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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Remarks:
source of read across approach
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V.
- Age at study initiation: 11 wks
- Weight at study initiation: males 322-375 g, females 205-238 g
- Housing: individual (after mating).
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 80/11) rodent maintenance diet ad libitum.
- Water: tap water ad libitum.
- Acclimation period: minimum 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30-70 %
- Air changes: 10 - 15 air changes per hour.
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
TESTING SOLUTION
The dose formulations were be prepared fresh daily using the test item as supplied by the Sponsor.
Test item was weighed into a glass beaker on a tared precision balance and approximately 80 % of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days at maximum.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear; this day was referred to as day 0 post coitum.
- After successful mating each pregnant female was caged: individual.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group (middle only) as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. During week 3 of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice toHarlan Laboratories Ltd and stored there at -20 ± 5 °C until analysis.
The samples were analyzed by HPLC coupled to an UV detector following an analytical procedure provided by the Sponsor.
Duration of treatment / exposure:
Males: minimum 4 weeks
Females: approximately 7 weeks
Frequency of treatment:
once every day
Details on study schedule:
Males and Females were treated with the test substance for 14 days prior to mating (10 ml/kg bw); after 14 days, one male was paired with one female and animals treated for 14 days at maximum; males and females were then seperated; males were subjected to necroscopy, whereas treatment of females continued until day 3 post partum
Males were sacrificed after they had been treated for at least 28 days. Dams and pups were sacrificed on day 4 post partum.
If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.
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:
11
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on available toxicological data of the test compound and of a closely related compound

Examinations

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: daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Males: pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; after pairing period weekly.
Females: pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; gestation days 0 – 7, 7 14 and 14 – 21 and days 1 - 4 of the lactation.
No food consumption was recorded during the pairing period.
Oestrous cyclicity (parental animals):
The number of implantation sites and corpora lutea was recorded for all dams with litters.
Sperm parameters (parental animals):
Parameters examined in male parental generations: testis weight, epididymis weight.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain

GROSS EXAMINATION OF DEAD PUPS
Dead pups were examined macroscopically.
Postmortem examinations (parental animals):
All animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4 % formaldehyde solution.
All parent animals and pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.
For the parent animals, special attention was directed at the organs of the reproductive system.
The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.
At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately. The ovaries (including oviduct) and uterus (including cervix and vagina) from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution. Slides of testes, epididymides and ovaries from all animals of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.

Postmortem examinations (offspring):
Dead pups were examined macroscopically.
Statistics:
The following statistical methods were used to analyze food consumption, body weights, organ weights and reproduction data:
- means and standard deviations of various data were calculated.
- the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- the Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
Fertility index [%] = (number of pregnenat females / number of females paired) x 100
Conception index [%] = (number of pregnenat females / number of females mated) x 100
Gestation index [%] = (number of femeales bearing live pups/ number of pregnant females) x 100
Offspring viability indices:
Viability index [%] = (number of live pups on Day 4 post-partum / number of pups born alive) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Red colored feces were recorded in males and females of all groups treated with the test item from the start of dosing until necropsy. Severity increased with increasing dose levels. No clinical signs were noted in males or females at any dose level.
Mortality:
no mortality observed
Description (incidence):
All animals survived the scheduled study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The overall differences in mean body weight gain for males at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were: +12 %, +12 %, +11 % and +9 % during the pre-pairing period and +3 %, +3 %, +2 % and +2 % during the pairing period (percentages refer to the body weight gain within the period).
The overall differences in mean body weight gain for females at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were: +6 %, +7 %, +5 % and +4 % during the pre-pairing period, +58 %, +54 %, +53 % and +56 % during the gestation period and +9 %, +6 %, +6 % and +5 % 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 food consumption at any dose level in males and females.
Organ weight findings including organ / body weight ratios:
no effects observed
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 occurred in individual males or females and were considered to be within the range of the normal background alterations.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no microscopic findings that could be attributed to treatment with the test item. All findings recorded were considered to be within the range of normal background alterations.
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Description (incidence and severity):
No effects on corpora lutea count were observed at any dose level. Mean number of corpora lutea per dam was 17.0, 17.6, 15.8 and 15.7 in order of ascending dose levels.
No effects on implantation rate and post-implantation loss were noted. An increase in the total number of post-implantation loss was noted at 300 mg/kg bw/day but due to the absence of any dosage relationship this was considered to be incidental. The overall number of implantations per dam was 14.4, 12.7, 12.6 and 12.7 in order of ascending dose level. The overall mean number of post-implantation loss per dam was 0.7, 1.1, 1.5 and 1.0 at the dose level of 0, 100, 300 and 1000 mg/kg bw/day.
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
No effect on mating performance or fertility was observed at any dose level. Mean (median) precoital times calculated for the first pairing period were 3.4 (3), 3.6 (3), 4.0 (3) and 4.5 (3) days in order of ascending dose levels.
Two females in the control group (nos. 52 and 55), two of the low dose group (nos. 59 and 65), one of the mid dose group (no. 70) and three of the high dose group (nos. 78, 81 and 87) were not pregnant -> Fertility Index = Conception Index = 81.8 %, 81.8 %, 90.9 % and 72.7 % in order of ascending dose levels.
No birth was recorded for one female at the dose level of 1000 mg/kg bw/day (no. 83). At termination on day 25 post coitum, only implantation sites were found in this female -> gestation index = 100, 100, 100, 87.5 % in order of ascending dose levels.
None of these parameters were statistically significantly different compared to the control group and in the range of the historical control data and therefore considered to be a result of biological variability.
No effects on duration of gestation were observed at any dose level. Mean duration of gestation was 21.7, 21.8, 21.8 and 21.9 days, in order of ascending dose levels.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No observations were noted in pups during the first litter check or during lactation at any dose level. Pups sex ratio was not affected by exposure to the test item at any dose level.
Mortality / viability:
no mortality observed
Description (incidence and severity):
The overall mean numbers of living pups per dam at first litter check were 13.8, 11.6, 11.1 and 11.7, whereas birth indices (number of pups borne alive as a percentage of implantations) were 95.4 %, 91.2 %, 88.1 % and 92.1 % at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively. There was no effect on postnatal loss at any dose level. At the dose level of 300 mg/kg bw/day, one female (no. 67) gave birth to two dead pups. Four pups of this dam died within the first four days after delivery. This isolated occurrence was considered to be incidental.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects on pup body weights were noted at any dose level.
Mean body weights of pups on day 1 post partum were: 5.8 g, 6.3 g, 6.0 g and 6.2 g, at the dose levels of 0, 100, 300 and 1000 mg/kg/day, respectively, body weight gain of pups during the first four days of the lactation period was +45.4 %, +47.9 %, +54.7 % and +43.0 %, respectively.
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
No findings were found in pups at any dose level.
Histopathological findings:
not specified

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Group
(mg/kg/day)

1
(0)

2
(100)

3
(300)

4
(1000)

Female numbers

45-55

56-66

67-77

78-88

Number of females paired

11

11

11

11

Number of females mated

11

11

11

11

Number of pregnant females (A)

9

9

10

8

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

9

9

10

7

(A)   Female nos. 52, 55, 59, 65, 70, 78, 81 and 87 were not pregnant

(B)   Female no 83 had only implantation sites

Applicant's summary and conclusion

Conclusions:
NOAEL (maternal/reproductive toxicity) > 1000 mg/kg bw/day
NOAEL (developmental toxicity) > 1000 mg/kg bw/day
Executive summary:

Substance was tested according to OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test) on wistar rats by oral gavage, at dose 100, 300, 1000 mg/kg bw/day, during minimum 4 weeks for males and approximately 7 weeks for females.

All animals survived the scheduled study period. Red colored feces were recorded in males and females of all groups treated with the test item from the start of dosing until necropsy. Severity increased with increasing dose levels. No clinical signs were noted in males or females at any dose level. There were no effects on food consumption at any dose level in males and females. There were no macroscopical findings that were considered to be related to treatment with the test item. All findings occurred in individual males or females and were considered to be within the range of the normal background alterations. There were no microscopic findings that could be attributed to treatment with the test item; all findings recorded were considered to be within the range of normal background alterations. No effects on corpora lutea count were observed at any dose level. No effects on implantation rate and post-implantation loss were noted. An increase in the total number of post-implantation loss was noted at 300 mg/kg bw/day but due to the absence of any dosage relationship this was considered to be incidental. No effect on mating performance or fertility was observed at any dose level. No birth was recorded for one female at the dose level of 1000 mg/kg bw/day (no. 83). No effects on duration of gestation were observed at any dose level.

There was no effect on postnatal loss at any dose level. At the dose level of 300 mg/kg bw/day, one female (no. 67) gave birth to two dead pups. Four pups of this dam died within the first four days after delivery. This isolated occurrence was considered to be incidental. No observations were noted in pups during the first litter check or during lactation at any dose level. Pups sex ratio was not affected by exposure to the test item at any dose level. No effects on pup body weights were noted at any dose level. No findings were found in pups at any dose level.

Conclusion

NOAEL (maternal/reproductive toxicity) > 1000 mg/kg bw/day

NOAEL (developmental toxicity) > 1000 mg/kg bw/day