Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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 421
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Description: White solid
Storage Conditions: Room temperature (20 ± 5 °C); avoiding strong oxidizing agents, strong acids, and strong bases
Specific details on test material used for the study:
white solid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
- Source: Harlan Laboratories, B.V. Kreuzelweg 53 5961 NM Horst / Netherlands
- Age at study initiation: 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: 8d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- 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:
polyethylene glycol
Remarks:
PEG 400 / 0.5% Tween 80
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): PEG 400 / 0.5% Tween 80
- Storage temperature of food: RT

- applied as a suspension

- Dose formulations were stored in the refrigerator (5 ± 3 °C) in glass beakers
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- according GLP
- method stability of test item in vehicle: gas chromatography
- formulations investigated during the study were found to comprise test item in the range of 81.4% to 112.7% and, thus, the required content limit of ±20% with reference to the nominal content was met.
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:
Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to the day 3 post partum.
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: 28d study
- 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

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
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
Historical control data:
historical data from 5 421/422 studies

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No test item-related clinical signs were noted in males and females at any dose level.
One male treated at 100 mg/kg bw/day showed a slight reddish secretion from the nose during the last two days of the after pairing period. In this time this male lost also 5 % of his body weight. No macroscopical abnormalities were found for this male. Due to the isolated occurrence, this finding was considered to be incidental.
Two females treated at 300 and one female treated at 1000 mg/kg bw/day showed slight hair loss on the left thigh or in the neck.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no test item-related deaths.
One female treated at 100 mg/kg bw/day died during the pre-pairing period as a consequence of an intubation error. One female treated at 1000 mg/kg bw/day was removed from the study due to a closed vagina.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In males treated at 1000 mg/kg bw/day, a slight reduction in mean body weight gain was observed during the pre-pairing period. However, this slight and transient reduction was considered not to be adverse.
There were no test item-related effects on mean body weights and mean body weight gain at 100 and 300 mg/kg bw/day.
The overall values of mean body weight gain at the dose levels of 0, 100, 200 and 1000 mg/kg bw/day were: + 12 %, + 11 %, + 11 % and + 9 % during the pre-pairing period, + 2 %, + 3 %, + 2 % and + 3 % during the pairing period and + 2 %, + 2 %, + 2 % and + 2 % during the after pairing period (percentages refer to the body weight gain within the period).

There were no effects on mean body weights and mean body weight gains at any dose level and any study phase in female animals.
The slight reduction in mean body weight gain in females treated at 1000 mg/kg bw/day observed during the gestation period was most probably based on the slightly lower mean number of fetuses and not a test item-related effect.
The overall values of mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were: + 10 %, + 8 %, + 10 % and + 8 % during the pre-pairing period, + 63 %, + 63 %, + 61 % and + 56 % during the gestation period and + 5 %, + 5 %, + 7 % and + 6 % during the lactation period (percentages refer to the body weight gain within the period).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no effects on mean food consumption at any dose level and in any study phase.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No changes in organ weights, which were considered to be test item-related, were noted at any dose level.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopical findings that were considered to be related to treatment with the test item. All findings that occurred were considered to be within the range of normal background alterations.
Macroscopical findings consisted of:
- One male treated at 300 mg/kg bw/day showed a seminal vesicle, which was reduced in size.
- In the female treated at 100 mg/kg bw/day that died of an intubation error, dark red discolorations of the lungs as well as a clear fluid in the thoracic cavity were found.
- One female treated at 300 mg/kg bw/day and two females treated at 1000 mg/kg bw/day showed a reddish or dark red discoloration of the ovaries.
- The female treated at 1000 mg/kg bw/day that was removed from the study due to her closed vagina additionally showed dilation of the uterus, which contained watery fluid and had a cervical cyst.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The histopathological evaluation of the reproductive organs did not reveal any relevant changes in the high-dose animals.
Special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure did not reveal any differences between control (group 1) and high dose (group 4) males.
Sperm staging: the stages were checked on completeness of cell populations, completeness of stages, and potential degenerative changes. An assessment of the stages of spermatogenesis in the unilaterally occurring minimal and focal degenerative tubules with atrophy in a few group 1 and 4 males was not possible.
All tubular changes in testes of male groups 1 to 4 were considered to be within the normal biological range.
All findings in testes and epididymides were considered to be incidental and unrelated to the test item. The prostate and seminal vesicles did also not reveal any test item-related morphological changes.
The remaining microscopical findings recorded in this study were to be within the range of normal background lesions which may be recorded in animals of this strain and age.

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No effects on implantation rate and post-implantation loss were noted.
At 1000 mg/kg bw/day, a slightly lower mean number on implantations was noted, which was not statistically significantly different compared to the control group. Since the value was in the range of the historical control data (11.4 - 13.7), this finding was considered to be not test item-related.
The overall number of implantations per dam was 13.4, 13.6, 14.1 and 12.7 in order of ascending dose levels. The overall mean number of post-implantation loss per dam was 0.7, 1.6, 0.9 and 1.1 at the dose level of 0, 100, 300 and 1000 mg/kg bw/day.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No effects on duration of gestation were observed at any dose level. Mean duration of gestation was 21.3, 21.6, 21.7 and 21.6 days, in order of ascending dose levels.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All mated females in the study were pregnant. As a result, fertility indices (number of females achieving pregnancy as a percentage of females paired) and conception rates (number of females achieving pregnancy as a percentage of females mated) were 100 % in the control group and at all dose levels.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: absence of maternal toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
No effects on pup body weights were noted at any dose level.
In animal no. 46, 54 (group 1), 63 (group 2) and 88 (group 4) all pups were incorrectly weighed on day 1 post partum due to a technical error. Due to the low incidence, the exclusion of these litters on this day has no impact on the study.
Mean body weights of pups on day 1 post partum were: 6.1 g, 6.8 g, 6.5 g and 6.5 g, at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day respectively, body weight gain of pups during the first four days of the lactation period was + 49.4 %, + 46.1 %, + 48.9 % and + 48.8 %, respectively.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Pups sex ratio was not affected by exposure to the test item at any dose level.
At first litter check, percentages of male pups were 52 %, 58 %, 50 % and 56 %, in order of ascending dose level.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There was no effect on mean value of living pups per dam at first litter check. Birth index was unaffected by treatment with the test item.
At first litter check, one pup in the control group and 3 pups from three different litters in animals treated at 300 mg/kg bw/day were found dead. The overall mean numbers of living pups per dam at first litter check were 12.6, 12.0, 13.2 and 11.6, whereas birth indices (number of pups born alive as a percentage of implantations) were 94.6 %, 88.2 %, 93.5 % and 91.2 % at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively. The lower number of pups at 1000 mg/kg bw/day was a consequence of the lower number of implanttions and not a test item-related effect.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
There was no effect on postnatal loss at any dose level.
One pup in the control group died on day 2 post partum. All pups from the test item-related groups survived up to day 4 post partum.
Other effects:
no effects observed
Description (incidence and severity):
No macroscopical abnormalities were noted in any pup at any dose level.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of developmental toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 2: P Animals Breeding for F1 Litters

Group (mg/kg bw/day)

1 (0)

2 (100)

3 (300)

4 (1000)

Female numbers

45 – 55

56 – 66

67 – 77

78 – 88

Number of females paired (A)

11

10

11

10

Number of females mated

11

10

11

10

Number of pregnant females

11

10

11

10

Numbers of females, which did not deliver any pups (B)

0

0

0

1

Number of females which reared their pups until day 4 post partum

11

10

11

9

(A)   Female no. 64 died on day 13 of the pre-mating period. Female no. 78 was removed from the study due to a closed vagina.

(B)   Female no. 86 had only one fetal resorption.

Applicant's summary and conclusion

Conclusions:
Based on these results, the NOAEL (No Observed Adverse Effect Level) for general and reproductive toxicity in males and females was considered to be 1000 mg/kg bw/day, the highest dose level tested.
Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the test substance on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition.

Four groups of 11 males and 11 females were treated by gavage with the test substance once daily. Males were treated over a 14 -day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to the day 3 post partum.

The following dose levels were used:

- Group 1: 0 mg/kg bw/day (control group)

- Group 2: 100 mg/kg bw/day

- Group 3: 300 mg/kg bw/day

- Group 4: 1000 mg/kg bw/day

A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (PEG 400 / 0.5 % Tween 80).

The following results were obtained:

Parent Animals

- Mortality and General Tolerability: There were no test item-related deaths. One female treated at 100 mg/kg bw/day died as a consequence of an intubation error during the pre-pairing period. No test item-related clinical signs were noted in males and females at any dose level.

- Food Consumption: There were no effects on mean food consumption at any dose level and in any study phase.

- Body Weights: In males treated at 1000 mg/kg bw/day, a slight reduction in mean body weight gain was observed during the pre-pairing period. However, this slight and transient reduction was considered not to be adverse. Body weights and body weight gain in females were not affected by the treatment with the test item up to and including the dose level of 1000 mg/kg bw/day.

- Reproduction and Breeding Data: No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.

- Organ Weights: No test item-related effects on organ weights were noted at any dose level.

- Macroscopical Findings and Histopathological Examinations: No findings related to the treatment with the test item were noted during macroscopical and histopathological examinations in males or females at any dose level.

Litter Data - F1 Pups

- Findings at First Litter Check and during Lactation: No test item-related findings were noted in pups at any dose-level. Pups sex ratio was not affected by the exposure to the test item at any dose level.

- Pup Weights to Day 4 Post Partum: No effects on pup body weights or body weight gain were noted at any dose level.

- Macroscopical Findings: No findings were noted in pups at any dose level.

Based on the results, the NOAEL (No Observed Adverse Effect Level) for general and reproductive toxicity in males and females was considered to be 1000 mg/kg bw/day, the highest dose level tested.