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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2012 - 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 422
Version / remarks:
22 Mar 1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
solid yellow

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks
- 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 exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): water
- Storage temperature of food: RT

- applied as a suspension
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- according 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
Doses / concentrationsopen allclose all
Dose / conc.:
110 mg/kg bw/day (actual dose received)
Dose / conc.:
330 mg/kg bw/day (actual dose received)
Dose / conc.:
1 110 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

Examinations

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 / No / No data
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
- 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 number and status at delivery
Pup viability/mortality
Sex ratio
Pup body weight data

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
All male and female animals of test group 2 (330 mg/kg bw/d) and 3 (1110 mg/kg bw/d) showed yellowish discolored feces from day 7 until the end of the entire study during premating, mating and postmating period.
All female animals of test group 2 (330 mg/kg bw/d) and 3 (1110 mg/kg bw/d) showed yellowish discolored feces during the whole gestation period.
One female animal (No. 113) of test group 1 (110 mg/kg bw/d) showed palpable mass through skin from gestation day 12 until the end of the period. This finding was assessed as being spontaneous in nature.
One sperm-positive F0 female of the test group 1 (No. 119) and 1 sperm-negative F0 female of the test group 3 (No. 137) did not deliver any F1 pups.
All female animals of test group 2 (330 mg/kg bw/d) and 3 (1110 mg/kg bw/d) showed yellowish discolored feces during the entire lactation period.
One female animal (No. 113) of test group 1 (110 mg/kg bw/d) showed palpable mass through skin during the whole lactation period. This finding was assessed as being spontaneous in nature.
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely in the present study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weight and mean body weight gain of the F0 females in test groups 1-3 (110, 330 and 1110 mg/kg bw/d) were comparable to the concurrent control throughout the entire premating, gestation and lactation periods.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption of the male and female F0 generation parental animals in all test substance-treated groups (110, 330 and 1110 mg/kg bw/d) was comparable to the concurrent control group during the entire study period, covering premating, gestation and lactation.
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related, adverse changes among clinical chemistry parameters were observed.
In females of test groups 2 and 3 (300 and 1000 mg/kg bw/d) glucose levels were lower compared to controls. This was the only changed parameter in these animals and therefore, the alteration was regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002).
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The weight decrease in absolute brain weight in males of test group 2 (330 mg/kg bw/day) was regarded to be incidental due to a missing dose response relationship and missing histopathologic findings in test group 3 (1110 mg/kg bw). The decrease in heart weights of males in all test groups was also regarded to be incidental as no histopathologic correlate could explain the weight decrease.
All other mean weight parameters did not show significant differences when compared to the control groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Several animals of test group 1, 2 and 3 revealed a yellow discoloration of the contents of the glandular stomach, small and large intestines. These findings are regarded to be treatment related.
All other gross lesions noted were single observations and they were regarded to have developed spontaneously and unrelated to compound and treatment.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The discoloration of the content in the digestive tract was regarded to be a consequence to the oral intake of the test substance which is of yellow color. Therefore, the gross findings in the remaining animals were not investigated in addition.
Only in some animals the gross findings “discoloration of contents” could be approved by light microscopy. This was regarded to be due to clearing of the stomach and intestine before histotechnical processing.
All other findings noted were either single observations or they were biologically equally distributed between control and treatment group. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 97.5% and 99% for test group 0 and 2, respectively. For test groups 1 and 3, the live birth indices were 100%.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The postimplantation loss in test group was 8.8% in test group 0, 8.9% in test group 1, 7.8% in test group 2 and 5.6% in test group 3. These findings 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.
Details on maternal toxic effects:
Regarding pathology, macroscopically yellow discoloration of the content of the digestive tract in numerous animals was observed. In single treated animals and single organs of the digestive tract these yellow pigments could also be observed histopathologically. Beside the discoloration no signs of toxicity in the respective tissues were noted. This finding is regarded to be a consequence to the oral intake of the yellow test substance and therefore treatment related but not adverse in nature.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 110 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects observed

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group.
Two male runts were seen in control group. One female runt was seen in test group 2 (330 mg/kg bw/d).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The viability index indicating pup mortality during lactation (PND 0 - 4) was 98% in the control, 100% (test group 1), 95 % (test group 2) and 99% (test group 3) and was in the normal range of biological variation inherent in the strain of rats used for this study.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 110 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 3: Significantly altered absolute organ weights

 

Male animals

Test group (mg/kg bw/day)

1

(110)

2

(330)

3

(1110)

Brain

99%

95%**

101%

* p ≤ 0.05, ** p ≤ 0.01

Table 4: Significantly altered relative organ weights

 

Male animals

Test group (mg/kg bw/day)

1

(110)

2

(330)

3

(1110)

Heart

86%**

87%**

89%*

* p ≤ 0.05, ** p ≤ 0.01

Table 5: Relevant gross findings

 

Male animals

Female animals

Test group

(mg/kg bw/day)

0

(0)

1

(110)

2

(330)

3

(1110)

0

(0)

1

(110)

2

(330)

3

(1110)

No of animals

10

10

10

10

10

10

10

10

Glandular stomach

Discoloration of contents

 

5

7

10

 

1

4

8

Jejunum

Discoloration of contents

 

 

2

9

 

 

1

2

Cecum

Discoloration of contents

 

 

 

 

 

 

 

2

Colon

Discoloration of contents

 

 

2

9

 

 

 

3

Table 6: Male fertility indices for F0 males

 

Test group 0

(0 mg/kg bw/d)

Test group 1

(110 mg/kg bw/d)

Test group 2

(330 mg/kg bw/d)

Test group 3

(1110 mg/kg bw/d)

Male fertility index [%]

100

90

100

90

* p ≤ 0.05; ** p ≤ 0.01

 

Table 7: Fertility indices for F0 females

 

Test group 0

(0 mg/kg bw/d)

Test group 1

(110 mg/kg bw/d)

Test group 2

(330 mg/kg bw/d)

Test group 3

(1110 mg/kg bw/d)

Female fertility index [%]

100

90

100

100

* p ≤ 0.05; ** p ≤ 0.01

 

Table 8: Sex ratio of live F1 pups

PND 0

Test group 0

(0 mg/kg bw/d)

Test group 1

(110 mg/kg bw/d)

Test group 2

(330 mg/kg bw/d)

Test group 3

(1110 mg/kg bw/d)

Live males [%]

55.1

55.1

44.1

55.8

Live females [%]

44.9

44.9

55.9

44.2

 

 

 

 

 

PND 4

 

 

 

 

Live males [%]

55.2

55.1

44.6

56.3

Live females [%]

44.8

44.9

55.4

43.7

 

Table 9: Number of males with no offspring and of not pregnant females

 

Male animals

Female animals

Test group

(mg/kg bw/day)

0

(0)

1

(110)

2

(330)

3

(1110)

0

(0)

1

(110)

2

(330)

3

(1110)

Number of animals

10

10

10

10

10

10

10

10

No offspring

 

1

 

1

 

 

 

 

Animal not pregnant

 

 

 

 

 

1

 

1

Applicant's summary and conclusion

Conclusions:
Thus, 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 1110 mg/kg bw/d in male and female Wistar rats.
Executive summary:

The test substance was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg bw/d (test group 0), 110 mg/kg bw/d (test group 1), 330 mg/kg bw/d (test group 2) and 1110 mg/kg bw/d (test group 3).

The objective of the study was to detect possible effects of the test substance on the integrity and performance of male and female reproductive systems including gonadal function, mating behavior, conception, gestation and parturition. Furthermore, it was intended to obtain information about the general toxicological profile including target organs and the no observed adverse effect level (NOAEL) after repeated oral administration. The duration of treatment covered a 2-week pre-mating and 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 two weeks thereafter in females.

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear.

A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals.

Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 andlactation days 1 - 4.

Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating and mating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, at the day of parturition (postnatal day [PND] 0) and on PND 4.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia with CO2and examined macroscopically for external and visceral findings.

Clinicochemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period.

Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5animals per sex and test group.

All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

The various analyses:

·        demonstrated the stability of the test substance in drinking water over a period of 7 days at room temperature

·        confirmed the overall accuracy of the prepared concentrations.

The following test substance-related adverse effects/findings were noted:

Test group 3: 1110 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

·        No test substance-related adverse findings were observed.

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

·        No test substance-related adverse findings were observed.

Test group 2: 330 mg/kg bw/d

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

·        No test substance-related adverse findings were observed.

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

·        No test substance-related adverse findings were observed.

Test group 1: 110 mg/kg bw/d

F0 PARENTALANIMALS

F0 PARENTALANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/ PATHOLOGY

·        No test substance-related adverse findings were observed.

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

·        No test substance-related adverse findings were observed.

Thus, under the conditions of this reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance and fertility was 1110 mg/kg bw/d.

The NOAEL for developmental toxicity in the F1 progeny was found to be 1110 mg/kg bw/d.

The NOAEL for general, systemic toxicity was 1110 mg/kg bw/d.