Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD 421
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx 11 weeks
- Weight at study initiation: (P) Males: 315 - 346 g; Females: 189-216 g
- Fasting period before study: no
- Housing: 5 animals/sex/cage (pre-mating, also males post mating), Females were caged together with males on a one-to-one-basis during mating and individually post mating.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%,
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 April 2013 To: 11 June 2013
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made
for specific gravity of the vehicle

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item is not soluble in water.
- Concentration in vehicle: adjusted to dose
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: max 14 days
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal
copulatory plug. This day was designated Day 0 post-coitum.
- Further matings after two unsuccessful attempts: no (not required in guideline)
- After successful mating each pregnant female was caged individually
Duration of treatment / exposure:
28 days (males)
42 - 52 days (females)
Frequency of treatment:
daily
Duration of test:
28 days (males)
42 - 52 days (females)

Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
it is referrred to the robust study summary for fertility as this is the routine place in IUCLID for entering a OECD 421 study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.
- Cage side observations included viability/morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily, at least conducted approximately 1 to 2 hours after dosing.


BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.


Ovaries and uterine content:
not examined
Fetal examinations:
not examined
Statistics:
yes
Indices:
Percentage live males at first Litter Check, Percentage live females at First Litter Check, Percentage of postnatal loss, Viability index
Clinical signs:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption (absolute and relative) was decreased during the first week of treatment and during lactation for females at 200 mg/kg bw/day.
Ophthalmological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Findings of note were recorded in the kidneys and thymus of females treated at 200 mg/kg bw/day and consisted of:
- Glomerular and tubular necrosis (marked) of the kidneys in 1/10 females, noted at necropsy as many reddish foci (both sides).
- Lymhoid atrophy (moderate) of the thymus in 2/10 females, noted at necropsy as gelatinous or reduced size. These two females also showed total litter loss.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg bw/day, there were possible treatment related macroscopic findings in kidneys and/or thymus for the two females that showed a total litter loss.
One female (no. 71) that had a total litter loss showed general pale discolouration, many reddish foci on the kidneys, gelatinous thymus, and the other (female no. 79) showed the thymus reduced in size.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
not specified
Description (incidence and severity):
One female (no. 71) had a total litter loss.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
not examined
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Details on maternal toxic effects:
Food consumption (absolute and relative) was decreased during the first week of treatment and during lactation for females at 200 mg/kg bw/day.

At 200 mg/kg bw/day, there were possible treatment related macroscopic findings in kidneys and/or thymus for the two females that showed a total litter loss.
One female (no. 71) that had a total litter loss showed general pale discolouration, many reddish foci on the kidneys, gelatinous thymus, and the other (female no. 79) showed the thymus reduced in size.

Findings of note were recorded in the kidneys and thymus of females treated at 200 mg/kg bw/day and consisted of:
- Glomerular and tubular necrosis (marked) of the kidneys in 1/10 females, noted at necropsy as many reddish foci (both sides).
- Lymhoid atrophy (moderate) of the thymus in 2/10 females, noted at necropsy as gelatinous or reduced size. These two females also showed total litter loss.
Dose descriptor:
NOEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
food consumption and compound intake
histopathology: non-neoplastic
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): At 200 mg/kg bw/day body weights of the pups were reduced on Days 1 and 4 of lactation.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
Developmental toxicity was observed at 200 mg/kg bw/day. This consisted of an increased number of dead pups at first litter check and postnatal loss, and a decreased number of living pups at first litter check and viability index. These findings were mainly caused by two litters of which all pups died on Day 1 or 2 of lactation (all twenty pups that were found dead at first litter check and 7 out of 12 pups that died later). One of these dams showed marked glomerular and tubular necrosis of the kidneys which probably had an impact on the animal’s health and could have resulted in her total litter loss on Day 1 of lactation. This could be regarded as an indirect effect on pup development for this litter, however this was not the case for the other pup deaths.
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
This consisted of an increased number of dead pups at first litter check and postnatal loss, and a decreased number of living pups at first litter check and viability index. These findings were mainly caused by two litters of which all pups died on Day 1 or 2 of lactation (all twenty pups that were found dead at first litter check and 7 out of 12 pups that died later). One of these dams showed marked glomerular and tubular necrosis of the kidneys which probably had an impact on the animal’s health and could have resulted in her total litter loss on Day 1 of lactation. This could be regarded as an indirect effect on pup development for this litter, however this was not the case for the other pup deaths.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Developmental toxicity was observed at 200 mg/kg bw/day.
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
no effects observed
Developmental effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Summary tables are contained in the attachment to this endpoint study record.

It is also referrred to the robust study summary for fertility as this is the routine place in IUCLID for entering a OECD 421 study.

Conclusions:
The test material was determined to have a maternal toxicity NOEL of 60 mg/kg bw/day, and a fetal developmental NOAEL of 60.0 mg/kg bw/day (bodyweight) in the rat under the conditions of the test.
Executive summary:

In this guideline (OECD 421) study conducted with GLP certification, the test material was found to provoke developmental toxicity in rats. The test was conducted at three dose levels (20, 60 and 200 mg/kg bw/day). 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. Treatment-related developmental toxicity was observed at 200 mg/kg bw/day. This consisted of an increased number of dead pups at first litter check and postnatal loss, and a decreased number of living pups at first litter check and viability index. The NOEL was set at the 60 mg/kg bw/day.

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Yellowish solid
- Analytical purity: 96.4 g/100g (HPLC)
- Purity test date: 2013
- Lot/batch No.: 0008150577
- Expiration date of the lot/batch: 21 January 2015
- Stability under test conditions: stable
- Storage condition of test material: At room temperature protected from light. Solutions were handled amber-coloured glassware or container wrapped in aluminium-foil.

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx 11 weeks
- Weight at study initiation: (P) Males: 315 - 346 g; Females: 189-216 g
- Fasting period before study: no
- Housing: 5 animals/sex/cage (pre-mating, also males post mating), Females were caged together with males on a one-to-one-basis during mating and individually post mating.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%,
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 April 2013 To: 11 June 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made
for specific gravity of the vehicle

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item is not soluble in water.
- Concentration in vehicle: adjusted to dose
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: max 14 days
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal
copulatory plug. This day was designated Day 0 post-coitum.
- Further matings after two unsuccessful attempts: no (not required in guideline)
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples (0.5 mL) were taken using a pipette with the tip cut off (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice.

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
28 days (males)
42 - 52 days (females)
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 13 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
200 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 study
- Rationale for animal assignment (if not random): Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.
- Cage side observations included viability/morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily, at least conducted approximately 1 to 2 hours after dosing.


BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
Parameters examined in male parental animals:
testis weight, epididymis weight, staging of spermatogenesis
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk. If possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All animals after 28 days.
- Maternal animals: All surviving animals at day 5-7 post partum
- Females which failed to deliver: Post-coitum Day 27 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).
- Females with total litter loss: within 24h after total litter loss

GROSS NECROPSY
- Gross necropsy consisted of external, thoracic and abdominal examination, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Epididymides and testes were weighed.
Histopathology investigation was peformed on the ovaries of the females of Groups 1 and 4, the testes and epididymides of the males of all groups, the additional slides of the testes of the males of all groups to examine staging of spermatogenesis and all gross lesions of all animals (all dose groups).
For animals not mated or not pregnant, the following organs were examined histopathologically: cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.
For animals with total litter loss, the mammary gland area was investigated.

The numbers of former implantation sites and corpora lutea were recorded for all paired females
Postmortem examinations (offspring):

GROSS NECROPSY
- Gross necropsy consisted of external examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS: not investigated
Statistics:
If the variables could be assumed to follow a normal distribution, the Dunnett-test (manyto-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
The Fisher Exact-test was applied to frequency data.
Reproductive indices:
Mating index (%), fertility index (%), conception index (%), gestation index (%), duration of gestation
Offspring viability indices:
Percentage live males at first Litter Check, Percentage live females at First Litter Check, Percentage of postnatal loss, Viability index

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg bw day, treatment related clinical signs consisted of piloerection (six females), hunched posture (three females), and one female (no. 71) also showed lethargy and pale appearance.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight gain was noted for males at 60 and 200 mg/kg bw/day. Females at 200 mg/kg bw/day showed a body weight loss during the first week of treatment; this recovered during the remainder of the study period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight gain was noted for males at 60 and 200 mg/kg bw/day. Females at 200 mg/kg bw/day showed a body weight loss during the first week of treatment; this recovered during the remainder of the study period.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):

At 200 mg/kg bw/day, absolute and relative weight of the epididymides was statistically significantly reduced. Additionally, terminal body weights were decreased for males at 60 and 200 mg/kg bw/day.
The statistically significant change between testes weights of treated and control animals were considered not to be a sign of toxicity as all values remained within normal limits and no dose response was observed.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg bw/day, there were possible treatment related macroscopic findings in kidneys and/or thymus for the two females that showed a total litter loss.
One female (no. 71) that had a total litter loss showed general pale discolouration, many reddish foci on the kidneys, gelatinous thymus, and the other (female no. 79) showed the thymus reduced in size.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in testes and epididymides of males treated at 200 mg/kg bw/day and consisted of:
- Intraluminal cell debris of the epididymides in 8/10 males (up to moderate degree).
- Oligospermia of the epididymides in 4/10 males (up to slight degree).
- Germ cell exfoliating into the lumen of seminiferous tubules of the testes (without degeneration) in 9/10 males (up to moderate degree).

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Findings of note were recorded in the kidneys and thymus of females treated at 200 mg/kg bw/day and consisted of:
- Glomerular and tubular necrosis (marked) of the kidneys in 1/10 females, noted at necropsy as many reddish foci (both sides).
- Lymhoid atrophy (moderate) of the thymus in 2/10 females, noted at necropsy as gelatinous or reduced size. These two females also showed total litter loss.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
Dose descriptor:
NOEL
Remarks:
reproductive performance (fertility)
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive indeces were not affected.

Target system / organ toxicity (P0)

Critical effects observed:
not specified
Lowest effective dose / conc.:
200 mg/kg bw/day (actual dose received)
System:
other: Detailed investigations on organ toxicity are not part of a screening study for reproductive toxicity.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
200 mg/kg bw - This consisted of an increased number of dead pups at first litter check and postnatal loss, and a decreased number of living pups at first litter check and viability index. These findings were mainly caused by two litters of which all pups died on Day 1 or 2 of lactation (all twenty pups that were found dead at first litter check and 7 out of 12 pups that died later). One of these dams showed marked glomerular and tubular necrosis of the kidneys which probably had an impact on the animal’s health and could have resulted in her total litter loss on Day 1 of lactation. This could be regarded as an indirect effect on pup development for this litter, however this was not the case for the other pup deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg bw/day body weights of the pups were reduced on Days 1 and 4 of lactation.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Effect levels (F1)

Dose descriptor:
NOEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
Remarks on result:
other: observation period postnatal days 1-4

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

REPRODUCTION DATA SUMMARY

  control 20mg/kg bw 60 mg/kg bw 200 mg/kg bw
Females paired 10 10 10 10
Females mated 9 10 10 10
Pregnant females 8 10 9 10
Females with living pups on Day 1 8 10 9 9
Mating index (%)
(Females mated / Females paired) * 100
90 100 100 100
Fertility index (%)
(Pregnant females / Females paired) * 100
80 100 90 100
Conception index (%)
(Pregnant females / Females mated) * 100
89 100 90 100
Gestation index (%)
(Females with living pups on Day 1 / Pregnant females) * 100
100 100 100 90

BODY WEIGHTS (GRAM), males 
 
    control 20mg/kg bw 60 mg/kg bw 200 mg/kg bw
PREMATING
DAY1
MEAN 327 327 326 331
WEEK1 ST.DEV 8.1 12.0 5.8 7.6
  N 10 10 10 10
DAY8 MEAN 348 346 340 335*
WEEK2 ST.DEV 10.8 13.0 7.6 9.2
  N 10 10 10 10
MATINGPERIOD
DAY1
MEAN 366 362 353 346**
WEEK1 ST.DEV 13.5 13.5 8.7 10.2
  N 10 10 10 10
DAY8 MEAN 375 374 362 353**
WEEK2 ST.DEV 15.9 15.0 8.1 12.2
  N 10 10 10 10
DAY15 MEAN 392 389 376 367**
WEEK3 ST.DEV 16.5 17.7 8.5 15.7
  N 10 10 10 10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

BODY WEIGHTS (GRAM), females
    control 20mg/kg bw 60 mg/kg bw 200 mg/kg bw
PREMATING
DAY1
MEAN 201 204 202 204
WEEK1 ST.DEV 7.1 9.0 5.7 10.3
  N 10 10 10 10
DAY8 MEAN 206 203 204 196
WEEK2 ST.DEV 10.4 6.9 8.3 10.0
  N 10 10 10 10
MATINGPERIOD
DAY1
MEAN 211 213 213 211
WEEK1 ST.DEV 7.4 8.1 9.5 7.4
  N 10 10 10 10
DAY8
WEEK2
MEANST.DEVN 231
---
1
  226
---
1
 
DAY15
WEEK3
MEANST.DEVN 249
---
1
     
DAY22
WEEK4
MEANST.DEVN 238
---
1
     
DAY29
WEEK5
MEANST.DEVN 240
---
1
     
POSTCOITUM
DAY0
MEAN 213 213 214 207
  ST.DEV. 10.9 7.8 9.3 7.7
  N 8 10 9 10
DAY4 MEAN 225 227 226 220
  ST.DEV. 10.2 9.2 7.0 8.5
  N 8 10 9 10
DAY7 MEAN 233 235 236 229
  ST.DEV. 8.6 11.0 6.1 8.7
  N 8 10 9 10
DAY11 MEAN 248 248 249 240
  ST.DEV. 13.2 12.6 7.3 7.3
  N 8 10 9 10
DAY14 MEAN 261 263 261 254
  ST.DEV. 11.1 12.1 7.0 9.3
  N 8 10 9 10
DAY17 MEAN 283 285 284 274
  ST.DEV. 14.8 9.5 8.3 9.3
  N 8 10 9 10
DAY20 MEAN 319 317 315 305
  ST.DEV. 21.0 12.1 13.1 16.3
  N 8 10 9 10
LACTATION  
DAY1 MEAN 243 247 244 230
  ST.DEV. 14.3 12.1 11.6 12.4
  N 8 10 9 10
DAY4 MEAN 261 260 257 252
  ST.DEV. 15.2 11.6 6.5 8.3
  N 8 10 9 8

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

CORPORA LUTEA AND IMPLANTATION SITES SUMMARY
    GROUP1 GROUP2 GROUP3 GROUP4
    CONTROL 20MG/KG 60MG/KG 200MG/KG
Corpora Lutea MEAN 13.5 12.8 13.4 13.2
ST.DEV 2.4 1.3 2.1 2.3
  N 8 10 9 10
Implantations MEAN 12.5 11.2 12.2 12.6
ST.DEV 1.7 2 1.6 2.5
  N 8 10 9 10

    CONTROL 20MG/KG 60MG/KG 200MG/KG
LITTERS          
  Total 8 10 9 10
           
DURATION OF GESTATION             
  MEAN (+) 21.5 21.3 21.7 21.4
  ST.DEV 0.5 0.5 0.7 0.7
  N 8 10 9 10
           
DEAD PUPS AT FIRST LITTER CHECK               
  LITTERS AFFECTED (#) 0 2 1 2
  TOTAL 0 2 4 20
           
LIVING PUPS AT FIRST LITTER CHECK               
  % OF MALES / FEMALES 48 / 52 59 / 41 45 / 55 46 / 54
  TOTAL 92 106 98 99
  MEAN 11.5 10.6 10.9 9.9
  ST.DEV 2 1.8 2.5 4.2
  N (litters) 8 10 9 10
           
POSTNATAL LOSS              
  % OF LIVING PUPS 1.1 0 1 12.1
  LITTERS AFFECTED (#) 1 0 1 4
  TOTAL (#) 1 0 1

12 ##

  VIABILITY INDEX (#) 98.9 100 99

87.9 ##

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

+/++ Steel-test significant at 5% (+) or 1% (++) level

Viability index = (Number of alive pups before planned necropsy / Number of pups born alive) *100

BODY WEIGHTS OF PUPS (GRAM)
GROUP1 GROUP2 GROUP3 GROUP4
  Sex   CONTROL 20MG/KG 60MG/KG 200MG/KG
MEAN 6.3 6.2 6.3

5.4 *

Male ST.DEV 0.6 0.7 0.7 0.5
  N (litters) 8 10 9 9
MEAN 6 6 6

5.1 *

Day 1 Female ST.DEV 0.7 0.6 0.6

0.6

  N (litters) 8 10 9

9

MEAN 6.2 6.1 6.1

5.3 *

M/F ST.DEV 0.6 0.6 0.7

0.6

    N (litters) 8 10 9 9
MEAN 9.4 9.2 9.3

7.5 **

Male ST.DEV 1 1.2 1.3

0.9

  N (litters) 8 10 9 8
MEAN 9 9 9

7.3 *

Day 4 Female ST.DEV 1.2 1.2 1.3 1.1
  N (litters) 8 10 9 8
MEAN 9.2 9.1 9.1 7.4 *
M/F ST.DEV 1.1 1.2 1.3 1
    N (litters) 8 10 9 8
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Applicant's summary and conclusion

Conclusions:
The substance does not affect fertility. It affects pup development at maternally toxic doses.
Executive summary:

In this guideline (OECD 421) study conducted with GLP certification, the test material was found to provoke no reproductive toxicity in rats. The test was conducted at three dose levels (20, 60 and 200 mg/kg bw/day). 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. No treatment related toxicity or treatment related reproductive toxicity was observed in the parental or offspring animals. The NOEL was set at the 60 mg/kg bw/day.