Alopecia

1/12

0/5

0/11

1/12

1/11

0/5

FEMALES – NON-PREGNANT

 

No macroscopic finding

NA

NA

1/1

NA

0/1

NA

Uterus

Hydrometra

NA

NA

0/1

NA

1/1

NA

Frequency of observation =number of animals with observations / number of animals examined

NA = not applicable

 

Table 12: Summary of organ weights

 

 

Body weight [g]

Organ weight [g]

Organ weight relative to body weight [%]

Organ weight relative to brain weight [%]

Group

 

 

Liver

Kidney

Liver

Kidney

Liver

Kidney

MALES – TREATMENT PERIOD

Control

Mean

448.4

11.31

2.70

2.604

0.622

518.31

123.81

SD

24.45

1.05

0.17

0.179

0.037

40.16

7.90

n

12

5

5

5

5

5

5

100 mg/kg bw/day

Mean

443.5

11.65

2.62

2.632

0.595

533.25

120.14

SD

29.02

1.86

0.22

0.235

0.039

70.18

7.46

n

12

5

5

5

5

5

5

±%

-1

3

-3

1

-4

3

-3

300 mg/kg bw/day

Mean

444.8

12.43

2.77

2.740

0.610

578.38

129.37

SD

34.75

0.84

0.32

0.201

0.057

43.08

19.33

n

12

5

5

5

5

5

5

±%

-1

10

3

5

-2

12

4

1000 mg/kg bw/day

Mean

430.7

12.07

12.82

2.768

0.647

555.54

129.84  

SD

34.57

1.25

0.30

0.263

0.082

43.19

14.08

n

12

5

5

5

5

5

5

±%

-4

7

4

6

4

7

5

 

NS

NS

NS

NS

NS

NS

NS

MALES – RECOVERY PERIOD

Control

Mean

448.8

11.68

2.54

2.599

0.566

436.09

116.69

SD

23.21

1.19

0.19

0.193

0.018

45.94

7.51

n

5

5

5

5

5

5

5

1000 mg/kg bw/day

Mean

458.2

11.93

2.82*

2.607

0.618

544.92

129.01

SD

27.07

0.94

0.13

0.187

0.049

41.69

9.65

n

5

5

5

5

5

5

5

±%

2

2

11

0

9

2

11

 

NS

NS

*

NS

NS

NS

NS

FEMALES – TREATMENT PERIOD

Control

Mean

248.8

7.92

1.78

3.183

0.716

392.68

88.44

SD

7.29

0.45

0.12

0.137

0.052

15.47

7.80

n

5

5

5

5

5

5

5

100 mg/kg bw/day

Mean

263.6

8.29

1.83

3.142

0.695

406.12

89.58

SD

8.88

0.63

0.09

0.152

0.026

40.55

4.22

n

5

5

5

5

5

5

5

±%

6

5

3

-1

-3

3

1

300 mg/kg bw/day

Mean

270.8*

9.45*

1.88

3.481

0.695

469.07*

93.36

SD

14.20

1.23

0.11

0.295

0.049

61.20

6.89

n

5

5

5

5

5

5

5

±%

9

19

6

9

-3

19

6

1000 mg/kg bw/day

Mean

264.0

9.00

1.94

3.404

0.737

447.20

96.67

SD

13.11

1.05

0.13

0.288

0.063

40.25

5.75

n

5

5

5

5

5

5

5

±%

6

14

9

7

3

14

9

 

DN

DN

NS

NS

NS

DN

NS

FEMALES – RECOVERY PERIOD

Control

Mean

255.6

6.99

1.56

2.738

0.614

350.45

78.40

SD

18.20

0.86

0.12

0.309

0.057

47.29

6.87

n

5

5

5

5

5

5

5

1000 mg/kg bw/day

Mean

264.6

7.17

1.67

2.705

0.630

371.57

86.39

SD

18.70

0.81

0.12

0.118

0.021

33.63

4.98

n

5

5

5

5

5

5

5

±%

4

3

7

-1

3

6

10

 

NS

NS

NS

NS

NS

NS

NS

* p < 0.05, **p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 13: Summary of histopathological findings

Organs

Observations

Incidence of observations per group

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Main group

Recovery group

 

 

Main group

 

Recovery group

MALES

Kidneys

Cyst

0/5

1/5

-

-

0/5

0/5

Liver

Centrilobular vacuolation in hepatocytes

0/12

0/5

0/12

3/12

7/12

0/5

Lungs

Alveolar emphysema

0/5

1/5

-

-

1/5

1/5

Hyperplasia of BALT

1/5

1/5

-

-

0/5

0/5

Thymus

Acute hemorrhage

1/5

0/5

-

1/1

1/5

0/5

FEMALES

Kidneys

Cyst

0/5

0/5

-

-

0/5

0/5

Liver

Centrilobular vacuolation in hepatocytes

0/12

0/5

0/11

0/11

3/12

0/5

Lungs

Alveolar emphysema

1/5

0/5

-

-

1/5

0/5

Hyperplasia of BALT

0/5

1/5

-

-

1/5

0/5

Thymus

Acute hemorrhage

1/5

0/5

-

-

1/5

0/5

Uterus with cervix

Dilatation

2/12

0/5

0/1

1/1

1/12

2/5

Incidence of observation =number of animals with observations / number of animals examined

BALT = bronchus associated lymphoid tissue

 

Table 14: Summary of clinical observation and fate of offspring

Observations/Fate of pup

Group [mg/kg bw/day]

Control

100

300

1000

Number of pups examined (liveborns)

139

120

144

116

No signs

N

97

107

101

82

%

70

89

70

71

Cold

N

38

10

29

33

%

27

8

20

28

No milk in the stomach

N

42

14

27

21

%

30

12

19

18

Swollen abdomen

N

0

1

1

0

%

0

1

1

0

Smaller than others

N

0

1

1

0

%

0

1

1

0

Cianotic

N

0

0

1

1

%

0

0

1

1

Dispnoe

N

0

0

1

0

%

0

0

1

0

Found dead

N

1

2

2

1

%

1

2

1

1

Missing

N

1

2

3

1

%

1

2

2

1

Interim euthanazia

N

42

33

44

37

%

30

28

31

23

Terminal euthanazia

N

95

83

95

87

%

68

69

66

75

 

Table 15: Summary data of reproductive ability

Values

 

Group [mg/kg bw/day]

 

 

Control

100

300

1000

 

MALES

No. of males paired

 

12

12

12

12

 

Copulatory index

%

100

92**

92**

100

 

FEMALES

Pre-coital interval (days)

Mean

1.2

0.6

2.2*

2.1

DN

SD

1.1

0.7

1.5

1.0

N

12

12

12

12

Conceiving days

Mean

2.2

1.6

3.2*

3.1

DN

SD

1.1

0.7

1.5

1.0

N

12

12

12

12

Fertility index

%

100

92**

100

92**

 

* p < 0.05 CH2, ** p < 0.01 CH2

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 16: Summary of extra uterine mortality and sex distribution of offspring – litter data

Values

 

 

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

 

Number of litters

 

N

12

11

12

11

 

Number of viable pups

 

 

 

 

 

on post-natal day 0

Total

N

11.6

10.8

11.7

10.5

NS

SD

1.7

2.9

2.5

2.5

Male

N

6.4

5.5

6.4

4.5

* DN

SD

1.7

1.9

2.0

1.8

Female

N

5.2

5.4

5.3

5.9

NS

SD

1.5

1.8

1.2

2.3

on post-natal day 4

Total

N

11.5

10.6

11.6

10.4

NS

SD

1.7

2.9

2.4

2.6

Male

N

6.4

5.4

6.3

4.5

NS

SD

1.7

1.9

2.0

1.8

Female

N

5.1

5.3

5.3

5.8

 

SD

1.5

1.9

1.2

2.5

on post-natal day 13

Total

N

7.9

7.5

7.9

7.9

NS

SD

0.3

1.2

0.3

0.8

Male

N

4.1

3.9

3.9

3.8

 

SD

0.5

0.8

0.7

1.3

Female

N

3.8

3.6

4.0

4.1

 

SD

0.6

0.7

0.4

1.3

* p < 0.05

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 17: Summary of anogenital distance of offspring

Group

 

Anogenital distance (AGD) on post-natal day 4

Absolute (mm)

Normalized (AGD/3√bw)

MALES

Control

Mean

6.27

2.85

SD

0.55

0.22

n

77

77

N

12

12

100 mg/kg bw/day

Mean

6.19

2.77*

SD

0.57

0.25

n

59

59

N

11

11

300 mg/kg bw/day

Mean

6.12

2.76*

SD

0.46

0.19

n

76

76

N

12

12

1000 mg/kg bw/day

Mean

6.24

2.79

SD

0.56

0.21

n

50

50

N

11

11

 

NS

DN

FEMALES

Control

Mean

3.31

1.52

SD

0.47

0.18

n

61

61

N

12

12

100 mg/kg bw/day

Mean

3.31

1.51

SD

0.47

0.19

n

58

58

N

11

11

300 mg/kg bw/day

Mean

3.29

1.49

SD

0.46

0.20

n

63

63

N

12

12

1000 mg/kg bw/day

Mean

3.16

1.44*

SD

0.37

0.15

n

64

64

N

11

11

 

NS

DN

* p < 0.05

NS = Not Significant

DN = Duncan´s multiple range test

n = number of offspring

N = Number of litters

Applicant's summary and conclusion

Conclusions:
The test material had no effect on reproductive performance.