Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Jul 2012 - 16 Jan 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Erlangen, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks
- Weight at study initiation: 285-329 g (males), 173-209 g (females)
- Housing: single housing in IVC cages, type III H (except for mating period)
- Diet: Altromin 1324 maintenance diet for rats and mice (Lot No. 0939), ad libitum
- Water: tap water (sulphur acidified to pH 2.8), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 55±10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
cotton seed oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle (cottonseed oil) was added to give the appropriate final concentration of the test item. The formulation vials were placed on a Vortex machine for a short period to ensure proper homogenistation of the formulation. The test item formulation was prepared freshly on each administration day before the administration procedure. The time of preparation and time of dosing was recorded for all dosing formulations. The vehicle was also used as control item.


VEHICLE
- Justification for use and choice of vehicle: The vehicle has been selected on the basis of the test item’s characteristics.
- Concentration in vehicle (nominal): 25, 75, and 250 mg/mL
- Amount of vehicle: 4 mL/kg bw
- Lot/batch no.: MKDJ0602V
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- In case 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:
Each dosing concentration was analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the low and high dose concentrations. Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation) from all groups (16 samples). The mean recoveries observed in low dose, middle dose and high dose groups were 102.5%, 101.4% and 101.9% of the nominal concentration. Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 and 5 (12 samples). The mean recovery observed for the low dose group was 104.1 and 109.4% of nominal value, and 106.0 and 95.1% for high dose group. Samples for stability analysis were taken in the first week of the study, 0 hours after the preparation and another sample 6 hours after the preparation (at room temperature), from high and low dose formulations (4 samples). After 6 h storage at room temperature recovery compared to starting value was 88.0% and 100.6%.
Duration of treatment / exposure:
Males: at least 28 days (beginning during 14 days of pre-mating period)
Females: maximum period of 54 days (14 days pre-mating until post-natal day 3)
Frequency of treatment:
once daily (7 days per 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:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL. The doses were selected on the basis of data from a Dose Range Finding Study, where 3 animals per sex and dose (100, 300, and 1000 mg/kg bw/day) were treated in the same way as in the main study. No mortality and clinical findings were observed. Slightly reduced body weight development was observed in 300 and 1000 mg/kg bw dose groups. 1/3 animals of the high dose group did not deliver pubs and lost them during pregnancy. Based on these results 100, 300, and 1000 mg/kg bw were chosen as appropriate dose levels for the main study.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality were recorded. Twice daily all animals were observed for morbidity and mortality. Females showing signs of abortion or premature delivery prior to the scheduled scarification of the animals were sacrificed and subjected to a thorough macroscopic examination.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure, and at least once a week thereafter.
- Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behavior were also recorded.
- Behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and on day 3 of the lactation period in 5 randomly selected females (only lactating females will be evaluated) outside the home cage using a functional observational battery of tests. Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups. Any animals prematurely sacrificed were weighed prior to the sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE :
Food consumption was measured weekly on the respective days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

WATER CONSUMPTION AND COMPOUND INTAKE : No data

HAEMATOLOGY/CLINICAL CHEMISTRY: Haematological parameters (differential blood count), clinical chemistry parameters and prothrombin time and activated partial thromboplastin time were examined in five males and five females randomly selected from each group at the end of the treatment period prior to or as part of the sacrifice of the animals. Clinical chemistry parameters included alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine, total protein (TP), albumin, urea, total bile acids (TBA), total cholesterol, glucose, sodium, and potassium.

URINALYSIS: An urinalysis was performed with samples collected from 5 randomly selected males prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance were recorded. Urinalysis parameters included specific gravity, nitrite, pH, protein, glucose, ketone bodies, urobilinogen, bilirubin, blood, leukocytes.
Oestrous cyclicity (parental animals):
Not examined.
Sperm parameters (parental animals):
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle for the evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
Litter observations:
The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing numbers on the back with the help of a permanent marker or by tattooing. In addition to the observations of parent animals, any abnormal behavior of the offspring was recorded.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on study day 29 or 30 (28 day total dosing period)
- Maternal animals: All surviving animals on post natal day 4. Females showing no evidence of copulation after the mating period were sacrificed 24 to 26 days after the last day of the mating period.

GROSS NECROPSY
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), and all organs showing macroscopic lesions of all adult animals were preserved.

HISTOPATHOLOGY / ORGAN WEIGHTS
All full histopathology was carried out on the organs and tissues of 5 randomly selected males/females of the control and high dose group. These examinations were extended to animals of all other dosage groups for treatment-related changes that were observed in the high dose group. Only organs and tissues of the other dosage groups showing changes in the high dose group were examined.
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) and all organs showing gross lesions were examined in all animals.
The following tissues were prepared for microscopic examination and weighed, respectively: brain (cerebrum, cerebellum and pons), heart, spinal cord, ovaries (females), liver, uterus with cervix (females), kidneys, vagina (females), adrenal glands, testes (males), stomach, epididymides, (males, unilateral), small and large intestines (including Peyer´s patches), prostate and seminal, vesicles with coagulating glands as a whole (males), thymus, urinary bladder, thyroid, lymphnodes (mesentric and axillary), spleen, peripheral nerve (e.g. sciatic nerve) with skeletal muscle, lungs and trachea, bone with bone marrow (sternum), mammary glands, pituitary gland, all gross lesions
Postmortem examinations (offspring):
SACRIFICE
- Dead pups and pups sacrificed on day 4 post-partum.

GROSS NECROPSY
- These animals were carefully examined externally for gross abnormalities.

HISTOPATHOLOGY / ORGAN WEIGTHS
- Not examined
Statistics:
Comparison of dosed with control animals of the main groups was perfomed using one-way ANOVA and a post-hoc Dunnett Test for body weight, food consumption, parameters of haematology, blood coagulation, clinical biochemistry and absolute and relative organ weights. The statistics were performed with GraphPad Prism 5.01 software (p<0.05 was considered as statistically significant).
Reproductive indices:
Number of corpora lutea and implantation sites, live pups (born on post-natal day 0) and pre- and post imlantation loss (%) were examined and calculated. In addition, the following indices were calculated:
Copulation Index (%) = (No. of rats copulated / No. of pairs) x 100
Fertility Index (%) = (No. of Females Pregant / No. of females copulated) x 100
Delivery Index (%) = (No. of dams with live newborns / No. of pregnant dams) x 100
Offspring viability indices:
Viability index (%) = (No. of live offspring at day 4/No. of live offspring at birth) X 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
piloerection and salivation in all dose groups (non-adverse)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
decreased bw gain (day 1-7 of pre-mating period (high dose) and 0-4 during lactation (all test doses)) in females, non-adverse; reduced food consumption (day 1-7, males, high dose), non-adverse
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
decreased bw gain (day 1-7 of pre-mating period (high dose) and 0-4 during lactation (all test doses)) in females, non-adverse; reduced food consumption (day 1-7, males, high dose), non-adverse
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
males: decreased spleen weight in high dose group, non-adverse; females: increased relative thyroid/parathyroid weights in low dose group, non-adverse
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No clinical symptoms were found in the control group. Slight piloerection was found in 5/10 high dose male animals (beginning on pre-mating day (PMD) 5), 3/10 medium dose male animals (beginning on mating day (MD) 3), and 1/10 low dose male animals (beginning on MD 14). Furthermore, 4/10 high dose female animals exhibited slight piloerection (beginning on PMD 7). Moving the bedding exhibited 3/10 male as well as 2/10 female high dose animals. Salivation was observed for 1/10 high and medium dose male animals as well as in 5/10 high dose and 1/10 low dose female animals. Besides these mentioned clinical symptoms alopecia at both forepaws and abnormal breathing were individually found in treatment groups.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In male animals, body weight increased with the progress of the study. No test item related influence on male body weight development or body weight gain could be detected. Food consumption was significantly decreased in the high dose group during pre-mating days 1-7.

In female animals, a statistically significant decreased body weight gain could be observed in the high dose group during pre-mating days 1-7 (3.6 compared to 9.9 g). Furthermore, during lactation days 0-4 a decrease in body weight was observed for treatment groups but not for control animals. However, this decrease in body weight was not dose dependent and not considered to be test item related. No change in food consumption could be measured for treatment groups when compared to the group.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No findings were reported after qualitative examination of testes, taking into account the tubular stages of the spermatogenic cycle.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No influence of the test substance was noted regarding the reproductive performance of the parental animals. Three females of the control group, one female treated at 100 mg/kg bw/day and one female treated at 1000 mg/kg bw/day were found not to be pregnant at terminal sacrifice, but in view of the group distribution this was not considered to be treatment-related.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In male animals, absolute weights of spleens were significantly decreased in high doe group when compared to the control group. Relative spleen weights were slightly decreased in the high dose group which was not statistical significant. Since no toxicological relevant finding could bewas found during histopathological analysis, this weight decrease is not considered to be not of toxicological relevance.

In female animals, relative thyroid/parathyroid weights were significantly increased in the low dose group (0.006±0.001 to 0.009±0.001). The same tendency could be found for absolute weights (0.018±0.003 g to 0.023±0.004 g). However, since no similar findings could be mentioned for the other test groups a test item relation cannot be stated.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Few specific gross pathological changes were recorded for the male and female animals and were not considered to be treatment-related. Among others, yellow spots on epididymides of male animals (1/10 high dose animals) or discoloured dark caecum (1/10 female control animals) were found.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No test item-related histopathological findings were noted in the reproductive organs and in the other organs evaluated in this study.

FUNCTIONAL OBSERVATIONS (PARENTAL ANIMALS)
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse and treatment-related effects were observed up to and including the highest tested dose level.
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse and treatment-related effects were observed up to and including the highest tested dose level.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
No treatment-related effect on the litter data was observed for parameters such as the total number of pups born or live pups on PND 0 and PND 4. No still births or runts were recognized.

BODY WEIGHT (OFFSPRING)
No change in litter weight was observed in the treated groups.

GROSS PATHOLOGY (OFFSPRING)
No gross external findings were observed in any group.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse and treatment-related effects were observed up to and including the highest tested dose level.

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Table 1: Clinical observations of the male animals.

Clinical finding

Control

Low dose

Mid dose

High dose

Total number of animals examined

10

10

10

10

Slight piloerection

-

1

3

5

Moderate piloerection

-

-

-

1

Salivation

-

-

-

1

Severe salivation

-

-

1

-

Alopecia at both forepaws

-

1

1

-

Slight alopecia at both forepaws and at thigh

-

-

1

-

Moving the bedding

 

-

-

-

3

Table 2: Clinical observations of the female animals.

Clinical finding

Control

Low dose

Mid dose

High dose

Total number of animals examined

10

10

10

10

Slight piloerection

-

-

-

4

Moderate piloerection

-

-

-

2

Piloerection

-

-

-

1

Slight alopecia at right forepaw

-

1

-

-

Alopecia at both forepaws

-

1

1

1

Salivation

-

1

-

5

Moderate salivation

-

1

-

-

Moving the bedding

 

-

-

1

5

Abnormal breathing

-

-

-

2

 


  Table 3: Body weights (g) of the male animals.

 

Day of study

 

Group

 

 

 

Control

Low dose

Mid dose

High dose

Premating

P1

Mean

332.5

332.5

332.9

329.3

SD

13.14

15.75

13.43

14.87

P7

Mean

351.2

350.4

350.8

344.5

SD

15.6

18.86

16.85

17.47

Mating and Post mating

MP1

Mean

366.1

365.6

366.6

357.1

SD

16.83

18.86

20.91

19.25

MP7

Mean

372.8

368.7

370.6

362.5

SD

18.4

23.63

23.13

20.78

MP14

Mean

380.6

377.6

376.3

372.8

SD

17.71

23.32

21.62

18.02

Table 4: Body weights (g) of the female animals.

 

Day of study

 

Group

 

 

 

 

 

 

Control

Low dose

Mid dose

High dose

Premating

P1

Mean

204.8

203.7

204.1

206.1

 

 

SD

9.72

10.2

10.32

9.72

 

P7

Mean

214.7

212.8

212.5

209.7

 

 

SD

10.98

10.11

14.71

13.74

 

P14

 

221.7

222.3

216.8

216.5

 

 

 

12.6

13.86

11.47

13.97

Gestation

GD0

Mean

223.67

222.67

221.44

218.33

 

 

SD

9.99

12.76

20.0

12.58

 

GD7

Mean

244.33

247.56

243.56

237.67

 

 

SD

9.61

18.79

17.97

15.91

 

GD14

Mean

274.33

277.44

271.22

266.67

 

 

SD

8.91

19.18

19.05

17.87

 

GD20

Mean

323.5

331.44

327.89

325.44

 

 

SD

10.86

21.40

23.12

10.11

Lactation

L0

Mean

264.71

267.22

264.0

265.11

 

 

SD

12.37

22.94

19.26

30.58

 

L4

Mean

265.71

262.78

256.7

262.89

 

 

SD

12.79

21.85

15.71

20.5


  Table 5: Pre- and Post Natal Data and Mean Reproductive Indices.

 

 

Group

 

 

 

 

 

Control

Low dose

Mid dose

High dose

Corpora lutea

Mean

16.71

14.56

18.2

16.89

 

SD

5.22

3.68

3.88

4.48

Implantation sites

Mean

9.86

11.22

11.7

11.67

 

SD

2.61

2.59

1.06

1.5

Live pups born on PND 0

 

9.57

9.44

11.3

11.0

 

 

2.57

2.24

1.34

2.06

% Pre Implantation loss

Mean

35.07

21.26

33.07

27.48

 

SD

24.65

16.69

15.7

15.16

% Post Implantation loss

Mean

2.88

14.66

3.59

6.22

 

SD

5.12

12.99

4.66

11.38

Viability Index (%)

Mean

100

100

100

100

 

SD

0

0

0

0

 

Applicant's summary and conclusion