Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

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:
22 March 1996
Deviations:
yes
Remarks:
Several deviations from the study protocol occurred but are not expected to influence the overall test results. Deviations included minor dosing errors and errors with data collection/test performance.
GLP compliance:
yes (incl. certificate)
Remarks:
Bayrisches Landesamt für Gesundheit und Lebensmittelsicherheit
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 10-11 wks
- Weight at study initiation: (P) Males: 241 - 276 g; Females: 167 - 193 g
- Housing: 2 animals / sex / cage in IVC cages (type III H, polysulphone cages) during the premating period for both males and females and during postmating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). Animals of the recovery groups were housed in groups of 2 animals / sex / cage. In each cage Altromin saw fibre was used as bedding.
- Diet: Free access to Altromin 1324 maintenance diet access to Altromin 1324 maintenance diet
- Water: Free access to tap water
- Acclimation period: 5 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulation was prepared with corn oil. The test item was weighed on a suitable precision balance into a plastic vial which was flooded with argon and the vehicle was added to give the appropriate final concentration of the test item. Formulations were vortexed for 2-3 minutes. The test item formulation was prepared at least once every ten days based on available stability data. Formulates were kept under magnetic stirring during the daily administration.

VEHICLE
- Concentration in vehicle: undiluted, ≤ 0.1 % of water content
- Amount of vehicle (if gavage): 2 mL/kg body weight
- Lot/batch no. (if required): MKBQ9948V
Details on mating procedure:
- M/F ratio per cage: 1:1 (male to female)
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: After the confirmation of mating, females were kept individually during gestation/lactation period and males were returned to its original cage.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mean recoveries observed for the low-dose group was between 96.4% and 100.8% of the nominal value, between 91.6% and 98.1% for the mid-dose group and between 92.5% and 100.0% of the nominal value for HD group. The mean recoveries observed in the low-, mid-, and high-dose groups were 98.2%, 95.8%, and 95.4% of the nominal concentration, respectively.

Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%. However one sample (no. 16, mid dose week 3, middle sampling location) did not meet this criterion with a recovery of 89.7%. This was not considered to have relevant impact on the study results as the recovery only narrowly missed defined acceptance criterion and as mean recovery of mid dose week 3 was within the range of 10%.

The coefficients of variation (COV) of the different sampling locations (top, middle, bottom) was between 0.7% and 4.5% in LD dose group, between 0.3% and 1.8% in MD dose group and between 1.1% and 2.6% in HD dose group. All samples were homogenous, as COV was below or equal 10%.
Duration of treatment / exposure:
The animals of the main groups were treated with the test item formulation or vehicle on 7 days per week for a period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
The 4 groups consisted of 10 male and 10 female Wistar rats and additional 6 male and 6 female Wistar rats in the control and high dose recovery group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on the results of a previous dose range finding study and in consultation with the sponsor.
- Rationale for animal assignment: Randomisation was performed with IDBS Workbook 9.4.0 software.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals of the main groups and the recovery groups. Clinical observations included check for spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, 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 behaviour were recorded if present.

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter, 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 on day 4 post-partum along with the pups. Any animals prematurely sacrificed were weighed prior to the sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Oestrous cyclicity (parental animals):
Not reported
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
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:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: 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. Additionally, any abnormal behavior of the offspring was recorded.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Males were sacrificed after completion of the mating period on treatment days 29 or 30.
- Maternal animals: Females along with their pups were sacrificed on post-natal day 4. Non-pregnant females of the main groups were sacrificed on study day 26 using the sperm-positive vaginal smear.

GROSS NECROPSY
- All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed on post-natal day 4.
- Dead pups and all surviving pups on post-natal day 4 were carefully examined for gross external abnormalities before terminal sacrifice. All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

GROSS NECROPSY
- All animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Statistics:
A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using an one-way ANOVA and a post-hoc Dunnett Test. Statistical comparisons of data acquired during the recovery period were performed with a Student’s t-Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry were statistically analyzed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics will be performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).
Reproductive indices:
Copulation index = no. of animals copulated/no. of pairs x 100
Fertility index = no. of pregnant females/no. of copulated females x 100
Offspring viability indices:
Delivery index = no. of dams with live pups born / no. of pregnant dams x 100
Viability index = no. of live offspring at day 4 / no. of live offspring at birth x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Moving the bedding was observed transiently in all high-dose males and females, including the high-dose recovery group, during the treatment period of this study. Furthermore, salivation was noted transiently in 8/10 males and 8/10 females of the high-dose group, 1/6 males and 4/6 females of the high-dose recovery group and in 1/10 males on one single day of the low-dose group. Moving the bedding and salivation were observed in short timely relation to dose application and thus were considered to be a sign of discomfort or a local reaction to the test item. These slight clinical signs were not considered as adverse systemic effects. They were not observed during the recovery period of this study.

Low incidences of slight clinical signs like local alopecia, crust or scratch in few animals of the control and/or dose groups were seen without dose dependency and were considered as incidental in nature.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Two high-dose females died prematurely during the course of treatment. On high-dose female was found dead on post-natal day 2 and the other was euthanised in a moribund condition for animal welfare reasons on post-natal day 2.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A treatment-related decrease in male body weight was observed at the high dose. During the mating/postmating period, high-dose males showed a slight, statistically significant loss of body weight (mean body weight change -0.40 g in week 1 and -0.60 g in week 2 compared to +96.0 g and +10.50 g in the control group). Considering the whole treatment period, there was a biologically and statistically significantly lower body weight gain in high-dose males when compared to controls (52% below controls). Thus, mean body weight of high-dose males was slightly but not statistically significantly lower at the end of the treatment period (7% below controls).

In females, transiently and slightly but statistically significantly lower body weight gain was noted in the 2nd week of gestation in the high-dose group when compared to the control group (23% below controls).

In the high-dose male recovery group, body weight development was similarly affected when compared to the main high-dose male group. From the second week of treatment onwards, body weight gain decreased with a statistical significant difference to controls of the recovery group. Body weight gain was 6% below controls in the 2nd week of treatment and 91% below controls in the 3rd week of treatment. In the last week of treatment a slight mean loss of 1.50 g body weight occurred when compared to a gain of 9.00 g in controls. Thus, mean body weight was slightly below controls at the end of the treatment period (7% below controls without statistical significance) and the beginning of the recovery period (8% below controls with statistical significance). After ceasing of treatment with the test item, body weight development was noted to recover in the high-dose male recovery group and showed no statistical or biological difference to controls in the first week of the recovery period. In the 2nd week of the recovery period body weight gain of the high-dose male recovery group was statistically significantly 119% above controls.

In the high-dose female recovery group, body weight gain was transiently slightly but statistically significantly higher in the 4th week of the treatment period. This was followed by a slight but statistically significant loss of mean body weight (- 2.50 g) when compared to a gain of 6.83 g in controls in the last week of treatment. These slight, transient and inconsistent effects were not considered to be in a toxicologically relevant range. During recovery period, body weight development was comparable between the dose group and controls. No effects of toxicological relevance on body weight development were noted in the male and female low-dose and mid-dose group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males and females, mean food consumption was comparable between the dose groups and the respective controls during the premating period of this study. There were no statistically or biologically significant differences. Furthermore, there was no effect of toxicological relevance on food consumption during gestation period of females. However, food consumption was biologically and statistically significantly reduced in high-dose females during lactation period. Food consumption was 43% below controls. There was no effect in low- and mid-dose females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ophthalmoscopic findings in any of the animals of this study.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Slightly but statistically significantly higher percentage of monocytes was noted in the high-dose males (5.66%) and females (3.96%) when compared to the respective control group (males: 1.54%, females: 2.08%). However, as values were within the normal range of biological variation, this isolated change with only slight differences to controls was not considered to be toxicologically relevant. Prothrombin time (PT) was noted to be marginally but statistically significantly higher in mid-dose males when compared to the control group (9% above controls). As values were within the normal range of variation, this marginal not dose dependent difference was considered as incidental in nature.

At the end of the recovery period, a statistically significant effect was noted on mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) with marginally lower values in the high-dose males recovery group (6% and 8% below controls, respectively) and the high-dose female recovery group (both 6% below controls). No such effect was noted at the end of the treatment period. Furthermore, red blood cell count and hemoglobin was comparable between the high-dose male and female recovery group and the respective controls. Differences to controls were marginal and did not adversely affect the health condition of the animals. There were no clinical signs of anemia such as pale skin or reduced spontaneous activity. Furthermore, there were no microscopic findings in the bone marrow at the end of the recovery period. Thus, these marginal and isolated changes of few red blood cell parameters were not considered as toxicologically relevant.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant but inconsistent changes were observed for specific enzymes in the blood. In males, alkaline phosphatase (AP) was statistically significantly, 58% below controls in the mid-dose group. For high-dose males, mean value was 44% below controls without achieving statistical significance. In females, AP was statistically significantly, 56% below controls in the low-dose group and 62% below controls in the high-dose group. Mean value was 34% below controls in the mid-dose female group without achieving statistical significance. Without dose dependency, differences to controls were considered as incidental and not related to the treatment with the test item. Furthermore, slightly and statistically significantly lower aspartate-aminotransferase (ASAT) in mid- (44% below controls) and high-dose (39% below controls) males also did not follow a dose dependent pattern and was not considered as toxicologically relevant with values in the normal range of variation. No statistically significant effect for ASAT was noted in females.

Slightly but statistically significantly lower mean value for sodium was noted in mid-dose males (15% below controls) but not in females. As this did not follow a dose dependent pattern this isolated change was not considered as toxicologically relevant.

There were no statistically significant changes in clinical biochemistry findings during the recovery period for any treatment level.
Urinalysis findings:
no effects observed
Description (incidence and severity):
All parameters of urinalysis of the dose groups at the end of the treatment and the recovery period were not considerably different to the corresponding control group and were within the normal range of variation.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
At the end of the treatment and the recovery period in males and females, no toxicologically relevant effects were observed in any of the tested parameters of the functional observation battery including gait, strength and reflexes.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The testes of all high-dose males were affected by tubular degeneration. Mainly elongated and round spermatids of stages V-VII were affected. More mature spermatids and spermia appeared to be unaffected. In some tubules, there were reabsorbed apoptotic bodies or Sertoli cell vacuolation. In the epididymides, only a minimal severity of cellular detritus (shedded epitehlia and/or pyknotic sperm cells) was recorded in most high-dose males. After the treatment-free recovery period, the findings in testes did not resolve.

In most mid- and high-dose animals, there was a minor severity of hepatocellular hypertrophy in the liver. In one decedent female, there was in addition a multifocal moderate necrosis that may be considered to be of agonal nature. No further hepatic lesion was recorded. Findings resolved after the treatment-free period. In male kidneys, the severity of tubular hyaline inclusions increased at the high-dose group. After evaluation by immunohistochemistry, there were no meaningful differences in the contents of α2-microglobulin between controls and test item-treated groups. In both decedent females, there was tubular cell necrosis. This finding affected mainly the distal tubules. No findings were noted after the treatment-free recovery period.

In one surviving high-dose female, there was an ulceration associated with squamous hyperplasia and inflammation in the forestomach. In another female, there was also forestomach squamous hyperplasia. Ulceration in the glandular stomach and squamous hyperplasia at the limiting ridge of the forestomach were deemed to be stress-related. These findings were associated with diffuse hypertrophy of the zona fasciculata in the adrenal cortex of two high-dose females, lymphoid atrophy of the spleen and lymph node, and bone marrow atrophy in high-dose females, and an increased atrophy of the thymus in both sexes of the high-dose group. No such differences were noted after the treatment-free recovery period.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Tubular degeneration was noted in the testes of one high-dose male. However, by microscopic examination it was deemed that sperm became damaged late during the study period, and hence, fertility was not affected during the mating period.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The copulation index (no. of animals copulated/no. of pairs x 100) was 100% in all groups including control. The fertility index (number of pregnant females/number of copulated females x 100) was comparable between the groups with an index of 90% in the control group, 100% in the low-dose group, 90% in the mid-dose group and 90% in the high-dose group.

A statistically and biologically significant effect was noted on the duration of gestation in the high-dose group. The gestation period was found to be prolonged in the high-dose group (23.89 days) when compared to 22.44 days of the control group. 6/9 dams of the high-dose group were noted with 24 days of gestation, 2/9 with 23 days and one dam (no. 91) showed a markedly prolonged gestation period of 25 days. In controls 1/9 dams were noted with 24 days of gestation, 2/9 dams with 23 days and the remaining ones with 22 days. This effect in the high-dose group was considered as adverse.

There were no effects of toxicological relevance on the number of corpora lutea and number of implantation sites in the dose groups when compared to the control group. Percentage of pre-implantation loss was within the normal range of biological variation.

Percentage of post-implantation loss was markedly and statistically significantly higher in the high-dose group when compared to the control group (high-dose group 61.31 % compared to 6.57 % in the control group).

Marginally and not statistically significantly higher post-implantation loss in the low-dose group (14.50 %) without dose dependency was considered as incidental.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (sperm measures)
reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There was no statistically or biologically significant effect on mean number of total pups (live and dead) delivered and sex ratio of pups. The mean number of still births was statistically significantly increased at the high dose (5.00 when compared to 0.11 in controls). Still births were seen in 6/8 hiigh-dose dams. The biologically increased number of still births was considered as an adverse effect of the treatment with the test item. Male and female pups were comparably affected by death at birth or in the post-natal period. Due to the increased number of still births at the high-dose level, the percentage of post-implantation loss was markedly and statistically significantly higher at the high dose (51.31%) when compared to the control group (6.57%).
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
A moderately and statistically significantly increased mortality of pups was observed in the high-dose group with a total mortality of 24.44%. No mortality occurred on PND 3 and PND 4. This effect on survival of pups was considered as related to the treatment with the test item.

Marginally higher mean mortality of 1.00% in the low-dose group and 1.11% in the mid-dose group was related to a single pup in a single dam in each the low-dose and the mid-dose group missing (attributed to cannibalism by the dam). Without dose dependency these single deaths were considered as incidental and within the normal range of biological variation.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Based on still births and reduced viability of pups in the high-dose group, moderately and statistically significantly lower total litter weight was noted in the high-dose group on post-natal day 4 (40 % below controls).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Reduced indication of suckling and coldness of pups was noted in the 6 live born pups of a high-dose dam which were found dead on the next day.

15/15 pups of one litter from a mid-dose dam were transiently noted with no indication of suckling on PND 0 but survived without further findings until terminal sacrifice. Without relevant impact on the pups, this transient finding was not considered as toxicologically relevant.

A single pup from the low-dose group which was found to be cannibalized on PND 1 was seen cold and blue the day before (PND 0). A single pup of the MD group which was found to be cannibalized on PND 1 was noted with dark skin and no indication of suckling on PND 0.

Few further external findings were observed in live pups throughout all groups. Transiently blue/dark snout in one control and one low-dose pup were considered to be spontaneous and not related to the treatment with the test item.

External findings of still born pups or pups found dead during the post-natal period irrespective of the dose group included discoloured skin, damaged tissues, squashed bodies and missing body parts due to cannibalism by the dam. Most still born pups from one high-dose dam were seen with a dark and bloated abdomen.

No other remarkable gross external abnormalities of toxicological relevance were observed in the groups.
Histopathological findings:
not examined
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
no
Relevant for humans:
not specified

Any other information on results incl. tables

Table 2: Mean Body Weight (g) - Males- Main Groups

Group

 

Premating

Mating and Postmating

Terminal Sacrifice

Day 1

Day 7

Day 14

Day 7

Day 14

C

Mean

297.10

314.80

327.50

337.10

347.60

353.70

SD

17.97

24.68

28.75

33.48

38.74

40.47

N

10

10

10

10

10

10

LD

Mean

302.50

319.50

332.60

341.40

346.89

355.70

SD

16.87

19.27

25.68

29.02

31.01

33.87

N

10

10

10

10

9

10

%

102

101

102

101

100

101

MD

Mean

300.50

318.10

330.30

339.40

344.90

347.80

SD

15.41

17.25

18.86

22.84

24.67

25.08

N

10

10

10

10

10

10

%

101

101

101

101

99

98

HD

Mean

298.70

315.30

323.70

323.30

322.70

326.40

SD

16.40

18.99

20.78

23.17

24.80

24.84

N

10

10

10

10

10

10

%

101

100

99

96

93

92

Table 3: Mean Body Weight (g) - Males - Recovery Groups

Group

 

Study Day

 

Day 1

Day 7

Day 14

Day 21

Day 28

Day 35

Day 42

CR

Mean

293.83

310.00

327.33

342.00

351.00

363.17

373.33

SD

11.87

17.13

18.64

19.71

24.41

25.36

23.92

N

6

6

6

6

6

6

6

HDR

Mean

300.67

319.67

325.50

326.83

325.33

334.00*

355.75

SD

14.72

16.77

19.37

19.50

20.53

19.41

26.74

N

6

6

6

6

6

6

4

Asterisks indicate statistically significant differences to control group C, with * p<0.05, ** p<0.01 and *** p<0.001

Table 4: Mean Body Weight (g) - Females - Main Groups

Group

 

Premating

Gestation

Lactation

Day 1

Day 7

Day 14

Day 0

Day 7

Day 14

Day 20

Day 0

Day 4

C

Mean

198.20

205.30

210.80

213.11

231.44

258.56

319.00

247.33

264.67

SD

10.28

10.11

12.24

11.06

11.86

15.29

24.74

15.31

15.86

N

10

10

10

9

9

9

9

9

9

LD

Mean

194.20

204.10

207.50

208.90

231.90

257.40

318.20

251.80

260.00

SD

5.65

6.26

8.00

7.40

7.95

10.96

18.02

17.50

12.84

N

10

10

10

10

10

10

10

10

10

%

98

99

98

98

100

100

100

102

98

MD

Mean

194.80

202.60

209.40

208.22

230.22

256.56

313.56

238.67

253.89

SD

9.82

13.05

14.03

12.96

13.45

12.20

14.30

17.90

15.78

N

10

10

10

9

9

9

9

9

9

%

98

99

99

98

99

99

98

96

96

HD

Mean

195.50

205.70

210.40

212.89

235.44

256.44

318.00

234.67

235.57*

SD

9.92

9.91

10.55

13.36

14.85

14.09

20.55

23.71

32.95

N

10

10

10

9

9

9

9

9

7

%

99

100

100

100

102

99

100

95

89

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

Table 5: Mean Body Weight (g) - Females - Recovery Groups

Group

 

Treatment Period

Recovery Period

 

Day 1

Day 7

Day 14

Day 21

Day 28

Day 35

Day 1

Day 7

Day 14

CR

Mean

195.33

200.00

207.67

213.00

213.33

220.17

223.00

223.00

223.17

SD

5.89

8.46

10.61

11.14

12.58

12.11

12.39

10.24

11.29

N

6

6

6

6

6

6

6

6

6

HDR

Mean

194.67

201.17

210.00

215.83

220.17

217.67

219.67

220.33

221.33

SD

6.62

6.31

7.29

8.18

7.41

8.31

11.11

9.83

11.36

N

6

6

6

6

6

6

6

6

6

Table 6: Mean Body Weight Gain (g) - Males - Main Groups

Group

 

Premating

Mating and Postmating

Premating Day 1 - Postmating Day 14

Premating Day 1 - Terminal Sacriface

Day 1-7

Day 7-14

Day PM14-7

Day 7-14

C

Mean

17.70

12.70

9.60

10.50

50.50

56.60

SD

7.36

4.99

5.15

6.04

21.76

23.69

N

10

10

10

10

10

10

LD

Mean

17.00

13.10

8.80

9.89

46.78

53.20

SD

5.08

8.66

5.14

5.09

16.35

18.25

N

10

10

10

9

9

10

MD

Mean

17.60

12.20

9.10

5.50*

44.40

47.30

SD

4.14

3.91

7.39

3.24

15.70

16.43

N

10

10

10

10

10

10

HD

Mean

16.60

8.40

-0.40**

-0.60***

24.00**

27.70**

SD

5.19

4.60

6.02

2.76

13.16

12.70

N

10

10

10

10

10

10

PM = Premating

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

Table 7: Mean Body Weight Gain (g) - Females - Main Groups

Group

 

Premating

Gestation

Lactation

Day 1-7

Day 7-14

Day 0-7

Day 7-14

Day 14-20

Day 0-20

Day 0-4

C

Mean

7.10

5.50

18.33

27.11

60.44

105.89

17.33

SD

7.11

5.50

4.82

4.81

10.88

16.95

5.66

N

10

10

9

9

9

9

9

LD

Mean

9.90

3.40

23.00

25.50

60.80

109.30

8.20

SD

5.92

4.72

4.06

5.80

11.27

17.01

12.37

N

10

10

10

10

10

10

10

MD

Mean

7.80

6.80

22.00

26.33

57.00

105.33

15.22

SD

5.47

7.36

4.66

4.97

8.38

9.06

9.40

N

10

10

9

9

9

9

9

HD

Mean

10.20

4.70

22.56

21.00*

61.56

105.11

-1.71*

SD

8.95

8.15

7.30

5.20

10.15

13.20

22.95

N

10

10

9

9

9

9

7

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

Table 8: Mean Body Weight Gain (g) - Females - Recovery Groups

Group

 

Treatment Period

Recovery Period

Day 1-7

Day 7-14

Day 14-21

Day 21-28

Day 28-35

Study Day
1-7

Study Day
7-14

CR

Mean

4.67

7.67

5.33

0.33

6.83

0.00

0.17

SD

3.56

5.20

1.86

2.34

3.43

3.79

5.49

N

6

6

6

6

6

6

6

HDR

Mean

6.50

8.83

5.83

4.33*

-2.5**

0.67

1.00

SD

6.47

6.79

4.26

3.01

4.85

5.54

3.69

N

6

6

6

6

6

6

6

Table 9: Mean Food Consumption (g) - Females - Main Groups

Group

 

Premating

Gestation

Lactation

Day 1-7

 Day 7-14

Day 0-7

Day 7-14

Day 14-20

Day 0-4

C

Mean

171.20

193.00

108.44

122.00

117.78

104.89

SD

11.39

16.54

11.96

11.87

14.28

19.88

N

5

5

9

9

9

9

LD

Mean

160.20

181.80

106.60

120.70

120.00

97.20

SD

11.73

12.85

14.03

9.19

11.47

19.16

N

5

5

10

10

10

10

MD

Mean

163.60

205.80

102.67

124.00

113.78

103.67

SD

7.89

54.97

10.22

10.81

8.57

16.86

N

5

5

9

9

9

9

HD

Mean

154.80

177.00

104.22

124.33

116.00

59.43**

SD

4.27

11.51

15.79

13.60

12.20

34.77

N

5

5

9

9

9

7

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

Table 10: Mean Haematology - Males and Females - End of Recovery

 

Control Male Recovery Group

High-dose Male Recovery Group

Control Female Recovery Group

High-dose Female Recovery Group

Mean corpuscular volume

54.93±1.49

51.40*±1.73

57.42±1.49

54.07***±0.51

Mean corpuscular hemoglobin

22.65±0.57

20.92**±0.43

22.17±0.67

20.75**±0.51

Asterisks indicate statistically significant differences to control group, with * p<0.05, ** p<0.01 and *** p<0.001

Table 11: Mean Clinical Chemistry - Males - End of Recovery

 

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

ASAT

(U/L)

90.76±25.55

79.20±13.83

50.50**±9.15

55.50**±10.42

AP

(U/L)

187.160±68.430

179.602±31.867

79.000**±18.215

104.388±26.046

Na

(mmol/L)

128.8±11.1

125.2±8.4

105.4*±12.8

119.6±8.0

Asterisks indicate statistically significant differences to control group, with * p<0.05, ** p<0.01 and *** p<0.001

Table 12: Mean Clinical Chemistry - Females - End of Recovery

 

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

ASAT

(U/L)

61.42±29.80

74.08±15.63

47.30±9.84

72.38±12.97

AP

(U/L)

200.495±61.472

88.606*±36.380

132.984±73.425

75.622*±25.135

Asterisks indicate statistically significant differences to control group, with * p<0.05, ** p<0.01 and *** p<0.001

Table 13: Mean Organ Weights - Males - End of Treatment

 

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Absolute organ weight (g)

Brain

2.0269±0.0478

2.0452±0.294

2.0026±0.0268

1.9253**±0.0424

Testes

3.5956±0.3541

3.7760±0.4294

3.5982±0.2928

2.6673**±0.2304

Prostate

2.5477±0.2627

2.5522±0.3813

2.3470±0.1889

2.1479**±0.2392

Spleen

0.7860±0.0490

0.8171±0.0870

0.6708*±0.0489

0.6586*±0.0656

Heart

1.1154±0.1048

1.0601±0.1034

0.9886±0.0516

0.9582*±0.0755

Pituitary

0.0110±0.0036

0.0087±0.0022

0.0079±0.0012

0.0074±0.0012

Liver

12.3815±1.6619

12.1581±1.9125

11.1299±1.4136

15.2790*±1.3653

Relative organ weight (%)

Testes

1.0211±00856

1.0692±0.1487

1.0351±0.0509

0.8197**±0.0759

Liver

3.3418±0.1895

3.4001±0.4337

3.3050±0.2397

4.6372***±0.1649

Asterisks indicate statistically significant differences to control group, with * p<0.05, ** p<0.01 and *** p<0.001

Table 14: Mean Organ Weights - Females - End of Treatment

 

Control

100 mg/kg bw/day

300 mg/kg bw/day

1000 mg/kg bw/day

Absolute organ weight (g)

Pituitary

0.0140±0.0020

0.0131±0.0017

0.0121±0.0014

0.0093*±0.0039

Liver

10.0647±0.9484

10.0518±1.0335

10.31515±0.6742

12.3377*±0.9702

Relative organ weight (%)

Thyroid/

Parathryroid

0.0096±0.0013

0.0084±0.0019

0.0097±0.0025

0.0416*±0.0032

Heart

0.3123±0.0149

0.3152±0.0261

0.2992±0.01403

0.3531**±0.0135

Liver

3.7511±0.2239

3.7842±0.2994

3.9252±0.2562

5.3964***±0.9823

Kidney

0.5789±0.497

0.7131*±0.0915

0.6162±0.0360

0.6626*±0.0308

Asterisks indicate statistically significant differences to control group C, with * p<0.05, ** p<0.01 and *** p<0.001

Table 15: Mean Organ Weights - Males and Females - End of Recovery

 

Control Male Recovery Group

High-dose Male Recovery Group

 

Control Female Recovery Group

High-dose Female Recovery Group

Absolute organ weight (g)

Liver

11.5479±1.5063

12.9948±1.4842

6.9666±0.5151

7.7119*±0.3717

Relative organ weight (%)

Heart

0.2694±0.0178

0.3127**±0.0210

0.2916±0.0211

0.3138±0.0256

Liver

3.0858±0.3053

3.6456*±0.2832

3.0849±0.1895

3.4386*±0.1894

Asterisks indicate statistically significant differences to control group, with * p<0.05, ** p<0.01 and *** p<0.001

Table 16: Mean Precoital Interval and Duration of Gestation

Group

 

Precoital Interval (Days)

Duration of Gestation (Days)

C

Mean

2.30

22.44

SD

1.16

0.73

N

10

9

LD

Mean

2.30

22.30

SD

1.06

0.48

N

10

10

MD

Mean

2.50

22.89

SD

1.08

0.33

N

10

9

HD

Mean

2.10

23.89***

SD

1.29

0.60

N

10

9

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

Table 17: Study Summary

OBSERVATIONS

Values

Groups

C

LD

MD

HD

Pairs started (N)

10

10

10

10

Females showing evidence of copulation (N)

10

10

10

10

Females achieving pregnancy (N)

9

10

9

9

Conceiving days 1 - 5 (N)

10

10

10

10

Conceiving days 6 - . . .(1) (N) and more

0

0

0

0

Pregnancy = 21 days (N)

0

0

0

0

Pregnancy = 22 days (N)

6

7

1

0

Pregnancy = 23 days (N)

2

3

8

2

Pregnancy = 24 days (N)

1

0

0

6

Pregnancy = 25 days (N)

0

0

0

1

Dams with live young born (N)

9

10

9

5

Dams with live young at day 4 pp (N)

9

10

9

4

Corpora lutea/dam (mean)

12.33

13.20

12.00

13.11

Implants/dam (mean)

10.56

11.00

11.44

11.78

Live pups/dam at birth (mean)

10.11

9.40

10.78

4.63

Live pups/dam at day 4 (mean)

10.11

9.30

10.67

3.63

Sex ratio (m/f) at birth (mean)

1.65

1.31

1.10

1.11

Sex ratio (m/f) at day 4 (mean)

1.54

1.65

1.11

1.40

Pup weight at birth (mean) (g)

6.32

6.48

6.75

6.08

Pup weight at day 4 (mean) (g)

11.38

11.81

11.53

9.49

Litter weight at birth (mean) (g)

64.16

59.92

71.67

44.98

Litter weight at day 4 (mean) (g)

112.99

106.37

120.90

67.60

ABNORMAL LIVE PUPS at birth

 

 

 

 

Dams with 0

8

8

6

3

Dams with 1

1

2

2

1

Dams with 2 and more

0

0

1

1

LOSS OF OFFSPRING

 

 

 

 

Pre-implantation (corpora lutea minus implantations)

 

 

 

 

Females with 0

3

2

6

4

Females with 1

1

2

2

1

Females with 2

1

1

0

2

Females with 3 and more

4

5

1

2

Pre-natal (implantations minus live births)

 

 

 

 

Females with 0

7

5

4

0

Females with 1

1

2

4

1

Females with 2

0

0

1

0

Females with 3 and more

1

3

0

7

Post-natal (live births minus alive at post natal day 4)

 

 

 

 

Females with 0

9

9

8

6

Females with 1

0

1

1

0

Females with 2

0

0

0

1

Females with 3 and more

0

0

0

1

Table 18: Pre- and Post-Natal Data - Summary

Group

 

Corpora Lutea (CL)

Implantation Sites (IS)

Live Pups on PND 0

Live Pups on PND 4

Pre Implantation Loss (%)

Post Implantation Loss (%)

C

Mean

12.33

10.56

10.11

10.11

13.26

6.57

SD

2.83

2.35

3.14

3.14

12.20

16.56

N

9

9

9

9

9

9

LD

Mean

13.20

11.00

9.40

9.30

17.77

14.50

SD

3.43

3.30

3.27

3.27

15.28

19.34

N

10

10

10

10

10

10

MD

Mean

12.00

11.44

10.78

10.67

4.23

6.07

SD

2.12

2.01

2.17

2.24

7.39

6.56

N

9

9

9

9

9

9

HD

Mean

13.11

11.78

4.63**

3.63***

9.35

61.31***

SD

1.76

1.09

4.24

4.21

9.95

35.76

N

9

9

8

8

9

8

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

Table 19: Litter Data - Summary

Group

 

PND 0

PND 4

Total No. of Pups

No. of Male

No. of Female

Sex Ratio (m/f)

Live Pups

Still Birth

Runt

No. of Male

No. of Female

Live Pups

Sex Ratio (m/f)

C

Mean

10.22

5.89

4.33

1.65

10.11

0.11

0.00

5.78

4.33

10.11

1.54

SD

2.86

2.57

1.66

0.95

3.14

0.33

0.00

2.73

1.66

3.14

0.83

N

9

9

9

9

9

9

9

9

9

9

9

LD

Mean

9.70

5.00

4.60

1.31

9.40

0.30

0.00

5.00

4.30

9.30

1.65

SD

3.13

1.70

2.59

0.80

3.27

0.67

0.00

1.70

2.71

3.27

1.42

N

10

10

10

9

10

10

10

10

10

10

9

MD

Mean

10.78

4.78

6.00

1.10

10.78

0.00

0.00

4.78

5.89

10.67

1.11

SD

2.17

2.11

2.00

1.30

2.17

0.00

0.00

2.11

2.03

2.24

1.29

N

9

9

9

9

9

9

9

9

9

9

9

HD

Mean

9.63

4.25

5.13

1.11

4.63**

5.00***

0.00

1.75**

1.88

3.63***

1.40

SD

2.88

1.83

2.03

0.90

4.24

4.63

0.00

2.19

2.42

4.21

1.17

N

8

8

8

8

8

8

8

8

8

8

4

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

Table 20: Mean Pup Survival Data

Group

 

Post Natal Day

0

1

2

Number of alive Males

Number of alive Females

Total

Number of alive Males

Number of alive Females

Total

Mortality

% Mortality

Number of alive Males

Number of alive Females

Total

Mortality

% Mortality

C

Mean

5.78

4.33

10.11

5.78

4.33

10.11

0.00

0.00

5.78

4.33

10.11

0.00

0.00

SD

2.73

1.66

3.14

2.73

1.66

3.14

0.00

0.00

2.73

1.66

3.14

0.00

0.00

N

9

9

9

9

9

9

9

9

9

9

9

9

9

LD

Mean

5.00

4.40

9.40

5.00

4.30

9.30

0.10

1.00

5.00

4.30

9.30

0.00

0

SD

1.70

2.72

3.27

1.70

2.71

3.27

0.32

3.16

1.70

2.71

3.27

0.00

0

N

10

10

10

10

10

10

10

10

10

10

10

10

10

MD

Mean

4.78

6.00

10.78

4.78

5.89

10.67

0.11

1.11

4.78

5.89

10.67

0.00

0.00

SD

2.11

2.00

2.17

2.11

2.03

2.24

0.33

3.33

2.11

2.03

2.24

0.00

0.00

N

9

9

9

9

9

9

9

9

9

9

9

9

9

HD

Mean

2.38

2.25

4.63

1.75

1.88

3.63

1.00

24.44*

1.75

1.88

3.63

0.00

0.00

SD

2.26

2.43

4.24

2.19

2.42

4.21

2.14

43.32

2.19

2.42

4.21

0.00

0.00

N

8

8

8

8

8

8

8

5

8

8

8

8

4

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

Table 20: Mean Pup Survival Data (Continued)

Group

 

Post Natal Day

Total Mortality (PND 0-4) %

3

4

Number of alive Males

Number of alive Females

Total

 Mortality

% Mortality

Number of alive Males

Number of alive Females

Total

Mortality

% Mortality

C

Mean

5.78

4.33

10.11

0.00

0.00

5.78

4.33

10.11

0.00

0.00

0.00

SD

2.73

1.66

3.14

0.00

0.00

2.73

1.66

3.14

0.00

0.00

0.00

N

9

9

9

9

9

9

9

9

9

9

9

LD

Mean

5.00

4.30

9.30

0.00

0.00

5.00

4.30

9.30

0.00

0.00

1.00

SD

1.70

2.71

3.27

0.00

0.00

1.70

2.71

3.27

0.00

0.00

3.16

N

10

10

10

10

10

10

10

10

10

10

10

MD

Mean

4.78

5.89

10.67

0.00

0.00

4.78

5.89

10.67

0.00

0.00

1.11

SD

2.11

2.03

2.24

0.00

0.00

2.11

2.03

2.24

0.00

0.00

3.33

N

9

9

9

9

9

9

9

9

9

9

9

HD

Mean

1.75

1.88

3.63

0.00

0.00

1.75

1.88

3.63

0.00

0.00

24.44*

SD

2.19

2.42

4.21

0.00

0.00

2.19

2.42

4.21

0.00

0.00

43.32

N

8

8

8

8

4

8

8

8

8

4

5

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

Table 21: Mean Litter Weight

 

PND 0

PND 4

Group

 

Pup Mean weight on Day 0 (g)

Pup Mean weight on Day 4 (g)

Total No. of Pups

Total Litter Weight (g)

Male Litter Weight (g)

Female Litter Weight (g)

Total Litter Weight (g)

Male Litter Weight (g)

Female Litter Weight (g)

C

Mean

6.32

11.38

10.22

64.16

37.13

27.02

112.99

65.29

47.70

SD

0.54

1.31

2.86

20.77

18.10

11.19

34.22

31.02

18.39

N

9

9

9

9

9

9

9

9

9

LD

Mean

6.48

11.81

9.70

59.92

32.69

27.23

106.37

59.11

47.26

SD

0.60

1.28

3.13

19.92

10.78

16.85

31.85

19.60

27.91

N

10

10

10

10

10

10

10

10

10

MD

Mean

6.75

11.53

10.78

71.67

32.93

38.73

120.90

55.97

64.93

SD

0.78

1.54

2.17

9.55

15.93

9.95

18.08

27.86

17.60

N

9

9

9

9

9

9

9

9

9

HD

Mean

6.08

9.49

9.63

44.98

23.46

21.52

67.60*

33.65

33.95

SD

0.62

1.06

2.88

16.18

10.57

12.60

21.32

15.61

19.10

N

5

4

8

5

5

5

4

4

4

Asterisks indicate statistically significant differences to control group C, with* p<0.05, ** p<0.01 and *** p<0.001.

Applicant's summary and conclusion

Conclusions:
A combined repeated dose toxicity study with a reproductive/developmental toxicity screening test was performed with the test substance according to OECD TG 422 and GLP at dose levels of 100, 300 and 1000 mg/kg bw/day. The NOAEL for general systemic toxicity and reproductive toxicity can be established at 300 mg/kg bw/day. General systemic toxicity was based on increased mortality, adversely reduced body weight, food consumption, and histopathology effects to the kidney and testes at the highest dose level. Developmental toxicity was based on an increase of still births, a prolonged gestation period, reduced delivery index, higher postimplantation loss, decreased litter weight, and a reduced viability index at the highest dose.