Reference type

Author

Year

Title

Bibliographic source

Testing laboratory

Report no.

Owner company

Company study no.

Report date

study report

MHW (Ministry of Health and Welfare), Japan

A combined repeated-dose/reproductive-developmental toxicity study of 1,3-Bis (aminomethyl) cyclohexane by oral administration in rats.

Japan Existing Chemical Data Base (JECDB)

Mitsubishi Safety Institute Ltd.

B041798

data published

7.8.1 Toxicity to reproduction: Toxicity to reproduction.001

subacute

no

Qualifier

Guideline

Deviations

according to

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

no

yes

yes

Identifier

Identity

CAS number

2579-20-6

- Name of test material (as cited in study report): 1,3-Bis (aminomethyl) cyclohexane or 1,3-Cyclohexanedimethanmine
- CAS No.: 2579-20-6
- Lot No.: 50303
- Purity: 99.98%
- Supplier: MITSUBISHI GAS CHEMICAL COMPANY, INC.
- Boiling point : 244°C
- Melting point/Freezing point: <-70°C
- Flash point: 113°C
- Specific gravity: 0.940-0.950 (20°C)
- Solubility: Soluble in water, alcohol, and n-hexane.
- Odor: Amine odor
- Physical state: Colorless liquid
- Storage condition of test material: Stored in a refrigerator (2.8 – 8.4°C).

rat

Crj: CD(SD)

male/female

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: 9 weeks of age
- Weight at study initiation: 307-370 g for males and 197-239 g for females
- Housing: bracket-type metallic wire-mesh cages (W 195 × D 235 × H 180 mm)
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.6 to 22.4°C
- Humidity (%): 52.9 to 64.9%
- Air changes (per hr): 6 to 20 times per hour
- Photoperiod (hrs dark / hrs light):12-hour lighting per day

oral: gavage

water

PREPARATION OF DOSING SOLUTIONS: Test substance was dissolved in olive oil for injection.
VEHICLE
- Justification for use and choice of vehicle: No data
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): No data
- Dosing volume: 5 mL/kg
- Stability (test solutions): For 8 days
- Storage condition of test solution: Stored in a refrigerator (2.8 – 8.4°C).

yes

Test suspensions at each concentration of initial and final preparations were analyzed by the GC method at Mitsubishi Safety Institute Ltd. Results showed that the concentration of the test article in each concentration was 92.0 to 108.5% of the nominal concentration and both values were within the acceptable range (concentration: percentage of the nominal concentration, 100 ± 10%)

(P) Males: 42 days including 14 days pre-mating (P)Females: Days including 14 days pre-mating, mating and gestation periods and the days until day 4 of lactation Female (no mating, satellite group): for 42 days

Daily: 7 times / week

0 (vehicle), 10, 60 and 300 mg/kg bw/day

Basis

actual ingested

Main group:12 females/dose (0, 10, 60, and 300 mg/kg bw/day), 7, 12, 12, and 7 males/dose ( 0, 10, 60, and 300 mg/kg bw/day)
Satellite group: 5 females/dose (0 and 300 mg/kg bw/day)
Recovery group: 5 males/dose and 5 females (satellite group)/dose (0 and 300 mg/kg bw/day )

yes, concurrent vehicle

- Dose selection rationale: A preliminary study was conducted to determine the doses to be employed. Three males and three female SD rats were administrated 0, 30, 100, 300, and 1000 mg/kg bw/day of the substance for 14 days. As a result, death or dying was observed in all males and females receiving 1000 mg/kg bw/day. Edema of the forestomach was observed in all dead animals receiving 300 mg/kg bw/day. No changes attributable to the test substance were observed on both sexes receiving 30 and 100 mg/kg bw/day. Therefore, the high dose was set at 300 mg/kg bw/day, and the middle and low dose were set at 60 and 10 mg/kg bw/day by using common ratio 5.

no

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Males and females: once before the start of administration, two times/day during the administration period, and once during the recovery period
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
All animals; once before the start of administration, once every week until Week 6 of the administration period.
BODY WEIGHT: Yes
- Time schedule for examinations:
Males in the main and recovery groups were weighed on Day 1, 8, 15, 22, 29, 36, 42, and 43 of administration, and males of recovery groups were weighed on Day 50 and 56. Female satellite groups were weighted same frequencies to male recovery groups. Females in the main groups were weighed on Day 1, 8 and 15 of administration and copulated females were weighed on Day 0, 7, 14 and 20 of gestation, and days 0 and 4 of lactation.
FOOD CONSUMPTION : Yes
- Food consumption (g/day/rat) for each animal determined from the difference of the of the previous day's feeding amount: Yes
Measurement of food consumption was conducted on all animals at the following frequencies:
Males in the main and recovery groups; on Day 1-8, 8-15, 15-22, 22-29, 29-36, 36-38, 43-50, and 50-52. Females in the satellite group; on Day 1-8, 8-15, 15-22, 22-29, 29-36, 36-42, 43-50, and 50-56. Females in the main group; on Day 1, 8 and 15 of administration, on Day 0, 7, 14 and 20 of gestation, and on Day 0 and 4 of lactation.
FOOD INTAKE: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: On Day 42 and 56 in males and satellite group females. On Day 4 of the lactation period in main group females.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals:5 animals/sex/group
- Parameters examined red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, reticulocyte count, platelet count, white blood cell count, differential white blood cell count, prothrombin time, activated partial thromboplastin time
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the next day of the final administration and on the final day of the recovery period
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked: ASAT (GOT), ALAT (GPT), γ-GT, ALP, total bilirubin, blood urea nitrogen, creatinine, glucose, total cholesterol, triglyceride, total protein, albumin, A/G ratio, calcium, inorganic phosphorus, sodium, potassium, chloride
URINALYSIS OF MALES: Yes
- Time schedule for collection of urine (males only): Day 38 of administration
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- How many animals: 5 animals/males/group
- Parameters checked: pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen
BLOOD HORMONE: No
NEUROBEHAVIOURAL EXAMINATION: Yes
Functional observation: Five males/dose at Week 6, and five females/dose during the lactation period. No tests conducted during the recovery period because no changes were observed during the administration period.
- Battery of functions tested:
1) Open field observation. Aerial righting reaction, arousal, urination, defecation, posture and body position, breathing, co-ordination movement, gait, tremor, clonic convulsion, tonic convulsion, stereotypy, and bizarre behavior.
2) Manipulative Test. Approach response, touch response, auditory response, tail pinch response, and aerial righting reflex
3) Measurement of Grip Strength. Following manipulative test, grip strength of forelimb and hind limb was measured by Digital force gauge MODEL-DPS-5 (IMADA CO., LTD.).
4) Measurement of Motor Activity.Motor activity was measured by a motor activity sensor for experimental animals SUPERMEX (Muromachi Kikai Co., Ltd.). The measurement was conducted for 1 hour, and measured values at 10-minute intervals and from 0 to 60 minutes was collected.

GROSS PATHOLOGY AND ORGAN WEIGHTS：Yes, brain, heart, liver, kidneys, adrenals, thymus, spleen, testes, and epididymis.
HISTOPATHOLOGY: Yes, cerebrum, puitality, thymus, lymph nodes (including mesenteric and mandibular lymph nodes), trachea, lung, stomach, intestinal tract (duodenum, jejunum, ileum, cecum, colon, rectum), thyroids, parathyroid, heart, liver, spleen, kidneys, adrenals, urinary bladder, testes, epididymis, seminal vesicles (including the coagulating gland), prostate (ventral lobe), ovaries, uterus, vagina, bone marrow (one side femur), Sciatic nerve (one side femur), spinal cord, and gross abnormalities site.

Parametric data such as grip strength, motor activity, body weight and gain, food consumption, urine volume, specific gravity, hematology, blood biochemistry, and absolute and relative organ weights were analyzed by Bartlett’s test for homogeneity of distribution. When homogeneity was recognized, one-way analysis of variance was performed. When a significant difference was observed, Dunnett’s multiple comparison test was conducted for comparison between control and treated groups. If not homogenous, analysis was performed using the Kruskal-Wallis ranking test. In consequence, if not homogenous, Dunnett’s type mean rank sum test was conducted to compare to control and individual treatment groups. Qualitative value as the pathological findings was analyzed by Wilcoxon test and Fisher’s exact test. Urinalyses data were analyzed by Kruskal-Wallis and Dunnet’s type mean rank test. Significance level was set at 0.05 compared with the control group and among the groups.

Endpoint

Effect level

Based on

Sex

Basis for effect level / Remarks

NOAEL

60 mg/kg bw/day (actual dose received)

act. ingr.

male/female

One male died in the 300 mg/kg bw/day group. At this dose, salivation was observed in both sexes, and decreased body weight gain was observed in males. The relative and absolute weights of the adrenal gland in males and relative weights of the kidneys and adrenal gland in females increased in the 300 mg/kg bw/day groups. Upon histopathological examination, inflammatory cell infiltration, focal hyperkeratosis, focal squamous cell hyperplasia, and ulceration in the forestomach in both sexes, and atrophy of seminiferous tubules of the testis in males were observed at 300 mg/kg bw/day.

yes

yes

no effects

not examined

yes

yes

no effects

no effects

yes

yes

yes

CLINICAL SIGNS AND MORTALITY: Mortality: One male animal died in the 300 mg/kg bw/day group.Clinical signs: Transient salivation was observed in all males and 16 females receiving 300 mg/kg bw/day, sporadically.
DETAILED CLINICAL OBSERVATIONS, MANIPULATIVE TEST, GRIP STRENGTH TEST AND LOCOMOTOR ACTIVITY MEASUREMENT: There were no changes related to the test substance in any group during the dosing.
BODY WEIGHT: Depression of body weight gains was observed in males receiving 300 mg/kg bw/day on Day 8 to 42. This change was recovered by withdraw. No changes in body weights were observed in both sexes receiving 10 and 30 mg/kg bw/day compared with the control groups.
FOOD CONSUMPTION: There were no changes related to the test substance in any groups during the dosing and recovery periods.
URINALYSIS: There were no changes related to the test substance in any groups at the end of the dosing period.
HAEMATOLOGY: Increases in reticulocyte count and increase tendencies in white blood cell count were observed in males receiving 300 mg/kg bw/day at end of the administration. No changes were observed in females receiving 300 mg/kg bw/day at end of the dosing and recovery periods compared with the control group.
CLINICAL CHEMISTRY: At the end of the dosing period, decreases in total protein level and increases in ALAT activity were observed in males and females receiving 300 mg/kg bw/day, respectively. ALP activity tended to increase at the end of the dosing period and significantly increased at the end of the recovery period in females at 300 mg/kg bw/day.
URINALYSES OF MALES: There were no changes related to the test substance.

ORGAN WEIGHTS: At the end of the dosing period, increases in absolute and relate adrenal weights were observed in males receiving 300 mg/kg bw/day and increases in relative kidney and adrenal weights were observed in females receiving 300 mg/kg bw/day. At the end of the recovery period, an increase in relative adrenal weight was observed in males receiving 300 mg/kg bw/day. No changes in absolute and relative organ weights were observed in both sexes receiving 10 and 60 mg/kg bw/day compared with the control group.

GROSS PATHOLOGY: At the end of the dosing period, thickening forestomach wall was observed in all males and females receiving 300 mg/kg bw/day and ulcer in the forestomach mucosa and adhesion to the liver were observed in each female receiving 300 mg/kg bw/day. For the genital system, small sized testes and epididymis were observed in two males receiving 300 mg/kg bw/day. These lesions were recovered at the end of the recovery period. Dark reddish changes in mucosa of the glandular stomach, abnormal contents (tar) in the duodenum, jejunum, and ileum, and dilatation in the cecum and ileum were observed in dead animals receiving 300 mg/kg bw/day.

HISTOPATHOLOGY: NON-NEOPLASTIC:
Stomach: Focal hyperplasia of squamous, focal hyperkeratosis, ulcer and focal inflammatory cell infiltration were observed in the forestomach of all males and females receiving 300 mg/kg bw/day. These lesions trended to recover at the end of the recovery period.
Testes: Atrophy of seminiferous tubule was observed in four males receiving 300 mg/kg bw/day and two males receiving 10 mg/kg bw/day. Diffuse hyperplasia of interstitial cell was observed in one male out of four males receiving 300 mg/kg bw/day. Atrophy of seminiferous tubule in the 10 mg/kg bw/day group was slight, and this lesion was not observed in the 60 mg/kg bw/day group. Therefore, this lesion was considered to be spontaneous. Atrophy of seminiferous tubule was observed in one male receiving 300 mg/kg bw/day at the end of the recovery period.
Epididymis: Cell debris in duct in two males, decreases in sperm of duct in two males, and atrophy in duct in one male were observed at 300 mg/kg bw/day.
In dead animals, there were focal hyperplasia of squamous and focal inflammatory cell infiltration in the forestomach, hemorrhage in the glandular stomach, atrophy in the spleen and thymus, and congestion and edema in the lungs.

Based on the decreased body weight gain and histopathological changes in the forestomach, the NOAEL for the male and female rat repeated dose toxicity of 1,3-cyclohexanedimethanamine was determined to be 60 mg/kg bw/day.

UUID

IUC5-cacf676c-1e5b-4a70-a9b9-477ceacbff28

Dossier UUID

0

Author

dra / National Institute of Health Sciences / Tokyo / Japan

Date

2017-01-06 15:59:51 JST

Remarks

Study result type

experimental result

Reliability

1 (reliable without restriction)

Rationale for reliability incl. deficiencies

OECD Test Guideline study under GLP condition