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Toxicological information

Toxicity to reproduction

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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:
August 1, 2005 to January 23, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to GLP and international test guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,3-Bis(aminomethyl)cyclohexane
- Physical state: liquid
- Stability under test conditions: confirmed stable (IR spectrophotometry)
- Storage condition of test material: Refrigeration (actual temperature: 2.8°C to 8.4°C, permissible range: 1°C to 10°C), shielded from light, nitrogen-sealed

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: (P) 9 wks; (F1) 4 days
- Weight at study initiation: (P) Males: 307-370 g; Females: 197-239 g
- Diet: ad libitum
- Water: ad libitum, except during fresh urine collection
- Acclimation period: From time of arrival 5 days quarantine, 8 days acclimation


ENVIRONMENTAL CONDITIONS-QUARANTINE ROOM
- Temperature (°C): 21.6-22.4
- Humidity (%): 52.9-64.9
- Air changes (per hr): 6 to 20
- Photoperiod (hrs dark / hrs light): 12/12

ENVIRONMENTAL CONDITIONS-TEST ROOM
- Temperature (°C): 21.8-23.7
- Humidity (%): 45.4-59.1
- Air changes (per hr): 6 to 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test material weighed and disolved in vehicle (purified water), measured by volume.

VEHICLE
- Concentration in vehicle: 2 - 60 mg/kg
- Amount of vehicle (if gavage): Dosed at 5 mL/kg.
Details on mating procedure:
- M/F ratio per cage:Male and female rats were cohabited day and night on a one-to-one basis
- Length of cohabitation: From Day 15 (initial day of mating) for the maximum of 14 days.
- Proof of pregnancy: Vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy: From the day after the initial day of mating, vaginal smears were collected every day in the morning, and examined for the presence of sperm. Copulation was confirmed by the presence of a vaginal plug or sperm in the vaginal smears.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Confirmation of stability and homogeneity:
The dosing solutions of 2 and 200 mg/mL were confirmed to be stable and homogeneous for 8 days in a refrigerator protected from light in a validation study by GC method (study No. B050280) conducted at the test facility.

Confirmation of concentration:
The concentration of the test substance in each dosing solution was measured at the first and final preparation by GC method. In the results, the actual mean concentration of each dosing solution ranged from 92.0 to 108.5% of the nominal concentration (permissible range: 90 to 110%). Since the concentrations of 2 and 12 mg/mL dosing solutions deviated from the criterion, the dosing solutions were reprepared and analyzed. The results of the reprepared solutions met the criterion.

Analytical method of dosing solutions
Each dosing solution was analyzed according to the analytical method validated in the study entitled “Validation of Analytical Method of 1,3-BAC Concentration in Preparation” (study No. B050280) conducted at the test facility.
Duration of treatment / exposure:
MALES: Administration was conducted from 14 days before mating to the day before necropsy (including the mating period; 42 days in total).

PREGNANT AND MATERNAL ANIMALS: Administration was conducted from 14 days before mating to Day 4 of lactation (including the mating period, gestation period, and delivery; the date of delivery was designated as Day 0 of lactation). In the non-delivered females, administration was conducted until the day before necropsy.

RECOVERY (satellite) FEMALES: Administration was conducted for 42 days without mating.


Frequency of treatment:
Once a day between 8:23 and 11:34
Details on study schedule:
Male and female rats were cohabited day and night on a one-to-one basis from Day 15 (initial day of mating) for the maximum of 14 days. From the day after the initial day of mating, vaginal smears were collected every day in the morning, and examined for the presence of sperm. Copulation was confirmed by the presence of a vaginal plug or sperm in the vaginal smears. The day on which evidence of copulation was found was designated as Day 0 of gestation. Based on the results, the following parameters were calculated.
(1) Days until copulation: Number of days from the start of mating to the detection of copulation
(2) Number of estrus stages without copulation
(3) Copulation index (%): (Number of animals with successful copulation/Number of animals cohabited) × 100
(4) Fertility index (%): (Number of pregnant animals/Number of animals with successful copulation) × 100

Doses / concentrations
Remarks:
Doses / Concentrations:
10, 60, 300 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
Dosing Period:
Control 7 males, 12 females;
10 mg/kg 12 males, 12 females;
60 mg/kg 12 males, 12 females;
300 mg/kg 7 males, 12 females,

Recovery Period:
Control and 300 mg/kg 5 males, 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosing conditions are recommended in the OECD guidelines.10, 60, and 300 mg/kg
In the dose-range finding study (No. B050279), the test substance was dosed orally by gavage to 3 SD rats/sex/group at 0, 30, 100, 300, and 1000 mg/kg for 14 days. As a result, all animals died or sacrificed moribund at 1000 mg/kg. In the 300 mg/kg group, mucosal edema in the forestomach was noted in both sexes as a major toxic and treatment related change. There were no test substance related changes in any animals at 30 or 100 mg/kg.
In consideration of these results and duration of the study period, the high dose for this study was set at 300 mg/kg, at which obvious toxicity was expected. The middle and low doses were set at 60 and 10 mg/kg, respectively, with a common ratio of about 5. The control group receiving the vehicle (purified water) alone was also set.


- Rationale for animal assignment (if not random):Animals were assigned to the groups by stratified-by-weight randomization method based on the body weight on the day before the first administration to make the group mean body weights even. Forty-eight males and 58 females were used in this study.

Examinations

Parental animals: Observations and examinations:
Observation during delivery and lactation:
All copulated females were allowed natural delivery. The observation of delivery was conducted twice daily (9:00 and 16:00) from Days 21 to 25 of gestation. The delivery completed by 9:00 was judged as a delivery on the corresponding day (the delivery day was regarded as Day 0 of lactation). The animals that completed delivery by 16:00 were observed for lactation the next day (the day after delivery was regarded as Day 0 of lactation). The females that did not deliver by 25 days after copulation were regarded as non-delivered females. The delivered animals (dams) were allowed to nurse new born infants until day 4 postpartum (Day 4 of lactation), and postpartum behavior such as lactation, nesting, and presence or absence of cannibalism was observed every day.

Observation after lactation :
At necropsy, the ovary and uterus of dams were removed and examined for the numbers of corpora lutea and implantations. Non-delivered females were examined in the same manner to detect implantation sites. Uterus without visible implantation sites was immersed in a 10 vol% ammonium sulfide solution to detect implantation sites. Non-delivered female with no implantation site was regarded as non-pregnant female. Based on the results, the following parameters were calculated.
(1) Gestation length: Days until completion of delivery from Day 0 of gestation
(2) Delivery index (%): (Number of pregnant animals delivered live offspring/Number of pregnant animals) × 100
(3) Implantation index (%): (Number of implantations/Number of corpora lutea) × 100
(4) Gestation index (%): (Total number of delivered offspring/Number of implantations) × 100
Oestrous cyclicity (parental animals):
From the first day of administration to the first day of mating, vaginal smears of all test females of each group were sampled in the morning and examined for estrus cycle. The mean estrus cycle and incidence of females with irregular estrus cycle (with the period of other than 4 to 6 days) were calculated.
Litter observations:
PARAMETERS EXAMINED
The number of delivered offspring (numbers of live offspring and stillborns), sex, and the presence or absence of external anomalies was examined on Day 0 of postpartum.
The body weight gain between days 0 and 4 postpartum was calculated from the litter mean weight.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for all dead offspring other than those found cannibalised.
Postmortem examinations (parental animals):
Refer to repeated dose toxicity study summary (IUCLID section 7.5.1) for full details regarding results seen in the parental animals.
Postmortem examinations (offspring):
External anomaly including that in the oral cavity was examined, and the offspring were euthanized in the same manner as the parental animals, and necropsied. Dead offspring except for those cannibalized were immersed and fixed in 10 vol% neutral phosphate-buffered formalin, and necropsied under a stereomicroscope.
Statistics:
Multiple comparison test:
Body weights, body weight gain, food consumption, hematology, blood chemistry, organ weights, behavioral test (grip strength and motor activity), number of corpora lutea, number of implantations, number of delivered offspring (numbers of live offspring and stillborns)

Kruskal-Wallis test and Dunnett-type multiple comparison test:
Urinalysis (pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen), days until copulation, number of estrus stages without copulation, mean estrus cycle, gestation length, implantation index, gestation index, live birth index, incidence of offspring with external anomaly, viability index on Day 4

Wilcoxon’s rank sum test:
Histopathological findings

Fisher’s exact probability test:
Incidence of females with irregular estrus cycle, copulation index, fertility index, delivery index, sex ratio (male/female), incidence of dams with externally anormal offspring
Offspring viability indices:
The number of delivered offspring (numbers of live offspring and stillborns), sex, and the presence or absence of external anomalies was examined on Day 0 of postpartum. Thereafter, clinical signs and mortality were observed daily. Based on the results, the following parameters were calculated.

(1) Live birth index (%): (Number of live offspring at birth/Total number of delivered offspring at birth) × 100
(2) Viability index on Day 4 (%): (Number of live offspring on Day 4/Number of live offspring at birth) × 100
All live offspring were weighed individually on days 0 and 4 postpartum. An electronic balance (PB3002-S: Mettler Toledo K.K.) was used for weighing. Moreover, the body weight gain between days 0 and 4 postpartum was calculated from the litter mean weight.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Bodyweight gain was supressed in males dosed at 300 mg/kg.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Bodyweight gain was supressed in males dosed at 300 mg/kg.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
In the 300 mg/kg group, 1 female (animal No. 50405) showed irregular estrus cycle; however, copulation was confirmed by cohabitation
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
no effects observed

Details on results (P0)

Refer to repeated dose toxicity study summary (IUCLID section 7.5.1) for full details regarding results seen in the parental animals

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One male (animal No. 00405) in the 300 mg/kg group was found dead. The animal showed irregular respiration and rale from 3 days before the death, decreases in locomotor activity, ptosis, and abnormal fur on the day before death, and died on Day 25 of dosing.
Salivation was sporadically observed in the 300 mg/kg group (11 males and 16 females) during the dosing period.
Rale and a decrease in locomotor activity were noted in 1 female at 300 mg/kg (animal No. 50409) during the gestation period, and a decrease in locomotor activity was noted continuously in this animal during the lactation period.
Loss of teeth was observed in 1 satellite female of the control group; however, it recovered 3 days later. No test substance-related abnormalities were noted in either sex at 10 or 60 mg/kg.
Salivation disappeared in the recovery males and satellite females at 300 mg/kg by termination of administration.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight gain suppression was noted in males at 300 mg/kg from Days 8 to 42. During the recovery period, it was comparable to that of the control group, indicating reversibility.
There were no significant differences from the control group in males receiving 10 or 60 mg/kg or test substance-treated females.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Increased absolute and relative adrenal weights were noted in males, and increased relative weights of kidney and adrenal were noted in females at 300mg/kg at the end of the dosing period. Increasing tendency in absolute adrenal weight and increased relative adrenal weight were noted in males at 300 mg/kg at the end of the recovery period.
Increased absolute liver weight was noted in females at 300 mg/kg at the end of the recovery period; however, since there were no changes in males at 300 mg/kg at the end of the dosing period, it was considered to be an incidental change.
There were no significant differences from the control group in either sex at 10 or 60 mg/kg.

GROSS PATHOLOGY (PARENTAL ANIMALS)
The following findings were noted at the scheduled necropsy: thickening of the forestomach wall in both sexes, ulcer of forestomach mucosa, or adhesion of the liver and forestomach in 1 female each in the 300 mg/kg group. The changes in the reproductive system were small testes and epididymides in 2 males (animal No. 00403: bilateral, No. 00406: unilateral) receiving 300 mg/kg. These changes were not noted in the animals necropsied at the end of the recovery period.
In the dead animal, dark reddish change of glandular stomach mucosa, tarry abnormal contents in the duodenum, jejunum, and ileum, and dilatation of the ileum and cecum were noted. In addition, dark reddish changes of the thymus and lung, and small spleen were noted in the dead animal.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Changes considered attributed to the test substance were noted in the stomach of both sexes and in the testes and epididymis of males at 300 mg/kg.In the stomach, focal squamous cell hyperplasia and hyperkeratosis and focal inflammatory cell infiltration were noted in the forestomach in all males and females at 300 mg/kg. The changes in 5 males and 9 females were accompanied by ulcer of the forestomach. Minimal focal squamous cell hyperplasia of the forestomach was noted in all males and females, and minimal focal inflammatory cell infiltration of the forestomach was noted in 3 males and 2 females in the 300 mg/kg group at the necropsy after the recovery period. However, their grades were less than those in the animals necropsied after the dosing period.

In the testis, atrophy of the seminiferous tubule was noted in 4 males at 300 mg/kg and in 2 males at 10 mg/kg. The change noted in 1 of the 4 males at 300 mg/kg was severe, and was accompanied by diffuse hyperplasia of interstitial cells. The atrophy of the seminiferous tubule noted at 10 mg/kg was minimal, and the change was not noted at 60 mg/kg. Accordingly, atrophy of the seminiferous tubule noted at 10 mg/kg was judged to be spontaneous. In 1 of the 5 animals receiving 300 mg/kg, atrophy of the seminiferous tubule was noted at the end of the recovery period.
In the duct of epididymis, cell debris was noted in 2 males, decreased sperm in 2 males, and atrophy in 1 male at 300 mg/kg. The changes in the epididymis were related to the pathological lesions of the testis, and were not noted at the end of the recovery period.
Besides, a variety of pathological changes were noted in each group including the control at the end of the dosing and recovery periods. However, these changes were not considered to be test substance-related, since there were no significant differences in their incidence among the groups and they develop nonspecifically in rats.

In the dead animal, the following changes were noted: focal squamous cell hyperplasia of the forestomach, focal inflammatory cell infiltration of the forestomach, hemorrhage in the glandular stomach, atrophy of the spleen, congestion and edema of the lung, atrophy of the thymus, and incidental cyst of the kidney.

The test substance had no effects on the reproductive parameters such as estrus cycle, copulation index, fertility index, gestation index, gestation length, numbers of corpora lutea or implantations, implantation index, delivery index, parturition, or maternal behavior.

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

In the observation of lactation, 1 dam receiving 300 mg/kg (animal No. 50405) showed lack of licking pups and retrieving on the day of delivery, and 4 males and 3 females out of 8 males and 9 females delivered by the dam died on the same day. In addition, the rest of delivered offspring except for 1 male died on Day 1 of lactation. There were no abnormalities after Day 1 of lactation.
There were no abnormalities in delivery or lactation in the other dams.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

In the examination of offspring, no changes attributed to the test substance were noted in the number of offspring, live birth index, sex ratio, viability index on Day 4, external features, clinical signs, body weights, or necropsy findings.

Applicant's summary and conclusion

Conclusions:
The no observed effect level of 1,3-Cyclohexanedimethanamine for reproductive/developmental toxicity was considered to be 300 mg/kg/day in parental animals and offspring.
Executive summary:

A combined repeated dose toxicity and reproductive toxicity screening study was conducted on the test substance 1,3-Bis(aminomethyl)cyclohexane (1,3-BAC). The study was performed according to OECD test guideline 422, and in compliance with GLP.

Male and female rats were dosed at levels 0 (vehicle control), 10, 60, and 300 mg/kg bw/day of 1,3-BAC as a solution in water; males were dosed for 42 days in total including the mating period, females were dosed from 14 days before mating until day 4 of lactation. The clinical and pathological effects on reproduction in both the parental generation and the offspring were recorded.

The test substance had no effects on the reproductive parameters of the parental animals, nor on the developmental parameters of the offspring. The no observed effect level of 1,3-Bis(aminomethyl)cyclohexane for reproductive / developmental toxicity was considered to be 300 mg/kg/day in parental animals and offspring.