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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
Adequacy of study:
key study
Study period:
2011-06-21 - 2011-08-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (GLP)
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4-methylcyclohexane-1,3-diamine
EC Number:
237-666-9
EC Name:
4-methylcyclohexane-1,3-diamine
Cas Number:
13897-55-7
Molecular formula:
C7H16N2
IUPAC Name:
4-methylcyclohexane-1,3-diamine
Constituent 2
Chemical structure
Reference substance name:
2-methylcyclohexane-1,3-diamine
EC Number:
237-667-4
EC Name:
2-methylcyclohexane-1,3-diamine
Cas Number:
13897-56-8
Molecular formula:
C7H16N2
IUPAC Name:
2-methylcyclohexane-1,3-diamine
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Methyl-Diamino-Cyclohexan (CAS No. 13897-55-7 and 13897-56-8; test substance No.: 10/0117-1)
- Physical state: liquid; colorless to yellowish, clear
- Analytical purity: 99.2%
- Lot/batch No.: 84407247G0
- Expiration date of the lot/batch: not specified (date of production: 2010-03-01)
- Stability under test conditions: the stability under storage conditions was determined by reanalysis
- Storage condition of test material: room temperature; store under N2

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Wistar Rat; Crl:WI(Han) from Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks at arrival, 11-12 weeks after acclimation
- Weight at study initiation: means of 320-322 for males and 203-204 g for females
- Housing: individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²). During overnight matings, male and female mating partners were housed together in Makrolon type M III cages. For motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material (the present supplier is documented in the raw data).
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: drinking water (from water bottles); ad libitum
- Acclimation period: ca 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
highly deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, the vehicle was filled up to the desired volume, subsequently released with a magnetic stirrer. The test substance preparations were produced at least once a week.

VEHICLE
- Concentration in vehicle: 0, 0.25, 1.0 and 2.5/2.0 mg/100 mL, respectively in the 0, 25, 100 and 250/200 mg/kg bw dose groups
- Amount of vehicle (if gavage): the administration volume was 10 mL/kg body weight.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: the animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning, for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): pregnant animals and their litters were housed together until PND 4 (end of lactation).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The test substance was completely miscible with highly deionized water and, thus, the test substance preparation was considered to be homogenous.
- The stability of the test substance in highly deionized water for a period of 7 days at room temperature was proven before the start of the study.
- The analyses (spectropic [H-NMR], chromatographic [HPLC / GC] and titration [GC/MS] methods) were carried out in compliance with the Principles of Good Laboratory Practice as a separate study at the test facility.
Duration of treatment / exposure:
38 days for males, 52 days for females; 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
Details on study schedule:
- Age at mating of the mated animals in the study: 13-14 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
25, 100 and 250/200 mg/kg bw per day (the high dose was reduced from 250 to 200 mg/kg bw from day 7 onwards)
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
None

Examinations

Parental animals: Observations and examinations:
See IUCLID Chap. 7.5.1 Repeated dose toxicity

OTHER:
- The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.
- On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.
- The day of littering was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 0) and on PND 4. Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly, and the determined body weight data were solely used for the calculations of the dose volume.
Sperm parameters (parental animals):
Parameters examined in all male parental generations: testis, cauda epididymis, prostate, epididymides, seminal vesicles weights
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: 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 (PND1-PND4), physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS: yes
Postmortem examinations (parental animals):
See IUCLID Chap. 7.5.1 Repeated dose toxicity
After sacrifice of the female animals the uterus and ovaries were removed and the implantation sites were counted. To determine the number of implantation sites in apparently non-pregnant animals, the uteri from those females were stained in 10% ammonium sulfide solution for about 5 minutes according to the method of SALEWSKI. Then, the respective uteri were rinsed carefully with fresh tap water. The implantation sites were recorded for the calculation of the post-implantation loss.
Postmortem examinations (offspring):
SACRIFICE
All surviving pups (sacrificed on PND 4 under isoflurane anesthesia with CO2)

GROSS NECROPSY
All stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically. All pups were discarded after their evaluation.
Statistics:
- Means, medians and standard deviations of each test group were calculated for several parameters.
- Number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, mean litter weight and number of pups delivered per litter were analyzed by simultaneous comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means.
- Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index and number of litters with affected pups at necropsy were analyzed by pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions
- Proportions of affected pups per litter with necropsy observations were analyzed by pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
Reproductive indices:
Male fertility index, female mating index, female fertility index, female gestation index, live birth index and post implantation loss.
Offspring viability indices:
Viability index, sex ratio

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
1) Mortality
- Female animals: 1 female animal of test group 3 (250 / 200 mg/kg bw/d) was found dead on study day 30 (gestation day 4; GD 4). 1 further female animal of test group 3 was sacrificed moribund on study day 20 (GD 4). 1 female animal of test group 2 (100 mg/kg bw/d) was also found dead on study day 36 (GD 21).
2) Clinical signs
- During gestation: Semi-closed eyelid in both eyes after treatment was observed in all animals of test group 3 from GD 0 onwards. In addition, 1 animal of test group 3 showed hypothermia on GDs 1 and 2, poor general state (moderate) from GD 1 to 4, labored respiration from GD 0 to 4 and piloerection from GD 1 to 4. Furthermore, 1 additional animal of test group 3 showed poor general state, labored respiration, respiratory sounds and piloerection during the first week of gestation. All findings were assessed as being related to treatment.
2 sperm-positive female animal of the test group 3 and one of test group 2 did not deliver F1 pups.
- During lactation: semi-closed eyelid in both eyes after treatment was observed in all animals of test group 3 from PND 0 onwards. The finding was assessed as being related to treatment.

BODY WEIGHT AND WEIGHT GAIN
- During gestation: mean body weight was significantly lower by -13% on GD 20 in the test group 3. The same was true for body weight change values between GD 7 and 14 (-21%), GD 14 and 20 (-44%) as well as for the entire gestation period (-31%).
- During lactation: mean body weight was significantly lower on PND 0 as well as on PND 4, with a maximum of -10% on PND 0 in the test group 3. However, body weight change was not impaired. All findings were assessed as being related to treatment.
No changes were observed in female animals of test groups 1 and 2 (25 and 100 mg/kg bw/d) over the entire administration period.

FOOD CONSUMPTION
- During gestation: after changing the dose levels to 200 mg/kg bw/d food consumption was significantly decreased in female animals during the gestation period, i.e. -14% on GD 20 and lactation period, i.e. -12% on PND 4. These findings were assessed as being related to treatment.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
1) Males reproduction data
- Male mating index: with the exception of 2 male animals of test group 0 (0 mg/kg bw/d; control group) and 2 of test group 1, mating was confirmed for all F0 parental males, which were placed with females to generate F1 pups. Thus, the male mating index was 100% in test groups 2 and 3, and 80% in test groups 0 and 1.
- Male fertility index: fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. Two control males, 2 male animals of test group 1, 1 male animal of test group 2 and 2 male animals of test group 3 did not generate F1 pups. Thus, the male fertility index ranged between 80% and 90% (80% in test groups 0, 1 and 3, and 90% in test group 2). These values 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
2) Females reproduction data
- female mating index: The female mating index calculated after the mating period for F1 litter was 80% for test groups 0 and 1 and 100% for test groups 2 and 3. The mean duration until sperm was detected (GD 0) was 3.8, 2.5, 2.2 and 3.7 days in test groups 0, 1, 2 and 3, respectively.
- Female fertility index: all sperm positive rats delivered pups or had implantation sites with the exception of 2 female animals of the control group, 2 of test group 1, 1 of test group 2 and 2 of test group 3. These animals were either sperm-negative or did not become pregnant. Thus, the female fertility index varied between 80% and 100% (100% in test groups 0 and 1, and 90% in test group 2 and 3).
- Gestation index: the gestation index reached only 75% in test group 3, 89% in test group 2, 88% in test group 1 and 100% in control group.
- Live birth index: the total number of delivered pups was significantly decreased. However, the relative amount of liveborn pups was not affected by the test substance, as indicated by live birth indices of 100% (test groups 0, 1 and 2) and 98% (test group 3). A single stillborn pup was only seen in test group 3 but assessed as being incidental.

GROSS PATHOLOGY
- The seminal vesicles of 4/10 male animals of test group 2 and 4/10 male animals of test group 3 showed slight enlargement.
- The mating pairs without litters did not show relevant gross lesions except for those animals with slightly enlarged seminal vesicles (see above). Since the number of mating pairs with no litters was equally distributed between control animals and test groups, the enlarged seminal vesicles were considered not to be the reason for reduced fertility in these animals. This assumption was supported by the fact, that all other mating pairs of test group 2 and 3 had a normal outcome of offspring although the male animals showed slightly enlarged seminal vesicles.
- 1 female animal of test group 3 which died ahead of schedule, showed a moderate dilation of duodenum, jejunum, ileum, cecum and colon and a severe dilation of the stomach. A further female animal of test group 3 was sacrificed in a moribund state and showed a severe dilation of jejunum and cecum. Dilation of the gastrointestinal tract in both animals was considered to be a postmortal and/or agonal change due to bacterial gas formation and was not considered to be treatment-related.
All other gross findings noted at necropsy were regarded as incidental and spontaneous in origin and were not related to the treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
- 2 female animal of test group 3 showed a diffuse thymic atrophy. Since the animals were sacrificed in a moribund state or were found dead, the thymic atrophy was considered to be the result of a general poor condition.
- Two female animals of test group 3 showed a minimal (grade 1) follicular hypertrophy/hyperplasia of thyroid glands. Since hypertrophy/hyperplasia was only minimal and since only 2 female animals were affected this finding was considered to be incidental in origin and not treatment-related. This conclusion was supported by the fact, that there was no significant increase in thyroid gland weights, in contrast, the relative weights of thyroid glands in females of test group 3 were slightly decreased. Neither macroscopic examination nor histopathologic examination of the animals that died ahead of schedule or were sacrificed in a moribund state revealed any pathological finding that would explain the death and the poor general condition, respectively.
All other histopathologic findings noted were either single observations, or were biologically equally distributed between control animals and treated rats. All of them were considered to be incidental and spontaneous in origin.

HISTORICAL CONTROL DATA (if applicable)
Historical control data were available and were used for the evaluation of the biological significance of the observed changes.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on mortality (pregnant female) in the 100 mg/kg dose group at the end of the gestation period
Dose descriptor:
NOAEL
Remarks:
male and non-pregnant female
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
mortality
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Highest dose evaluated

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
The viability index as indicator for pup mortality between PND 0 and 4 was 99% for the control, 100% for test groups 1 and 2 and 98% for test group 3 (1 pup was cannibalized). These findings were assessed to be incidental and not related to treatment.
The mean number of delivered pups per dam was significantly decreased in test group 3 (mean value of 9.0). The finding was assessed as being incidental as the calculation based on only 6 litters and negatively influenced by one dam, which delivered only 4 pups (for comparison, 1 female animal of group 1 also had only 5 pups but the mean value of test group 1 was 12.7). In addition, the lower bound for this parameter in the historical control data is very close to 9.0, i.e. 9.3). For all test groups the rate of liveborn and stillborn pups reflect the normal range of biological variation inherent in the strain used in this study.

CLINICAL SIGNS (OFFSPRING)
The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.
The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between test groups.

BODY WEIGHT (OFFSPRING)
Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the concurrent control values.

GROSS PATHOLOGY (OFFSPRING)
Dextrocardia was observed in 1 pup of test group 1. A discolored liver (dark red) was observed in each 1 pup of test group 2 and 3. These findings were assessed as being spontaneous in nature and without biological relevance.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Highest dose evaluated

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage of the test substance to male and female Wistar rats revealed signs of systemic toxicity at a dose level of 250/200 mg/kg bw/d.

-Female animals (dams): it was not possible to assess the cause of a single premature death at a dose level of 100 mg/kg bw/d. Applying a precautionary principle a link between this observation and test substance administration was assumed.

- Male animals: the coagulating glands and seminal vesicles of male animals of test groups 2 and 3 showed minimal to mild dilation with flattening of lining epithelial cells. Findings in coagulating glands correlated well with increased absolute and relative weights, as they were weighted together with seminal vesicles, whereas histopathological findings of seminal vesicles correlated to macroscopically enlarged glands and increased absolute and relative weights. Minimal to mild dilation with flattening of lining epithelial cells in dorsolateral lobes of prostate glands was additionally seen in males of test groups 1 to 3, as well as increased size of ventral lobes of prostate glands of animals of test groups 1 to 3. Histopathological findings of the prostate glands correlated also with increased absolute and relative weights, although there was no clear dose-response relationship especially in absolute weight parameters (probably because of the reduced body weight of test group 3 males).

As the study results did not reveal any impact of the substance on reproduction, it has been concluded, that increased filling of accessory sex glands did not influence reproductive functionality and was therefore considered to be a treatment-related, but non-adverse finding.

Epididymides (test group 3), and epididymal tails (test group 2 and 3) showed slight, but significantly increased relative weights. Although there were no corresponding histopathologic findings in this organ, a treatment-related effect was assumed, since the mean relative weights of epididymal tails were beyond historical control data.

Because the weight change of epididymides and epididymal tails did not have any impact on reproduction, it has been concluded, that increased relative weights of epididymides and epididymal tails did not influence reproductive functionality and was therefore considered to be non-adverse.

Applicant's summary and conclusion