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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 22, 2011 - August 3, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted in compliance with GLP and according to OECD Guideline 422 and EPA OPPTS 870.3650. This RSS is based on draft report and will be updated when the final study report is available.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Combined Repeated Dose and Reproductive / Developmental Toxicity Screening Test (Precursor Protocol of GL 422)
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3650 Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N-(butan-2-yl)-4-({4-[(butan-2-yl)amino]cyclohexyl}methyl)cyclohexan-1-amine
EC Number:
679-514-8
Cas Number:
154279-60-4
Molecular formula:
C21H42N2
IUPAC Name:
N-(butan-2-yl)-4-({4-[(butan-2-yl)amino]cyclohexyl}methyl)cyclohexan-1-amine
Constituent 2
Reference substance name:
4,4'-methylenebis(N-secbutylcyclohexanamine)
IUPAC Name:
4,4'-methylenebis(N-secbutylcyclohexanamine)
Details on test material:
- Name of test material (as cited in study report): MTDID 25575
- Physical state: Clear colourless liquid
- Analytical purity: 98.69 Wt%
- Batch No.: 30103 260308 031
- Expiration date of the batch: 30 March 2012
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
other: Crl:WI (Han) (outbred, SPF-Quality)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, sulzfeld, Germany
- Age at study initiation: (F0) approximately 15 weeks
- Weight at study initiation: Males: 378-432 g; Females: 233-240 g
- Fasting period before study:
- Housing: In groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm). This was also applicable for recovery animals throug hout the complete study period. During the mating the main females were caged together with main males on a one-to-one-basis in Macrolon plast ic cages (MIII type, height 18cm). Post-mating the main males were housed in their home cages with a maximum of 5 animals/cage. Main females we re individually housed in Macrolon plastic cages (MIII type, height 18cm). Pups were kept with the dam until termination in Macrolon plastic cages (M III type, height 18cm).
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: At leas 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): A temperature of 21.0 ± 3.0 °C (actual range (REES): 19.0 - 20.8 °C)
- Humidity (%): Relative humidity of 40-70% (actual range (REES): 48-86%).
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours darkness and 12 hours artificial fluorescent light per day.

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
Details on mating procedure:
- M/F ratio per cage: One male and one female per cage.
- Length of cohabitation: Once mating occurred, the males and females were separated.
- Proof of pregnancy: Mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated as day 0 of pregnancy.
- After successful mating each pregnant female was caged individualle in Macrolon plastic cages (MIII type, height 18 cm).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatments phase, according to a validated method (NOTOX Project 495946). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
Duration of treatment / exposure:
Main males and Recovery males were exposed for 29 days, including at least 2 weeks of exposure prior to mating and during the mating period
for Main males. The Main females were exposed for 42-45 days prior to mating, during mating, during the post coitum and lactation periods, and
up to the day prior to scheduled necropsy. Females 69, (Group 1) 81, 85 (Group 3) and 92 (Group 4) were not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
nominal conc.
0, 1, 3 and 10 mg/kg bw/day
No. of animals per sex per dose:
Main males and main females: 10 animals/sex/dose. Recovery (0 and 10 mg/kg bw/day): 10 males/dose.
Control animals:
yes
Details on study design:
- Dose selection rationale: Dese levels were based on the dose range finding study (NOTOX Project 495982).
Positive control:
Not reported.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily, detailed clinical observations were made in all animals, at least immediately 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.

FOOD CONSUMPTION:
- Time schedule: Weekly, except for males and females which were housed together for mating. Food consumpition of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

Oestrous cyclicity (parental animals):
Not reoprted.
Sperm parameters (parental animals):
Not reported.
Litter observations:
STANDARDISATION OF LITTERS: No

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 and physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals of the Main males were sacrificed after at least 28 days of dose administration. The recovery males were sacrificed after the 14-day and 28-day recovery periods, respectively.
- Maternal animals: All surviving Main females which delivered were sacrificed on lactation days 5-6.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-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:
for each group, the following calculations were performed:
Mating index (%): (Number of females mated/ Number of females paired) x 100
Fertility index (%): (Number of pregnant females/ Number of females paired) x 100
Conception index (%): (Number of pregnant females/ Number of females mated) x 100
Gestation index (%): (Number of females bearing live pups/Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Percentage live males at First Litter Check: (Number of live male pups at First Litter Check/ Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check: (Number of live female pups at First Litter Check/ Number of live pups at First Litter Check) x 100
Percentage of postnatal loss Days 0-4 of lactation: (Number of dead pups on Day 4 of lactation/ Number of live pups at First Litter Check) x 100
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 generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See section "Details on results".
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See section "Details on results".
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See section "Details on results".
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See section "Details on results".
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
See section "Details on results".

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

ORGAN WEIGHTS (PARENTAL ANIMALS)

GROSS PATHOLOGY (PARENTAL ANIMALS)

HISTOPATHOLOGY (PARENTAL ANIMALS)

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 1 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Based primarily on skeletal muscle degeneration and urothelial hyperplasia of the urinary bladder.
Dose descriptor:
NOAEL
Effect level:
>= 3 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based primarily on skeletal muscle degeneration and urothelial hyperplasia of the urinary bladder.
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
>= 10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects at the highest tested dose level.

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant effects were observed. See section "Details on results (offspring)":
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
No toxicologically relevant effects were observed. See section "Details on results (offspring)":
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant effects were observed. See section "Details on results (offspring)":
Sexual maturation:
not specified
Description (incidence and severity):
Pups surviving to planned termination were killed by decapitation on Days 5-6 lactation.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant effects were observed. See section "Details on results (offspring)":
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
- One pup of the control group and three pups at 1 mg/kg were found missing or found dead during the first days of lactation. Pups missing were m ost likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-rel ated trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS (OFFSPRING)
- Incidental clinical symptoms of pups consisted of blue discolouration of the abdomen and absence of milk in the stomach. The nature and incidenc e of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological
relevance.

BODY WEIGHT (OFFSPRING)
- The apparent lower mean body weight of male and female pups at 10 mg/kg on Day 1 and 4 of the lactation phase occurred without statistical signif icance and the change was minor in nature. This was considered to be related to the slightly lower parental body weight, and to be of no toxicologi cal significance.

GROSS PATHOLOGY (OFFSPRING)
- Incidental macroscopic findings of pups that were found dead included beginning autolysis and absence of milk in the stomach. The nature and inc idence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological
relevance.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
Developmental
Generation:
F1
Effect level:
>= 10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects at the highest tested dose level.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
To evaluate possible toxicological effects on reproduction/development of MTDID 25575 (trade name Clearlink 1000) study according to OECD Guideline 422 in rats was conducted. Test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 1, 3 and 10 mg/kg/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42-45 days). The study was conducted incompliance with GLP.

Treatment with MTDID 25575 up to 10 mg/kg was well tolerated by the animals during the exposure period. Except for slightly lower body weights for males and females during the treatment/post-coitum/lactation period, there were no in-life findings indicative of toxicity.

Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

No toxicologically relevant effects on gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy) were observed.


No reproduction or developmental toxicity was observed up to the highest dose level tested (10 mg/kg). Based on test results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 1 mg/kg/day (males) or 3 mg/kg/day (females), based primarily on skeletal muscle degeneration and urothelial hyperplasia of the urinary bladder.
Reproduction NOAEL: at least 10 mg/kg/day
Developmental NOAEL: at least 10 mg/kg/day

The results of this study would not lead to the classification for reproduction or developmental toxicity according to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.