Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct 2015 - Dec 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Remarks:
OECD TG 422

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
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:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No. of test material: 30-043-11
- Purity test date: Apr 01, 2015
- Content: w(Mw =404.16 g/mol) = 82.8 g/100 g
w(Mw = 488.32 g/mol) = 100.0 g/100 g
- Expiry date: 02 Jan 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of test substance in rat diet was demonstrated for a period of 8 days at room temperature. At the beginning (during premating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration
for potential homogeneity analyses. These samples were used as a concentration control at the same time. All measured values for the test substance were in the expected range of the target concentrations (90 - 110%), demonstrating the correctness of the preparations.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The required quantity of test substance was weighed in a beaker depending on the test group and thoroughly mixed with a small amount of food. Then further amounts of food were added to this premix and thoroughly mixed. Afterwards, further amounts of
food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing of this final mix was carried out for about 3 minutes in a laboratory mixer.

FORM AS APPLIED IN THE TEST (if different from that of starting material): with diet

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Animals were free from any clinical signs of disease
- Age at study initiation: 13 - 15 weeks
- Weight at study initiation: males: 334.0 g - 374.2 g; females: 194.3 g - 223.5 g
- Housing: individually during study period (during overnight matings male and female mating partners were housed together; pregnant animals and their litters were housed together until PND 13); grouped (up to 5 animals per sex and cage) during pretreatment
- Diet: ad libitum (from the day of supply to the day before necropsy); ground Kliba maintenance diet mouse-rat “GLP” (supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical and for microbiological contaminants. The drinking water was regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms. On the basis of the analytical findings the drinking water was found to be suitable. With regard to the analytical findings of chemical and microbiological contaminants and the duration of application, the diet was found to be suitable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 06 Oct 2015 To: 02 Dec 2015 (males); 21 and 31 Dec 2015 (females)

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The required quantity of test substance was weighed in a beaker depending on the test group and thoroughly mixed with a small amount of food. Then further amounts of food were added to this premix and thoroughly mixed. Afterwards, further amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing of this final mix was carried out for about 3 minutes in a laboratory mixer.

DIET PREPARATION
- Rate of preparation of diet (frequency): not specified; The stability of test substance in rat diet was demonstrated for a period of 8 days at room temperature.
- Mixing appropriate amounts with (Type of food): ground Kliba maintenance diet mouse-rat “GLP” (supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Storage temperature of food: not specified; The stability of test substance in rat diet was demonstrated for a period of 8 days at room temperature.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight (from about 16.00 h until 06.30 - 09.00 h of the following morning) for a maximum of 2 weeks
- A vaginal smear was prepared after each mating and examined for the presence of sperm.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (gestation day (GD) 0)
- After successful mating each pregnant female was caged (how): Pregnant females were provided with nesting material (cellulose wadding) towards the end of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. These samples were used as a concentration control at the same time. At these time points, one sample from the mid concentration was additionally taken for concentration control analysis. The samples were analyzed via HPLC-MS system with external standard quantification. The analytical investigations of the test substance preparations were carried out in compliance with the Principles of Good Laboratory Practice.

All determined mean concentrations were in the range from 90 % - 110 % of the nominal concentration.
Duration of treatment / exposure:
males: 37 days; females: 66 days
The duration of treatment covered a 2-weeks premating period and mating in both sexes (mating pairs were from the same test group), 8 days postmating in males and approximately 4 weeks postmating in one female (pregnant by stain) as well as the entire gestation and approximately 3 weeks of lactation period in females up to one day prior to the day of scheduled sacrifice of the animals.
Frequency of treatment:
continuously (via diet)
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 ppm
Remarks:
high-dose level;
for females during lactation: 500 ppm;
= mean intake of approx. 54 mg/kg bw/d (males), approx. 78 mg/kg bw/d (females)
Dose / conc.:
300 ppm
Remarks:
mid-dose level;
for females during lactation: 150 ppm;
= mean intake of approx. 17 mg/kg bw/d (males), approx. 23 mg/kg bw/d (females)
Dose / conc.:
100 ppm
Remarks:
low-dose group;
for females during lactation: 50 ppm;
= mean intake of approx. 6 mg/kg bw/d (males), approx. 8 mg/kg bw/d (females)
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: In a dose finding study male and female rats were treated with the test substance (600 ppm and 1800 ppm) via the diet for a period of 14 days. No effects were observed at 600 ppm. At 1800 ppm treatment-related findings like discolored feces, decreased body weight and decreased food and water consumption occured. In two further studies the test substance was administered via gavage at doses of 10, 50 and 250 mg/kg bw/d. Marked effects in respiratory and gastrointestinal tract, which are typical for irritating substances, up to mortality were observed from a dose of 50 mg/kg bw/d . Thus, an application of the test substance via the diet was chosen for this study.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes (for mortality, for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity, parturition and lactation behavior of the dams)
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration (day 0) and at weekly intervals during the administration period
- Examined parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning) until sacrifice with the following exceptions for the females:
• During the mating period, the females were weighed on the day of positive evidence of sperm (GD0) and on GD 3, 7, 10, 14, 17 and 20.
• Females with litter were weighed on the day of parturition (PND 1), PNDs 4, 7, 10 and 13.
• Body weight was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: once a week with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
• Food consumption of the females with evidence of sperm was determined on gestation days (GD) 0- 3, 3 - 7, 7 - 10, 10 - 14, 14 - 17 and 17 - 20.
• Food consumption of the females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13.
- Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observation battery and motor activity assessment (see "OTHER")

OTHER:
Functional observation battery (FOB):
- A functional observational battery was performed in the first five parental male animals per test group and the first five females with litter per group (in order of delivery) at the end of the administration period starting at about 10.00 h on study day 31 (males) and 45 (females).
- The FOB started in a randomized sequence with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests.
- Examined parameters:
• Home cage observations: posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations: behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/ pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/ stereotypes, gait, activity/ arousal level, feces excreted within 2 minutes (consistency/color), urine excreted within 2 minutes (amount/color), rearing within 2 minutes, other findings
• Sensory motor tests/reflexes: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test

Motor activity assessment:
- measured from 14:00 h onwards on the same day as the FOB was performed (at the end of the administration period) in the first five surviving parental males and the first five surviving females with litter (in order of delivery) per group
- The animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts were counted over 12 intervals for 5 minutes per interval.

Estrous cycle:
- In all parental females, estrous cycle length and normality was evaluated by daily analysis of vaginal smear during a minimum of 2 weeks prior to mating and throughout the pairing period until the female exhibited evidence of copulation. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.
- For all females in a pool of up to 50 animals, estrous cycle normality was evaluated before the randomization.

Male reproduction data:
- The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.

Female reproduction and delivery data
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.

Thyroid hormones (males only)
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: all surviving males at termination
- Parameters checked in table 3 were examined.
Oestrous cyclicity (parental animals):
- In all parental females, estrous cycle length and normality was evaluated by daily analysis of vaginal smear during a minimum of 2 weeks prior to mating and throughout the pairing period until the female exhibited evidence of copulation. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.
- For all females in a pool of up to 50 animals, estrous cycle normality was evaluated before the randomization.
Sperm parameters (parental animals):
Parameters examined in parental males:
testes weight, epididymides weight, glans penis weight, prostate weight, weight of seminal vesicles with coagulating glands, weight of Bulbourethal gland (Cowper´s gland); histopathological examination of epididymides, prostate gland, seminal vesicles, testes; spermatogenic staging profiles
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4 pups/sex/litter as nearly as possible); excess pups were sacrificed under isoflurane anesthesia by decapitation, blood was sampled for determination of thyroid hormone concentrations and pups were examined externally, eviscerated and their organs were assessed macroscopically.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, presence of gross anomalies, postnatal mortality, weight gain, anogenital distance (AGD), presence of nipples/areolae in male pups (on PND 13), blood thyroid hormone concentrations, gross necropsy

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, on study day 37
- Maternal animals: All surviving animals, on study day 56/66

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 4 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 (surplus pups after litter standardization) and 13 (remaining pups after litter standardization) days of age.
- These animals were subjected to postmortem examinations (macroscopic and microscopic (if required) as follows: the pups were examined externally and eviscerated, and the organs were assessed macroscopically.

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

HISTOPATHOLOGY
Thyroid glands/parathyroid glands of one male and one female pup per litter at 13 days of age were fixed in neutral buffered 4% formaldehyde solution for possible further processing (if required).
Statistics:
see table 5
Reproductive indices:
Male reproduction data:
- The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.
- mating and fertility indices were calculated for F1 litters (for formulas see "Any other information on materials and methods")

Female reproduction and delivery data
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.
- mating, fertility and gestation indices, live birth index, postimplantation loss were calculated for F1 litters (for formulas see "Any other information on materials and methods")
Offspring viability indices:
- viability index, survival index (for formulas see "Any other information on materials and methods")

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
high dose group (1000 ppm)
- Decreased body weights in the males during the entire mating and postmating period as well as decr eased terminal body weight at necropsy (up to 6% below control)
- Decreased body weight change in the males during premating (up to 38% below control)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- food consumption of the high-dose parental males statistically significantly below the control during the entire premating period (up to 11%)
- food consumption of the high-dose parental females statistically significantly below the control during the premating days 0 - 7 (about 10%)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Relative reticulocyte counts in males of test group 3 (1000 ppm) lower compared to controls. No other red blood cell parameter was changed in these individuals. Therefore, regarded as not adverse.
- Absolute eosinophil counts in males of test group 2 (300 ppm) higher compared to controls, but the values were not dose-dependently changed.
- Absolute eosinophil counts in females of test groups 1, 2 and 3 (100, 300 and 1000 ppm) lower compared to controls. However, the means in the test groups were within the historical control range, whereas that one of the controls was slightly above this range (absolute eosinophil counts 0.07-0.18 Giga/L).
- Red blood cell (RBC) counts in females of test group 3 (1000 ppm) increased, but the values were within the historical control range (RBC 7.71-8.65 Tera/L).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- In females of test group 3 (1000 ppm) increased sodium values. Among the electrolyte levels (sodium, potassium, chloride) sodium was the only altered parameter and therefore its change was regarded as treatment-related, but not adverse.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
- insufficient maternal care during PND 1 - 2 and a complete litter loss on PND 3 of one high-dose female
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- decrease in spleen weight of males of test groups 2 and 3 (300 and 1000 ppm) which was not thought to be treatment-related due to the fact that the relative organ weight was not significantly changed and no histopathologic finding was observed that could explain the weight decrease.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings (see Attachment) occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All findings (see Attachment) occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. The stages of spermatogenesis in the testes of males of test group 3 (1000 ppm) were comparable to those of the controls.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

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

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
ca. 17 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
= 300 ppm
Sex:
male
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
ca. 23 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
= 300 ppm
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
ca. 54 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
= 1000 ppm
Sex:
male
Basis for effect level:
other: no adverse effects observed up to the highest dose tested
Dose descriptor:
NOAEL
Remarks:
fertility and reproductive performance
Effect level:
ca. 78 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
= 1000 ppm
Sex:
female
Basis for effect level:
other: no adverse effects observed up to the highest dose tested

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- complete litter loss of one high-dose female (1000 ppm) on PND 3 (10 cannibalized and 2 dead pups)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- The mean body weights of the high-dose (1000 ppm) male and female pups and both sexes combined were statistically significantly below the concurrent control values on PND 1 (about 14%, 15% and 14%, respectively), in male pups and both sexes combined on PND 7 (about 15%, respectively) and in male pups on PND 10 (about 13%).
- Mean body weight change of the high-dose (1000 ppm) female pups and both sexes combined was statistically significantly below the concurrent control values during PND 4 - 7 (about 15%, respectively).
- 2 male and 1 female runt in the control, 2 male and 7 female runts in the low-dose group, 1 male and 2 female runts in the mid-dose group, 5 male and 13 female runts in the high-dose group.
- 3 male and 9 female runts in the litter of one high-dose female which had a complete litter loss on PND 3.
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:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
- Statistically significantly increased percentage (97.2% vers. 6.3% in control) of male pups having areolae on PND 13 in the 1000 ppm group.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- A few pups showed spontaneous findings at gross necropsy, such as discolored liver lobe (pale), dilated ureter, dilated renal pelvis and post mortem autolysis; no relation to dosing and/or findings also present in the historical control data at comparable or even higher incidences; therefore considered as not treatment-related
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 17 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
= 300 ppm
Sex:
male
Basis for effect level:
body weight and weight gain
other: sexual maturation
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 23 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
= 300 ppm
Sex:
female
Basis for effect level:
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.:
300 ppm
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes

Any other information on results incl. tables

Table 6: Summary delivery report

Sex:Female

 

 

 

Test Group 0/ F

0 ppm

 

Test Group 1/ F

100 ppm

Test Group 2/ F

300 ppm

Test Group 3/F

1000ppm

No. of females at start

N

10

 

10

10

10

No. of females mated

N

10

f-

10

10

10

 

%

100.0

 

100.0

100.0

100.0

Pregnant

N

10

f-

10

10

10

 

%

100.0

 

100.0

100.0

100.0

Dead

N

0

 

0

0

0

Without delivery

N

0

 

0

1

0

- Pregnant

N

0

 

0

1

0

- Not pregnant

N

0

 

0

0

0

-- Delivering

N

10

f-

10

9

10

 

%

100.0

 

100.0

90.0

100.0

-- With liveborn pups

N

10

f-

10

9

10

Gestation Index

%

100.0

 

100.0

90.0

100.0

Gestation days

Mean

22.4

n

22.6

22.4

21.9

 

S.d.

0.5

 

0.7

0.5

0.3

 

N

10

 

10

9

10

-- With stillborn pups

N

1

f+

0

1

0

 

%

10.0

 

0.0

11.1

0.0

-- With all pups stillborn

N

0

f+

0

0

0

 

%

0.0

 

0.0

0.0

0.0

Table 7: Summary litter report - pup status

Sex:Female 

Test

Group 0/ F

0 ppm

 

Test

Group 1/ F

100 ppm

Test

Group 2/ F

300 ppm

Test

Group 3/F

1000ppm

Total Number of Pregnant

Females

N

10

 

10

10

10

Total number of litters

N

10

 

10

9

10

With liveborn pups

N

10

f-

10

9

10

 

%

100.0

 

100.0

100.0

100.0

With stillborn pups

N

1

f+

0

1

0

 

%

10.0

 

0.0

11.1

0.0

With all pups stillborn

N

0

f+

0

0

0

 

%

0.0

 

0.0

0.0

0.0

Implantation Sites

N

115

 

126

105

120

 

Mean

11.5

x-

12.6

10.5

12.0

 

S.d.

1.2

 

2.2

4.6

1.8

 

N

10

 

10

10

10

Pups delivered

N

111

 

110

101

114

 

Mean

11.1

x-

11.0

11.2

11.4

 

S.d.

1.1

 

2.7

3.7

1.8

 

N

10

 

10

9

10

Postimplantation Loss

Mean%

3.2

x+

12.8

13.6

5.0

 

S.d.

6.9

 

14.3

31.7

4.4

 

N

10

 

10

10

10

Pups liveborn

N

109

 

110

100

114

 

%

98.2

 

100.0

99.0

100.0

 

Mean

10.9

x-

11.0

11.1

11.4

 

S.d.

1.1

 

2.7

3.7

1.8

 

N

10

 

10

9

10

Pups stillborn

N

2

 

0

1

0

 

%

1.8

 

0.0

1.0

0.0

 

Mean

0.2

x+

0.0

0.1

0.0

 

S.d.

0.6

 

0.0

0.3

0.0

 

N

10

 

10

9

10

Perinatal Loss

Mean%

1.7

x+

0.0

0.9

0.0

 

S.d.

5.3

 

0.0

2.8

0.0

 

N

10

 

10

9

10

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

f=FISHER-EXACT; x=WILCOX

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the test substance caused signs of systemic toxicity (decreased food consumption and decreased body weight parameters in male and female parental animals) at a concentration of 1000 ppm in the diet. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 300 ppm for male (about 17 mg/kg bw/d) and female Wistar rats (about 23 mg/kg bw/d).
The NOAEL for fertility and reproductive performance was 1000 ppm for male (about 54 mg/kg bw/d) and female (about 78 mg/kg bw/d) Wistar rats, the highest concentration tested.
The NOAEL for developmental toxicity in the offspring was 300 ppm, based on decreased pup body weights and an increased number of areolae in male pups at PND 13.
Executive summary:

In an OECD 422 study, the test compound was administered daily as a constant homogeneous addition to the food in different concentrations to groups of 10 male and 10 female Wistar rats to screen for potential systemic, reproductive and developmental toxicity. After a two-week premating period, these parental animals were mated and the females were allowed to give birth and bring up the offspring until sacrifice on PND 4 or PND 13.

Analyses confirmed the overall accuracy of the prepared concentrations and the homogeneity of the test substance in the diet. The stability of these preparations was also demonstrated over a period of 8 days under ambient conditions.

In the clinical examinations of the parental animals F0 no clinical symptoms were caused by the test compound up to the high concentration of 1000 ppm. In the subsequent investigations including the detailed clinical observation (DCO), the functional observational battery (FOB) and the measurement of motor activity (MA) no treatment related differences to control were observed at any concentration.

Food consumption of the high-dose F0 males was statistically significantly below the concurrent control values during the entire premating period (up to 11%) and in the high-dose females during premating days 0 - 7 (about 10%).

The mean body weights of the high-dose parental males were statistically significantly below the concurrent control values during the mating and postmating period as well as the terminal body weights. (up to 6%).

Additionally the mean body weight change of the high-dose parental males was statistically significantly below the concurrent control values during premating (up to 38%).

In summary the affected food consumption and body weight parameters of the rats of the high dose group point to systemic toxicity. This trend is only borderline; however, the total of minor changes is considered as an adverse effect of treatment. Concerning clinical pathology no treatment-related, adverse effects were observed up to a concentration of the compound of 1000 ppm in the diet. Regarding pathology all findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. Regarding fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups (100, 300, and 1000 ppm) during the entire study. All F0 parental animals proved to be fertile. Mating behavior, conception, implantation and parturition were not influenced.

Regarding developmental toxicity, the mean body weights of the high-dose (1000 ppm) male and female pups were statistically significantly below the concurrent control values on PND 1. Additionally, lower weights were noted in male pups and both sexes combined on PND 7 (about 15%) as well as in male pups on PND 10 (about 13%). The mean body weight change of the high-dose (1000 ppm) female pups and both sexes combined was statistically significantly below the concurrent control values during PND 4 - 7 (about 15%).

In summary, the decreased pup body weight parameters in the high-dose group are considered as an adverse effect of treatment. One high-dose female (1000 ppm) delivered very small pups (all pups are runts at PND 1) and had a complete litter loss on PND 3 due to an insufficient maternal care. Between PND1 and PND3 ten pups of this high-dose female where cannibalized and two pups died. At necropsy the two died pups had an empty stomach.

In test group 3 (1000 ppm) the number of runts was increased (18 vers. 3 runts in the control group), the viability index indicating pup mortality during lactation (PND 0 - 4) was decreased (89.1% vers. 99.2% in control) and the number of cannibalized/dead pups was increased during PND 1 - 4. Although a complete litter loss as finding (with a low incidence) was present in the historical control data of the facility, a treatment related cause of these changes cannot be excluded.

The percentage (97.2% vers. 63.3% in control) of male pups having areolae on PND 13 was statistically significantly increased in test group 3 (1000 ppm). The percantages of the historical controls range between 0% and 81.7%. This finding was assessed as an adverse treatment related change.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.