Registration Dossier
Registration Dossier
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EC number: 250-063-5 | CAS number: 30125-47-4
Toxicological information
Repeated dose toxicity: oral
Administrative data
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- August 2011 - June 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Cross-referenceopen allclose all
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- August 2011 - June 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 422
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks
- Weight at study initiation:
- Fasting period before study: no
- Housing: individually, following exceptions: During overnight matings, male and female mating partners were housed together. Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7d
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: August 9th, 2011 To:October 6th, 2011 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- - applied as a suspension in water
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - according to GLP
- stability of the test substance in drinking water for a period of 7 days at room temperature was proven before the start of the study
- method stability of test item in drinking water: UV/VIS spectroscopy - Details on mating procedure:
- - Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight for maximum 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy - Duration of treatment / exposure:
- The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males,
and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females. - Frequency of treatment:
- daily
- 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: range finder test 300 and 1000 mg/kg bw, no effects up to 1000 mg/kg bw
- Rationale for animal assignment (if not random): Randomization - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
FOOD CONSUMPTION
- weekly
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 4
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the course of FOB
- Dose groups that were examined: all
HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of administration period
- Anaesthetic used for blood collection: Yes, anaesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany)
- Animals fasted: Yes
- How many animals: 5/sex/dose
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of administration period
- Animals fasted: Yes
- How many animals: 5/sex/dose
URINALYSIS: Yes
- Time schedule for collection of urine: males: after mating, females: 1 day before end of administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: male: postnatal day 0, female: 10d after gestation
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No - Fetal examinations:
- - External examinations: Yes, all pubs
- Soft tissue examinations: necropsy observations macroscopically
- Skeletal examinations: No
- Head examinations: No
- other: Sex ratio, Pup body weight data, Pup clinical observations - Statistics:
- Blood parameters:
For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose
group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians
For parameters with unidirectional changes:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
Urinalysis parameters: WILCOXON-test (one-sided)
Food consumption: DUNNETT-test (twosided)
fertility indices: FISHER'S EXACT test
Proportions of affected pups per litter with necropsy observations: WILCOXON-test
Weight parameters: KRUSKAL-WALLIS test - Indices:
- Pup viability/mortality; Live birth index
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- - All female animals of test group 3 (1000 mg/kg bw/d) showed yellowish discolored feces at the end of the study.
- In one female animal of test group 1 (100 mg/kg bw/d) abdominal distension was observed on study days 49 and 50. - Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- - One female of the low dose group (100 mg/kg bw/d) was sacrificed moribund on study day 50 (the animal was not pregnant). It revealed dilation of uterus and cervix with a cloudy fluid content. This was histopathologically diagnosed as a diffuse inflammation with dilation and was regarded to be the cause for the infertility of this mating pair and also the reason for sacrificing this animal. It was a single case and not regarded to be treatment-related but spontaneous in origin.
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- - During the first premating week as well as in the first gestation week the food consumption in female animals of test group 3 (1000 mg/kg bw/d) was significantly increased. These findings were assessed as spontaneous in nature and not test substance-related.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- - Functional observational battery: Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test substance-treated groups and controls, were without a dose-response
relationship or occurred in single rats only, these observations were considered to have been incidental.
- Motor activity measurement: Deviations to the control were only noted in test group 1 (100 mg/kg bw/d), i.e. increased value in interval 6 in female animals. As no significant deviations were noted with regard to the overall motor activity and no dose-response relationship was observed, the findings were assessed as being incidental. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - The weight increase in absolute and relative spleen weight in females of test group 2 (300 mg/kg bw/d) was regarded to be incidental due to a missing dose-response relationship and missing histopathologic findings in test group 3 (1000 mg/kg bw/d).
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- - 7 females of test group 3 (1000 mg/kg bw/d) revealed a yellow discoloration of the glandular stomach contents. 3 females of test group 3 (1000 mg/kg bw/d) showed the same discoloration of the contents of the jejunum.
- One female animal of test group 1 (100 mg/kg bw/d) revealed a yellow discoloration of the lung (regarded to be test substance that was aspired subsequently to the gavage procedure or due to a gavage error into the trachea) and the mediastinal lymph nodes (regarded to be the physiologic clearing route of the lung).
These discolorations were caused by the test substance but were not regarded to be a treatmentrelated adverse finding. - Neuropathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- - 2 females of test group 3 (1000 mg/kg bw/d) revealed histopathological yellow crystalline particles within the alveoli, often close to or within macrophages, rarely inside multinucleated giant cells. The same finding was also observed for the female animal of test group 1 that revealed the macropscopic finding “discoloration” in the lung. These yellow particles were regarded to be test substance that was aspired subsequently to the gavage procedure. Therefore, these discolorations in lung and mediastinal lymph node were caused by the test substance but were not regarded to be a treatment-related adverse finding.
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Number of abortions:
- not examined
- Pre- and post-implantation loss:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The mean postimplantation loss was highest in test group 2 (300 mg/kg bw/d), i.e. 28.6% compared to the control group (13.8%). As no dose-response relationship was observed, the finding was assessed as not being related to treatment.
- Total litter losses by resorption:
- not examined
- Early or late resorptions:
- not examined
- Dead fetuses:
- not examined
- Changes in pregnancy duration:
- no effects observed
- Changes in number of pregnant:
- effects observed, non-treatment-related
- Description (incidence and severity):
- 1 female of the control group, 2 females of test group 1 (100 mg/kg bw/d), 3 females of test group 2 (300 mg/kg bw/d) and 1 female of test group 3 (1000 mg/kg bw/d) were either sperm-negative or did not become pregnant. These findings were assessed to be incidental and not related to treatment as no dose-response relationship was observed.
- Other effects:
- no effects observed
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Basis for effect level:
- other: no adverse effects observed up to and including the limit dose
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- (instead of fetal body weight changes, the body weight of the pups was assessed)
Mean pup body weights were significantly lower on PND1 in test groups 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d).
One female runt was seen in test group 2 (300 mg/kg bw/d) and 2 male and 3 female runts in test group 3 (1000 mg/kg bw/d).
These values were within the range of the biological variation inherent in the strain of rats used for this study. - Reduction in number of live offspring:
- effects observed, non-treatment-related
- Description (incidence and severity):
- one stillborn pup in test group 1 (100 mg/kg bw/d)
- Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- no effects observed
- Changes in postnatal survival:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The viability index as indicator for pup mortality between PND 0 and 4 was 99% for test groups 1 and 3 (100 and 1000 mg/kg bw/d; 1 pup of 1 female in group 1 and 3 was found dead).
These findings were assessed to be incidental and not related to treatment. - External malformations:
- no effects observed
- Skeletal malformations:
- no effects observed
- Visceral malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- A situs inversus of the heart was observed in one pup of test group 3 (1000 mg/kg bw/d). The finding was assessed as being spontaneous in nature and without biological relevance.
- Other effects:
- no effects observed
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects: no effects
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects observed up to and including the limit dose
- Abnormalities:
- not specified
- Developmental effects observed:
- no
- Conclusions:
- Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage to male and female Wistar rats did not reveal signs of toxicity. The NOAEL for reproductive performance, fertility and developmental toxicity was set to 1000 mg/kg bw/d in male and female Wistar rats.
Table 1: Summary of female reproduction and delivery data
|
ORAL ADMINISTRATION(GAVAGE) - F0 FEMALES (Fl LITTER) SUMMARY OF FEMALE REPRODUCTION AND DELIVERY DATA |
|
|
|||
|
|
TEST GROUP 0 0 MG/KG BW/D |
TEST GROUP 1 100 MG/KG BW/D |
TEST GROUP 2 300 MG/KG BW/D |
TEST GROUP 3 1000 MG/KG BW/D |
|
Females on Study |
N |
10 |
10 |
10 |
10 |
|
Females Mated |
N |
10Fi |
10 |
8 |
10 |
|
Female Mating Index |
% |
100 |
100 |
80 |
100 |
|
Mating days until day 0 pc |
MEAN |
3.8D |
1.9 |
2.3 |
4.2 |
|
|
S.D. |
3.39 |
1.66 |
1.49 |
3.33 |
|
|
N |
10 |
10 |
8 |
10 |
|
days 1 to4 |
N |
9 |
9 |
8 |
9 |
|
|
% |
90 |
90 |
100 |
90 |
|
days 5 to8 |
N |
0 |
1 |
0 |
0 |
|
|
% |
0.0 |
10 |
0.0 |
0.0 |
|
days 9 to 14 |
N |
1 |
0 |
0 |
1 |
|
|
% |
10 |
0.0 |
0.0 |
10 |
|
days 15 to 21 |
N |
0 |
0 |
0 |
0 |
|
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
|
Females Pregnant |
N |
9Fi |
8 |
7 |
9 |
|
Female Fertility Index |
% |
90 |
80 |
88 |
90 |
|
Duration of Gestation (Days) |
MEAN |
22.1D |
22.0 |
21.8 |
21.9 |
|
|
S.D. |
0.35 |
0.00 |
0.45 |
0.33 |
|
Implantation sites |
TOTAL |
101 |
90 |
71 |
123 |
|
|
MEAN |
11.2 D |
11.3 |
10.1 |
13.7 |
|
|
S.D. |
3.49 |
2.60 |
6.15 |
2.06 |
|
|
N |
9 |
8 |
7 |
9 |
|
Postimplantation Loss |
TOTAL |
5 |
1 |
3 |
2 |
|
|
MEAN |
0.6 D |
0.1 |
0.4 |
0.2 |
|
|
S.D. |
0.88 |
0.35 |
0.79 |
0.44 |
|
|
N |
9 |
8 |
7 |
9 |
|
% Postimplantation Loss |
MEAN |
13.8 D |
1.6 |
28.6 |
1.4 |
|
|
S.D. |
32.82 |
4.42 |
48.80 |
2.83 |
|
|
TEST GROUP 0 0 MG/KGBW/D |
TEST GROUP 1 100 MG/KG BW/D |
TEST GROUP 2 300 MG/KG BW/D |
TEST GROUP 3 1000 MG/KG BW/D |
|
Females with Liveborn |
N |
8Fi |
8 |
5 |
9 |
Gestation Index |
% |
89 |
100 |
71 |
100 |
with Stillborn Pups |
N |
0Fi |
1 |
0 |
0 |
|
% |
0.0 |
13 |
0.0 |
0.0 |
with all Stillborn |
N |
0Fi |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Delivered |
MEAN |
12.0 D |
11.1 |
13.6 |
13.4 |
|
S.D. |
0.93 |
2.80 |
2.07 |
1.81 |
|
TOTAL |
96 |
89 |
68 |
121 |
Liveborn |
N |
96Fi |
88 |
68 |
121 |
Live Birth Index |
% |
100 |
99 |
100 |
100 |
Stillborn |
N |
0Fi |
1 |
0 |
0 |
|
% |
0.0 |
1.1 |
0.0 |
0.0 |
Table 2: Summary of litter data
|
TEST GROUP0 0 MG/KGBW/D |
TEST GROUP 1 100 MG/KG BW/D |
TEST GROUP 2 300 MG/KG BW/D |
TEST GROUP 3 1000 MG/KG BW/D |
|
(Total Number of) Litters |
N |
8 |
8 |
5 |
9 |
Litters with LivebornPups |
N |
8Fi |
8 |
5 |
9 |
|
% |
100 |
100 |
100 |
100 |
Litters with StillbornPups |
N |
0Fi |
1 |
0 |
0 |
|
% |
0.0 |
13 |
0.0 |
0.0 |
Litters with all Stillborn Pups |
N |
0Fi |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Delivered |
TOTAL |
96 |
89 |
68 |
121 |
|
MEAN |
12.0 D |
11.1 |
13.6 |
13.4 |
|
S.D. |
0.93 |
2.80 |
2.07 |
1.81 |
Pups Liveborn |
N |
96Fi |
88 |
68 |
121 |
|
% |
100 |
99 |
100 |
100 |
Pups Stillborn |
N |
0Fi |
1 |
0 |
0 |
|
% |
0.0 |
1.1 |
0.0 |
0.0 |
Pups Died |
N |
0Fi |
1 |
0 |
1 |
|
% |
0.0 |
1.1 |
0.0 |
0.8 |
Pups Sacrificed Moribund |
N |
0Fi |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Cannibalized |
N |
0Fi |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Accidental Death |
N |
0 |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Sacrificed, Maternal Death |
N |
0 |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
|
TEST GROUP0 0 MG/KGBW/D |
TEST GROUP 1 100 MG/KG BW/D |
TEST GROUP 2 300 MG/KG BW/D |
TEST GROUP 3 1000 MG/KG BW/D |
|
Pups dead day 0 |
N |
0 |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
days 1 to 4 |
N |
0 |
1 |
0 |
1 |
|
% |
0.0 |
1.1 |
0.0 |
0.8 |
Pups Surviving days O to 4 |
N |
96Fi |
87 |
68 |
120 |
Viability Index |
% |
100 |
99 |
100 |
99 |
Table 3: Summary of necropsy observations
|
TEST GROUP0 0 MG/KGBW/D |
TEST GROUP 1 100 MG/KG BW/D |
TEST GROUP 2 300 MG/KG BW/D |
TEST GROUP 3 1000 MG/KG BW/D |
|
Litters Evaluated |
N |
8 |
8 |
5 |
9 |
Pups Evaluated |
N |
95 |
89 |
68 |
120 |
Live |
N |
95 |
88 |
68 |
120 |
Stillborn |
N |
0 |
1 |
0 |
0 |
HEART, SITUS INVERSUS |
|
|
|
|
|
Pup Incidence |
N |
0 |
0 |
0 |
1 |
|
% |
0.0 |
0.0 |
0.0 |
0.8 |
Litter Incidence |
N |
0Fi |
0 |
0 |
1 |
|
% |
0.0 |
0.0 |
0.0 |
11 |
Affected Pups/Litter |
MEAN% |
0.0Wi |
0.0 |
0.0 |
0.7 |
|
S.D. |
0.00 |
0.00 |
0.00 |
1.96 |
TOTAL PUP NECROPSY OBSERVATIONS |
|
|
|
|
|
Pup Incidence |
N |
0 |
0 |
0 |
1 |
|
% |
0.0 |
0.0 |
0.0 |
0.8 |
Litter Incidence |
N |
0Fi |
0 |
0 |
1 |
|
% |
0.0 |
0.0 |
0.0 |
11 |
Affected Pups/Litter |
MEAN% |
0.0Wi |
0.0 |
0.0 |
0.7 |
|
S.D. |
0.00 |
0.00 |
0.00 |
1.96 |
Statistics: D=Dunnett-test (two-sided); Fi =Fisher's exact test (one-sided); Wi=Wilcoxon-test (one-sided)
* : p<=0.05 ** : p<=0.01
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- August 2011 - June 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks
- Weight at study initiation:
- Fasting period before study: no
- Housing: individually, following exceptions: During overnight matings, male and female mating partners were housed together. Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7d
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: August 9th, 2011 To:October 6th, 2011 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- - applied as a suspension in water
- Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: maximum 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): pregnant animals and litter together - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - according to GLP
- stability of the test substance in drinking water for a period of 7 days at room temperature was proven before the start of the study
- method stability of test item in drinking water: UV/VIS spectroscopy - Duration of treatment / exposure:
- The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males,
and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females. - Frequency of treatment:
- daily
- Details on study schedule:
- - Age at mating of the mated animals in the study: 13-14 weeks
- 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: range finder test 300 and 1000 mg/kg bw, no effects up to 1000 mg/kg bw
- Positive control:
- no
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
FOOD CONSUMPTION
- weekly
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 4
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the course of FOB
- Dose groups that were examined: all
HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of administration period
- Anaesthetic used for blood collection: Yes, anaesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany)
- Animals fasted: Yes
- How many animals: 5/sex/dose
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of administration period
- Animals fasted: Yes
- How many animals: 5/sex/dose
URINALYSIS: Yes
- Time schedule for collection of urine: males: after mating, females: 1 day before end of administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: male: postnatal day 0, female: 10d after gestation
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity - Sperm parameters (parental animals):
- stages of spermatogenesis were examined in histopathology
- 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, postnatal mortality up to day 4, presence of gross anomalies
GROSS EXAMINATION OF DEAD PUPS:
yes, all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically - Postmortem examinations (parental animals):
- GROSS PATHOLOGY: Yes
1. Adrenal glands
2. All gross lesions
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal gland
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Target organs
43. Testes (modified Davidson’s solution)
44. Thymus
45. Thyroid glands
46. Trachea
47. Urinary bladder
48. Uterus
49. Vagina
HISTOPATHOLOGY
ORGAN WEIGHTS: Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus
HISTOPATHOLOGY: Yes, control and high dose group, gross lesions in all animals
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Liver
16. Lung
17. Lymph nodes (mesenteric and axillary lymph nodes)
18. Ovaries
19. Oviducts
20. Peyer’s patches
21. Prostate
22. Rectum
23. Sciatic nerve
24. Seminal vesicles
25. Spinal cord (cervical, thoracic and lumbar cords)
26. Spleen
27. Stomach (forestomach and glandular stomach)
28. Testes
29. Thymus
30. Thyroid glands
31. Trachea
32. Urinary bladder
33. Uterus
34. Vagina - Postmortem examinations (offspring):
- SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: All pups delivered from the F0 parents were examined as soon as possible on the day of birth
GROSS NECROPSY
All surviving pups (sacrificed on PND 4 under isoflurane anesthesia with CO2), all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically. - Statistics:
- Blood parameters:
For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose
group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians
For parameters with unidirectional changes:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
Urinalysis parameters: WILCOXON-test (one-sided)
Food consumption: DUNNETT-test (twosided)
fertility indices: FISHER'S EXACT test
Proportions of affected pups per litter with necropsy observations: WILCOXON-test
Weight parameters: KRUSKAL-WALLIS test - Reproductive indices:
- Male reproduction data:
- Male mating index
- Male fertility index
Female reproduction and delivery data:
- Female mating index
- Female fertility index
- Gestation index
- Live birth index
- Post implantation loss - Offspring viability indices:
- Pup number and status at delivery
Pup viability/mortality
Sex ratio
Pup body weight data - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- - All male animals of test group 3 (1000 mg/kg bw/d) showed yellowish discolored feces towards the end of the study, i.e. on study days 33 and 34. Also all female animals of test group 3 (1000 mg/kg bw/d) showed yellowish discolored feces at the end of the study.
- In one female animal of test group 1 (100 mg/kg bw/d) abdominal distension was observed on study days 49 and 50. - Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One female animal of test group 1 (100 mg/kg bw/d) was sacrificed moribund on study day 50 (the animal was not pregnant). It revealed dilation of uterus and cervix with a cloudy fluid content. This was histopathologically diagnosed as a diffuse inflammation with dilation and was regarded to be the cause for the infertility of this mating pair and also the reason for sacrificing this animal. It was a single case and not regarded to be treatment-related but spontaneous in origin.
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- - During the first premating week as well as in the first gestation week the food consumption in female animals of test group 3 (1000 mg/kg bw/d) was significantly increased. These findings were assessed as spontaneous in nature and not test substance-related.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - In males of test groups 1 and 2 (100 and 300 mg/kg bw/d), creatinine values were lower compared to controls, but the means were not dose-dependently decreased. Therefore, this alteration was regarded as incidental and not treatment-related.
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- - Functional observational battery: Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test substance-treated groups and controls, were without a doseresponse
relationship or occurred in single rats only, these observations were considered to have been incidental.
- Motor activity measurement: Deviations to the control were only noted in test group 1 (100 mg/kg bw/d), i.e. increased value in interval 9 in male animals and increased value in interval 6 in female animals. As no significant deviations were noted with regard to the overall motor activity and no doseresponse relationship was observed, the findings were assessed as being incidental. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- - 2 females of test group 3 (1000 mg/kg bw/d) revealed histopathological yellow crystalline particles within the alveoli, often close to or within macrophages, rarely inside multinucleated giant cells. The same finding was also observed for the female animal of test group 1 that revealed the macropscopic finding “discoloration” in the lung. These yellow particles were regarded to be test substance that was aspired subsequently to the gavage procedure. Therefore, these discolorations in lung and mediastinal lymph node were caused by the test substance but were not regarded to be a treatment-related adverse finding.
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Reproductive function: oestrous cycle:
- not examined
- Reproductive function: sperm measures:
- no effects observed
- Description (incidence and severity):
- Histopathological examination of the stages of spermatogenesis did not reveal any adverse effects.
- Reproductive performance:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - 2 control males, 2 male animals of test group 1, 3 male animals of test group 2 and 1 male animal of test group 3 did not generate F1 pups. Thus, the male fertility index ranged between 70% and 90%, what reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- 1 control female, 2 females of test group 1, 3 females test group 2 and 1 female of test group 3 were either sperm-negative or did not become pregnant
- 1 control female and 2 females of test group 2 (300 mg/kg bw/d) were pregnant after mating but had not delivered viable pups (implantation sites only). Thus, the female fertility index varied between 80% and 90%. These findings were assessed to be incidental and not related to treatment as no dose-response relationship was observed. - Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Remarks on result:
- other: no adverse effects observed up to and including the highest tested dose
- Clinical signs:
- no effects observed
- Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- - one pup of a female of test group 1 and one pup of a female of test group 3 (100 and 1000 mg/kg bw/d) were found dead. These findings were assessed to be incidental and not related to treatment.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - Mean pup body weights were significantly lower on PND1 in test groups 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d).
- One female runt was seen in test group 2 (300 mg/kg bw/d) and 2 male and 3 female runts in test group 3 (1000 mg/kg bw/d).
These values were within the range of the biological variation inherent in the strain of rats used for this study. - 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
- Anogenital distance (AGD):
- not examined
- Nipple retention in male pups:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - A situs inversus of the heart was observed in 1 pup of test group 3 (1000 mg/kg bw/d). The finding was assessed as being spontaneous in nature and without biological relevance.
- Histopathological findings:
- not examined
- Other effects:
- no effects observed
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Remarks on result:
- other: no adverse effects observed up to and including the highest tested dose
- Reproductive effects observed:
- no
- Conclusions:
- Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage to male and female Wistar rats did not reveal signs of toxicity. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d in both sexes. The NOAEL for reproductive performance, fertility and developmental toxicity was set to 1000 mg/kg bw/d in male and female Wistar rats.
Table 1: Summary of female reproduction and delivery data
|
|
TEST GROUP0 0 MG/KGBW/D |
TEST GROUP 1 100 MG/KG BW/D |
TEST GROUP 2 300 MG/KG BW/D |
TEST GROUP 3 1000 MG/KG BW/D |
Females on Study |
N |
10 |
10 |
10 |
10 |
Females Mated |
N |
l0Fi |
10 |
8 |
10 |
Female Mating Index |
% |
100 |
100 |
80 |
100 |
Mating days until day 0 pc |
MEAN |
3.8 D |
1.9 |
2.3 |
4.2 |
|
S.D. |
3.39 |
1.66 |
1.49 |
3.33 |
|
N |
10 |
10 |
8 |
10 |
days 1 to 4 |
N |
9 |
9 |
8 |
9 |
|
% |
90 |
90 |
100 |
90 |
days 5 to 8 |
N |
0 |
1 |
0 |
0 |
|
% |
0.0 |
10 |
0.0 |
0.0 |
days 9 to 14 |
N |
1 |
0 |
0 |
1 |
|
% |
10 |
0.0 |
0.0 |
10 |
days 15 to 21 |
N |
0 |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Females Pregnant |
N |
9Fi |
8 |
7 |
9 |
Female Fertility Index |
% |
90 |
80 |
88 |
90 |
Duration of Gestation (Days) |
MEAN |
22.1 D |
22.0 |
21.8 |
21.9 |
|
S.D. |
0.35 |
0.00 |
0.45 |
0.33 |
Implantation sites |
TOTAL |
101 |
90 |
71 |
123 |
|
MEAN |
11.2 D |
11.3 |
10.1 |
13.7 |
|
S.D. |
3.49 |
2.60 |
6.15 |
2.06 |
|
N |
9 |
8 |
7 |
9 |
Postimplantation Loss |
TOTAL |
5 |
1 |
3 |
2 |
|
MEAN |
0.6 D |
0.1 |
0.4 |
0.2 |
|
S.D. |
0.88 |
0.35 |
0.79 |
0.44 |
|
N |
9 |
8 |
7 |
9 |
% Postimplantation Loss |
MEAN |
13.8 D |
1.6 |
28.6 |
1.4 |
|
S.D. |
32.82 |
4.42 |
48.80 |
2.83 |
|
N |
9 |
8 |
7 |
9 |
Females withLiveborn |
N |
8Fi |
8 |
5 |
9 |
Gestation Index |
% |
89 |
100 |
71 |
100 |
with Stillborn Pups |
N |
0Fi |
1 |
0 |
0 |
|
% |
0.0 |
13 |
0.0 |
0.0 |
with all Stillborn |
N |
0Fi |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Delivered |
MEAN |
12.0 D |
11.1 |
13.6 |
13.4 |
|
S.D. |
0.93 |
2.80 |
2.07 |
1.81 |
|
TOTAL |
96 |
89 |
68 |
121 |
Liveborn |
N |
96Fi |
88 |
68 |
121 |
Live Birth Index |
% |
100 |
99 |
100 |
100 |
Stillborn |
N |
0Fi |
1 |
0 |
0 |
|
% |
0.0 |
1.1 |
0.0 |
0.0 |
Table 2: Summary of litter data
|
TEST GROUP0 0 MG/KGBW/D |
TEST GROUP 1 100 MG/KG BW/D |
TEST GROUP 2 300 MG/KG BW/D |
TEST GROUP 3 1000 MG/KG BW/D |
|
(Total Number of) Litters |
N |
8 |
8 |
5 |
9 |
Litters with LivebornPups |
N |
8Fi |
8 |
5 |
9 |
|
% |
100 |
100 |
100 |
100 |
Litters with StillbornPups |
N |
0Fi |
1 |
0 |
0 |
|
% |
0.0 |
13 |
0.0 |
0.0 |
Litters with all Stillborn Pups |
N |
0Fi |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Delivered |
TOTAL |
96 |
89 |
68 |
121 |
|
MEAN |
12.0 D |
11.1 |
13.6 |
13.4 |
|
S.D. |
0.93 |
2.80 |
2.07 |
1.81 |
Pups Liveborn |
N |
96Fi |
88 |
68 |
121 |
|
% |
100 |
99 |
100 |
100 |
Pups Stillborn |
N |
0Fi |
1 |
0 |
0 |
|
% |
0.0 |
1.1 |
0.0 |
0.0 |
Pups Died |
N |
0Fi |
1 |
0 |
1 |
|
% |
0.0 |
1.1 |
0.0 |
0.8 |
Pups Sacrificed Moribund |
N |
0Fi |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Cannibalized |
N |
0Fi |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Accidental Death |
N |
0 |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
Pups Sacrificed, Maternal Death |
N |
0 |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
|
TEST GROUP0 0 MG/KGBW/D |
TEST GROUP 1 100 MG/KG BW/D |
TEST GROUP 2 300 MG/KG BW/D |
TEST GROUP 3 1000 MG/KG BW/D |
|
Pups dead day 0 |
N |
0 |
0 |
0 |
0 |
|
% |
0.0 |
0.0 |
0.0 |
0.0 |
days 1 to 4 |
N |
0 |
1 |
0 |
1 |
|
% |
0.0 |
1.1 |
0.0 |
0.8 |
Pups Surviving days O to 4 |
N |
96Fi |
87 |
68 |
120 |
Viability Index |
% |
100 |
99 |
100 |
99 |
Table 3: Summary of necropsy observations
|
TEST GROUP0 0 MG/KGBW/D |
TEST GROUP 1 100 MG/KG BW/D |
TEST GROUP 2 300 MG/KG BW/D |
TEST GROUP 3 1000 MG/KG BW/D |
|
Litters Evaluated |
N |
8 |
8 |
5 |
9 |
Pups Evaluated |
N |
95 |
89 |
68 |
120 |
Live |
N |
95 |
88 |
68 |
120 |
Stillborn |
N |
0 |
1 |
0 |
0 |
HEART, SITUS INVERSUS |
|
|
|
|
|
Pup Incidence |
N |
0 |
0 |
0 |
1 |
|
% |
0.0 |
0.0 |
0.0 |
0.8 |
Litter Incidence |
N |
0Fi |
0 |
0 |
1 |
|
% |
0.0 |
0.0 |
0.0 |
11 |
Affected Pups/Litter |
MEAN% |
0.0Wi |
0.0 |
0.0 |
0.7 |
|
S.D. |
0.00 |
0.00 |
0.00 |
1.96 |
TOTAL PUP NECROPSY OBSERVATIONS |
|
|
|
|
|
Pup Incidence |
N |
0 |
0 |
0 |
1 |
|
% |
0.0 |
0.0 |
0.0 |
0.8 |
Litter Incidence |
N |
0Fi |
0 |
0 |
1 |
|
% |
0.0 |
0.0 |
0.0 |
11 |
Affected Pups/Litter |
MEAN% |
0.0Wi |
0.0 |
0.0 |
0.7 |
|
S.D. |
0.00 |
0.00 |
0.00 |
1.96 |
Statistics: D=Dunnett-test (two-sided); Fi =Fisher's exact test (one-sided); Wi=Wilcoxon-test (one-sided)
* : p<=0.05 ** : p<=0.01
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- specific investigations: other studies
- Type of information:
- experimental study
- Adequacy of study:
- other information
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 422
- GLP compliance:
- not specified
- Type of method:
- in vivo
- Endpoint addressed:
- other: metabolomics
- Species:
- rat
- Sex:
- male/female
- Dose / conc.:
- 100 mg/kg bw/day
- Dose / conc.:
- 300 mg/kg bw/day
- Dose / conc.:
- 1 000 mg/kg bw/day
- Details on study design:
- - K-EDTA samples were taken from the retroorbital sinus in all rats under isoflurane anesthesia on study day 7, 14 and 28 for mass spectrometry-based metabolite profiling analysis
- GC–MS (gas chromatography–mass spectrometry) and LC–MS/MS (liquid chromatography–MS/MS) were used for broad profiling and hormone measurement
- SP-LC–MS/MS (solid phase extraction-LC–MS/MS) was applied for the determination of catecholamine and steroid hormone levels - Details on results:
- Based on metabolome analysis, the NOAEL was defined at 1000 mg/kg body weight/day (male and female rats).
- Conclusions:
- The metabolomic NOAEL corresponds to the toxicological NOAEL established in an OECD Guidline 422 study.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 012
- Report date:
- 2012
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
Test material
- Reference substance name:
- 3,4,5,6-tetrachloro-N-[2-(4,5,6,7-tetrachloro-2,3-dihydro-1,3-dioxo-1H-inden-2-yl)-8-quinolyl]phthalimide
- EC Number:
- 250-063-5
- EC Name:
- 3,4,5,6-tetrachloro-N-[2-(4,5,6,7-tetrachloro-2,3-dihydro-1,3-dioxo-1H-inden-2-yl)-8-quinolyl]phthalimide
- Cas Number:
- 30125-47-4
- Molecular formula:
- C26H6Cl8N2O4
- IUPAC Name:
- 4,5,6,7-tetrachloro-2-[2-(4,5,6,7-tetrachloro-1,3-dioxo-2,3-dihydro-1H-inden-2-yl)quinolin-8-yl]-1H-isoindole-1,3(2H)-dione
- Test material form:
- solid: nanoform
- Details on test material:
- - Physical state/ appearance: solid / yellow
- Shape of particles: spherical
- Aspect ratio: 1.5
- Particle size distribution: 62.7 nm
- Crystal structure: crystalline
- Surface area of particles: 29.6 m^2/g
- Surface treatment: no
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks
- Weight at study initiation: not specified
- Fasting period before study: no
- Housing: individually, following exceptions: During overnight matings, male and female mating partners were housed together. Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7d
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: August 9th, 2011 To: October 6th, 2011
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on oral exposure:
- -Applied as a suspension in water
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - according to GLP
- stability of the test substance in drinking water for a period of 7 days at room temperature was proven before the start of the study
- method stability of test item in drinking water: UV/VIS spectroscopy - Duration of treatment / exposure:
- The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females
- Frequency of treatment:
- daily
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: range finder test 300 and 1000 mg/kg bw, no effects up to 1000 mg/kg bw
- Positive control:
- no
Examinations
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
FOOD CONSUMPTION
- weekly
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 4
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the course of FOB
- Dose groups that were examined: all
HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of administration period
- Anaesthetic used for blood collection: Yes, anaesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany)
- Animals fasted: Yes
- How many animals: 5/sex/dose
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of administration period
- Animals fasted: Yes
- How many animals: 5/sex/dose
URINALYSIS: Yes
- Time schedule for collection of urine: males: after mating, females: 1 day before end of administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: male: postnatal day 0, female: 10d after gestation
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
1. Adrenal glands
2. All gross lesions
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal gland
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Target organs
43. Testes (modified Davidson’s solution)
44. Thymus
45. Thyroid glands
46. Trachea
47. Urinary bladder
48. Uterus
49. Vagina
HISTOPATHOLOGY: Yes, control and high dose group, gross lesions in all animals
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Liver
16. Lung
17. Lymph nodes (mesenteric and axillary lymph nodes)
18. Ovaries
19. Oviducts
20. Peyer’s patches
21. Prostate
22. Rectum
23. Sciatic nerve
24. Seminal vesicles
25. Spinal cord (cervical, thoracic and lumbar cords)
26. Spleen
27. Stomach (forestomach and glandular stomach)
28. Testes
29. Thymus
30. Thyroid glands
31. Trachea
32. Urinary bladder
33. Uterus
34. Vagina - Statistics:
- Blood parameters:
For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose
group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians
For parameters with unidirectional changes:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
Urinalysis parameters: WILCOXON-test (one-sided)
Food consumption: DUNNETT-test (twosided)
fertility indices: FISHER'S EXACT test
Proportions of affected pups per litter with necropsy observations: WILCOXON-test
Weight parameters: KRUSKAL-WALLIS test
Results and discussion
Results of examinations
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- - All male animals of test group 3 (1000 mg/kg bw/d) showed yellowish discolored feces towards the end of the study, i.e. on study days 33 and 34. Also all female animals of test group 3 (1000 mg/kg bw/d) showed yellowish discolored feces at the end of the study.
- In one female animal of test group 1 (100 mg/kg bw/d) abdominal distension was observed on study days 49 and 50. - Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One female animal of test group 1 (100 mg/kg bw/d) was sacrificed moribund on study day 50 (the animal was not pregnant). It revealed dilation of uterus and cervix with a cloudy fluid content. This was histopathologically diagnosed as a diffuse inflammation with dilation and was regarded to be the cause for the infertility of this mating pair and also the reason for sacrificing this animal. It was a single case and not regarded to be treatment-related but spontaneous in origin.
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- - During the first premating week as well as in the first gestation week the food consumption in female animals of test group 3 (1000 mg/kg bw/d) was significantly increased. These findings were assessed as spontaneous in nature and not test substance-related.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - In males of test groups 1 and 2 (100 and 300 mg/kg bw/d), creatinine values were lower compared to controls, but the means were not dose-dependently decreased. Therefore, this alteration was regarded as incidental and not treatment-related.
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- - Functional observational battery: Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test substance-treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental.
- Motor activity measurement: Deviations to the control were only noted in test group 1 (100 mg/kg bw/d), i.e. increased value in interval 9 in male animals and increased value in interval 6 in female animals. As no significant deviations were noted with regard to the overall motor activity and no dose-response relationship was observed, the findings were assessed as being incidental. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- - The weight increase in absolute and relative spleen weight in females and in relative spleen weight in males of test group 2 (300 mg/kg bw/d) was regarded to be incidental due to a missing dose-response relationship and missing histopathologic findings in test group 3 (1000 mg/kg bw/d).
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- - 2 males and 7 females of test group 3 (1000 mg/kg bw/d) revealed a yellow discoloration of the glandular stomach contents. 3 females of test group 3 (1000 mg/kg bw/d) showed the same discoloration of the contents of the jejunum.
- One female animal of test group 1 (100 mg/kg bw/d) revealed a yellow discoloration of the lung (regarded to be test substance that was aspired subsequently to the gavage procedure or due to a gavage error into the trachea) and the mediastinal lymph nodes (regarded to be the physiologic clearing route of the lung).
These discolorations were caused by the test substance but were not regarded to be a treatment-related adverse finding. - Neuropathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- - 2 females of test group 3 (1000 mg/kg bw/d) revealed histopathological yellow crystalline particles within the alveoli, often close to or within macrophages, rarely inside multinucleated giant cells. The same finding was also observed for the female animal of test group 1 that revealed the macropscopic finding “discoloration” in the lung. These yellow particles were regarded to be test substance that was aspired subsequently to the gavage procedure. Therefore, these discolorations in lung and mediastinal lymph node were caused by the test substance but were not regarded to be a treatment-related adverse finding.
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
Effect levels
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Remarks on result:
- other: no adverse effects observed up to and including the limit dose
Target system / organ toxicity
- Critical effects observed:
- no
Applicant's summary and conclusion
- Conclusions:
- Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage to male and female Wistar rats did not reveal signs of toxicity. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d in both sexes.
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