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Administrative data

Description of key information

Repeated dose toxicity, 42 days' dosing to rats (male and female); NOEL = 60 mg/kg/day

Repeat dose toxicity by inhalation (13 weeks, 5 days per week, 6 hours per day) in male and female rats; NOAEL = 0.0586µg/L.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
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: Test was conducted according to GLP and international guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Species:
rat
Strain:
Crj: 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
- Weight at study initiation: (P) Males: 307-370 g; Females: 197-239 g
- Housing:
For males and females except during the gestation and lactation periods: Autoclave-sterilized stainless steel hanging type mesh cages (195W × 325D × 180H mm, Tokiwa Kagaku Kikai Co., Ltd.) were used and replaced once every 12 to 15 days.
For females during the gestation and lactation periods: Autoclave-sterilized polycarbonate cages (265W × 426D × 200H mm, Tokiwa Kagaku Kikai Co., Ltd.) were used and replaced once every 6 to 7 days.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: From time of arrival 5 days quarantine, 8 days acclimation


ENVIRONMENTAL CONDITIONS-QUARANTINE ROOM
- Temperature (°C): 21.6-22.4
- Humidity (%): 52.9-54.9
- 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
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was accurately weighed for each dose level and dissolved in the vehicle (purified water) with a measuring cylinder. The dosing solutions were prepared once every 4 to 8 days and stored in a refrigerator (actual value: 4.9 – 6.9°C, permissible range: 1 - 10°C) protected from light, up to 8 days. They were prepared under yellow light in the preparation room.

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
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
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: 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.
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups: Administration was conducted for 42 days without mating.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
(1) Home cage observation
Movement of animals in cages was observed for 1 minute.
Tremor, clonic convulsion, tonic convulsion, aspiration

(2) Hand-held observation
Animals were gently held from the back and removed from the cages, and were observed.
Reactivity on removal from the cages, reactivity to handling, aggression, skin (trauma, color of skin), fur (soiled fur), eyes (exophthalmos, palpebral closure), conjunctiva (color of conjunctiva), secretion, lacrimation, salivation, piloerection, pupil size

(3) Open field observation
Animals were observed for 2 minutes after being placed in the center of the open field (in advance, the floor of the field was cleaned with a wet cloth).
Rearing, arousal, urination, defecation, posture/body position, breathing, co-ordination movement, gait, tremor, clonic convulsion, tonic convulsion, stereotypy, bizarre behavior

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical signs were observed twice a day (before and after dosing) during the dosing period, and once a day in the other periods


BODY WEIGHT: Yes
- Time schedule for examinations:
The test and recovery males were weighed on Days 1, 8, 15, 22, 29, 36, 42, and 43. The recovery males were also weighed on Days 50 and 56. The satellite females were weighed in the same manner as the test males. The test females were weighed on Days 1, 8, and 15, Days 0, 7, 14 and 20 of gestation, and Days 0 and 4 of lactation. Electronic balances (PB-3002-S: Mettler Toledo K.K., EB-3200S: Shimadzu Corporation) were used.
Body weight gains were calculated based on the values on Day 1 for all males and satellite females. For test females, body weight gains in the pre-mating, gestation, and lactation periods were calculated based on the values on Day 1, Day 0 of gestation, and Day 0 of lactation, respectively.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was measured between Days 1 to 8, 8 to 15, 22 to 29, 29 to 36, 36 to 38, 43 to 50, 50 to 52 for the test and recovery males, and between Days 1 to 8, 8 to 15, 15 to 22, 22 to 29, 29 to 36, 36 to 42, 43 to 50, 50 to 56 for the satellite females. Food consumption of the test females was measured at the same time as body weights. However, food consumption was not measured for either sex during the mating period from Days 15 to 22. After the completion of copulation, the measurement for males was started from the nearest measurement day. An electronic balance (PB-3002-S: Mettler Toledo K.K.) was used for weighing. Gross weight of each feeder was weighed, and the mean daily food consumption of each animal was calculated for each period.

HAEMATOLOGY: Yes
- Animals fasted: Yes
- How many animals: All surviving animals in the recovery groups; 5 males and 5 females from the test groups

Parameters:Methods
(1):Red blood cell count:Isovolumetrically sphered two optical cytometric laser FCM
(2):Hemoglobin concentration:Modified cyanmethemoglobin method
(3):Hematocrit:Isovolumetrically sphered two optical cytometric laser FCM
(4):Mean corpuscular volume (MCV):Calculated from (1) and (3)
(5):Mean corpuscular hemoglobin (MCH):Calculated from (1) and (2)
(6):Mean corpuscular hemoglobin concentration (MCHC):Calculated from (2) and (3)
(7):Reticulocyte count:RNA-stained laser FCM
(8):Platelet count:Isovolumetrically sphered two optical cytometric laser FCM
(9):Prothrombin time (PT):Scattered light detection method
(10):Activated partial thromboplastin time (APTT):Scattered light detection method
(11):White blood cell count:Peroxidase cytometric FCM and baso/lobularity laser FCM
(12):Differential leukocyte ratio and count:Microscope examination after Wright's staining


All surviving animals were fasted as follows: from around 16:00 of the day before the scheduled necropsy (Day 42) for test males, Day 56 for recovery males and satellite females, and Day 4 of lactation for test females. In the test groups, 5 test males were used in ascending order of animal number, and 5 test females with an earlier delivery date were used in ascending order of the animal number. As for recovery males and satellite females, all animals were used. At the scheduled necropsy, blood was collected from the posterior vena cava of the above mentioned animals under anesthesia by intraperitoneal injection of sodium thiopental (Nembutal®; Dainippon Pharmaceutical Co., Ltd.). The blood samples were used for the following items. For items 9 and 10, plasma samples obtained after centrifugation of blood (at 12,000 rpm, about 12,000 g, at 4°C for 3 minutes) were mixed with 3.2 w/v% trisodium citrate and used. The blood samples treated with EDTA-2K were used for the other parameters.
Although the non-delivered female was fasted from 16:00 of the day before the scheduled necropsy described in Section 7.10.8.2, blood collection and further measurement in this animal were not performed.
The residual samples were discarded after termination of the examinations


CLINICAL CHEMISTRY: Yes
- Animals fasted: Yes
A portion of the collected blood samples at the scheduled necropsy was left to stand at room temperature for more than 30 minutes, and the serum was separated by centrifugation (at 3,000 rpm, 1,600 g, at about 4°C for 10 minutes). The following parameters were measured with the serum samples on the days of blood collection. The remaining sera were kept frozen in a -80°C freezer (permissible range: below -60°C) and discarded by the completion of this study.

(1):AST (GOT):UV-rate method (modified JSCC method)
(2):ALT (GPT):UV-rate method (modified JSCC method)
(3):γGT:γ-Glutamil-p-nitroanilid substrate method (modified SSCC method)
(4):ALP:p-Nitrophenyl phosphate substrate method (modified JSCC method)
(5):Total bilirubin:Enzymatic method (BOD method)
(6):Urea nitrogen:Enzymatic-UV method (Urease-LEDH method)
(7):Creatinine:Enzymatic method (Creatininase-POD method)
(8):Glucose:Enzymatic method (HK-G6PDH method)
(9):Total cholesterol:Enzymatic method (CO-HDAOS method)
(10):Triglyceride:Enzymatic method (GPO-DAOS method with elimination of free glycerin)
(11):Total protein:Biuret method
(12):Albumin:BCG method
(13):A/G ratio:Calculated from (11) and (12)
(14):Calcium:OCPC method
(15):Inorganic phosphorus:Enzymatic method (PNP-XOD-POD method)
(16):Sodium (Na):Ion selective electrode method
(17):Potassium (K):Ion selective electrode method
(18):Chlorine (Cl):Ion selective electrode method

URINALYSIS: Yes
- Time schedule for collection of urine:
Fresh urine samples were collected with metabolic cages from 5 males in each group (the animals were selected in ascending order of the animal number) from around 7:00 on Day 38, and the following parameters were examined. The fresh urine samples were collected before dosing, and the containers were tightly closed when sufficient volumes were collected.
According to the result, no abnormalities were detected in any parameters; therefore, examinations of sediments, accumulated urine, and urine of recovery animals were not performed. The residual samples were discarded after the termination of the examination.
:Parameters:Methods
(1):pH:Paper test (Multistix®, Bayer Medical Ltd.)
(2):Protein:Paper test (Multistix®, Bayer Medical Ltd.)
(3):Glucose:Paper test (Multistix®, Bayer Medical Ltd.)
(4):Ketones:Paper test (Multistix®, Bayer Medical Ltd.)
(5):Bilirubin:Paper test (Multistix®, Bayer Medical Ltd.)
(6):Occult blood:Paper test (Multistix®, Bayer Medical Ltd.)
(7):Urobilinogen:Paper test (Multistix®, Bayer Medical Ltd.)


NEUROBEHAVIOURAL EXAMINATION: Yes
Detailed clinical observation
(1) Home cage observation
Movement of animals in cages was observed for 1 minute.
Tremor, clonic convulsion, tonic convulsion, aspiration
(2) Hand-held observation
Animals were gently held from the back and removed from the cages, and were observed.
Reactivity on removal from the cages, reactivity to handling, aggression, skin (trauma, color of skin), fur (soiled fur), eyes (exophthalmos, palpebral closure), conjunctiva (color of conjunctiva), secretion, lacrimation, salivation, piloerection, pupil size
(3) Open field observation
Animals were observed for 2 minutes after being placed in the center of the open field (in advance, the floor of the field was cleaned with a wet cloth).
Rearing, arousal, urination, defecation, posture/body position, breathing, co-ordination movement, gait, tremor, clonic convulsion, tonic convulsion, stereotypy, bizarre behavior

Function tests
(1) Sensor reactivity to stimuli
The following items were examined in the open field.
Approach response, touch response, auditory response, tail pinch response, aerial righting reaction
(2) Grip strength
A digital force gauge (DPS-5, Imada Co., Ltd.) was used.
Grip strength of forelimb and hindlimb

A motor activity-measuring device (SUPERMEX, Muromachi Kikai Co., Ltd.) was used. On Day 41, animals of both sexes were acclimated to polycarbonate cages after observation upon administration, and were transferred to new polycarbonate cages just before measurement. Data were tabulated every 10 minutes from the start of the measurement to 1 hour after measurement.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Pathological examinations
Organ weight
In the test group, the following organs were weighed in 5 males selected in ascending order of the animal number, and 5 females with an earlier delivery date selected in ascending order of the animal number (bilateral organs were weighed together). As for the recovery males and satellite females, the organs of all animals were weighed. The testes and epididymides were weighed for all males. Electronic balances (AW120: Shimadzu Corporation, AG-204: Mettler Toledo K.K.) were used for weighing. Body weights were measured on the day of necropsy and were used to calculate the relative organ weights (ratio to body weight). However, organ weights of the dead animal and non-delivered females were not weighed.
Brain, heart, liver, kidneys, adrenals, thymus, spleen, testes, epididymides

Necropsy
The animals subjected to the above examinations were euthanized after blood collection, by exsanguination from the abdominal aorta under anesthesia and subjected to necropsy. The non-delivered females were euthanized in the same manner, and subjected to necropsy 26 days after confirmation of copulation. The dead animal was necropsied immediately upon discovery.

HISTOPATHOLOGY: Yes
Histological examination
The following organs/tissues of all animals were removed and preserved in 10 vol% neutral phosphate-buffered formalin. The testes and epididymides except for those of dead animal were fixed in Bouin’s solution and preserved in 10 vol% neutral phosphate-buffered formalin.
Brain, pituitary, thymus, lymphnodes (mandibular and mesenteric), trachea, lungs, stomach, intestines (duodenum, jejunum, ileum, cecum, colon, rectum), thyroids and parathyroids (bilateral), heart, liver, spleen, kidneys (bilateral), adrenals (bilateral), urinary bladder, testes (bilateral), epididymides (bilateral), seminal vesicle including coagulating gland, prostate, ovaries (bilateral), uterus, vagina, bone marrow (femur, unilateral), sciatic nerve (unilateral), spinal cord, gross lesion

In the control and 300 mg/kg groups, the following organs/tissues were collected: the above-mentioned organs/tissues of 5 test males selected in ascending order of the animal number, and 5 test females with earlier delivery date selected in ascending order of the animal number, and dead animal. Gross lesions in all groups including the control were also collected. They were embedded in paraffin by standard method to prepare hematoxylin- and eosin-stained sections, and were examined by microscopy. Consequently, changes suggested to be test substance-related were noted in the stomach, duodenum, jejunum, ileum, and adrenals of both sexes, and testes and epididymides of males. Therefore, these organs were examined microscopically in 5 animals of both sexes in the 10 and 60 mg/kg groups which were selected in the above-mentioned manner, and in all recovery animals.

Other examinations:
Observation and examination items for reproductive/developmental toxicity:
Examination on reproductive function:
Estrus cycle:
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.

Mating:
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

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

Observation and examination items for offspring:
Observation of offspring:
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

Body weights:
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.

Necropsy:
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:
Statistical analyses were performed on the items listed below. They were not conducted on the clinical observations, behavioral results (detailed clinical observations, sensory reactivity to stimuli), or necropsy findings.

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
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
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 WEIGHT GAIN
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.


FOOD CONSUMPTION

Decrease in food consumption was noted in females and satellite females at 300 mg/kg on Day 7 of gestation and Day 22, respectively; however, the change was transient. The food consumption of males in the 300 mg/kg group was slightly lower than that of the control group; however, no significant differences were noted.
There were no significant differences from the control group in either sexes receiving 10 or 60 mg/kg during the dosing period or in any animals during the recovery period.

HAEMATOLOGY
Increased reticulocyte ratio and increasing tendency of white blood cell count were noted in males at 300 mg/kg at the end of the dosing period.
Increased platelet count and decreased segmented neutrophils were noted in males at 300 mg/kg at the end of the recovery period; however, since there were no differences at the end of dosing period, it was considered to be an incidental change.
In females, there were no significant differences from the control group at the end of the dosing or recovery period.

CLINICAL CHEMISTRY
Decreased total protein was noted in males and increased ALAT (GPT) activity was noted in females in the 300 mg/ kg group at the end of the dosing period. ALP activity in females at 300 mg/kg showed increasing tendency at the end of the dosing period, and showed increases at the end of the recovery period.
Moreover, increased glucose and triglyceride were noted in males at 10 mg/kg, and decreased ASAT (GOT) activity was noted in females at 60 mg/kg at the end of the dosing period. However, since the changes were not dose-dependent, they were considered to be incidental.


URINALYSIS (male)
Negative results of protein and ketones increased at 300 mg/kg. However, since they were not positive (toxic) changes, they were judged to be incidental.

NEUROBEHAVIOUR
No test substance-related changes were noted in either sex during the dosing period.


ORGAN WEIGHTS
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


HISTOPATHOLOGY: NON-NEOPLASTIC
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. They are shown in the table below.

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.

OTHER FINDINGS
Necropsy findings
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.
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
neuropathology
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
histopathology: non-neoplastic
Critical effects observed:
not specified
Conclusions:
The no observed effect level of 1,3-Cyclohexanedimethanamine for repeated dose toxicity was considered to be 60 mg/kg/day in both sexes, based on the following major toxicological changes noted at 300 mg/kg: death of male, suppressed body weight gain in males, salivation in both sexes, effects on the forestomach in both sexes, and effects on the testes and epididymides. These changes disappeared or tended to recover during the 2-week recovery period.
Executive summary:

To assess the repeated dose oral toxicity and reproductive/developmental toxicity,1,3 -Cyclohexanedimethanamine was dosed to both sexes of SD rats [Crl:CD(SD)] at 10, 60, and 300 mg/kg. In addition, the reversibility of the induced toxic changes was evaluated. The male rats were dosed from 14 days before mating to the day before necropsy (including the mating period; 42 days in total), and the female rats were dosed from 14 days before mating to Day 4 of lactation (including the mating period, gestation period, and delivery). Each test group consisted of 12 males (recovery animals included) and 12 females (5 females each were added for recovery test in the control and 300 mg/kg groups). The control group received the vehicle (purified water) only.

 

As major toxicological changes, the following changes were noted at 300 mg/kg: death of a male, suppressed body weight gain in males, salivation in both sexes. In the histological examination, focal squamous epithelial hyperplasia and keratosis, focal inflammatory cell infiltration, and ulcer were found in the forestomach in both sexes, and atrophy of the seminiferous tubule was found in males. Besides, the following changes were also noted at 300 mg/kg: increased reticulocyte and white blood cell counts and decreased total protein in males, increased ALAT (GPT) and ALP activities in females, increased absolute and relative adrenal weights in males, and increased relative kidney and adrenal weights in females. These changes disappeared in the 2-week recovery period or tended to recover. There were no test substance effects including neurologic symptoms in the detailed clinical observation, functional test, or motor activity observation.

In conclusion, the no observed effect level of 1,3-Cyclohexanedimethanaminefor repeated dose toxicity was considered to be 60 mg/kg/day in both sexes.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2015 - April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at ambient temperature



Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 67 to 74 days
- Weight at study initiation: Males: 347 to 461g; Females 229 to 296g
- Fasting period before study: No
- Housing: Polycarbonate cages with steel mesh lid.
- Diet: Ad libitum, removed overnight before blood sampling and during exposure
- Water: Ad libitum (except during exposure).

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 40-70%
- Photoperiod: 12 hours light / 12 hours dark

IN-LIFE DATES: From: 07 January 2016 To: 27 April 2016 (Main group) or 23 May 2016 (Recovery group)
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
> 0.6 - < 2.9 µm
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow through snout only chamber, aluminium alloy construction comprising a base unit, three animal exposure sections, a top section and a pre chamber.
- Method of holding animals in test chamber: Polycarbonate snout-only restraint tube.
- Source and rate of air: In-house compressed air system, breathing quality. 5 - 10L/minute.
- System of generating aerosols: Stainless steel concentric jet atomizer (manufactured in-house). Test substance was delivered to teh generator via a polyethylene feed line from a syringe driven by a syringe pump at constant rate.
- Air flow rate: Extract airflow = 80L/minute per system.
- Method of particle size determination: Cascade impaction (Marple 290 series impactor in 298 configuration).
- Treatment of exhaust air: Filtered locally

TEST ATMOSPHERE
- Brief description of analytical method used: Test samples collected in a solvent trap (solvent = water) then stabilised using a 10% ammonia solution. Samples were further diluted as required using 1% ammonia solution prior to analysis by LC-MS/MS.
- Samples taken from breathing zone: Yes (sample taken from an unused animal exposure port).

VEHICLE (if applicable)
Test item was formulated in purified water. Groups 2 and 3 used 0.1% (v/v) 1,3-BAC in water, Group 4 used 1% (v/v) 1,3-BAC in water and Group 5 used 3% (v/v) 1,3-BAC in water.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were analysed by LC-MS/MS
Instrumental conditions:
Analytical column: Poroshell C18 EC, 2.6µm, 4.6 x 100 mm, 2.7µm
Column Temperature: 50°C
Mobile phase A: Perfluorooctanoic acid (0.41 g) in 1000 mL Methanol / water 10/90 v/v
Mobile phase B: Perfluorooctanoic acid (0.41 g) in 1000 mL Methanol / water 90/10 v/v
Linear gradient:
Time (min) %A %B
0.0 30 70
5.0 0 100
8.0 0 100
8.1 30 70
10.0 30 70

Flow rate: 0.5 mL/min
Monitored ions: 143.2 - 126.2
Ionisation: Turboionspray – positive ion mode
Source temperature: +350°C
Injection volume: 10 μL
Retention time: 4.3 minute

The LC-MS/MS system was calibrated using external standards. Peak area data acquired by the data capture software using a 2nd order fit with 1/x weighting was subjected to least squares regression analysis.

Duration of treatment / exposure:
6 hours per day, 5 days per week for 13 weeks
Frequency of treatment:
Daily, 5 days per week
Dose / conc.:
0.02 other: µg/L
Remarks:
Target exposure level
Dose / conc.:
0.06 other: µg/L
Remarks:
Target exposure level
Dose / conc.:
0.6 other: µg/L
Remarks:
Target exposure level
Dose / conc.:
2 other: µg/L
Remarks:
Target exposure level
No. of animals per sex per dose:
10; an additional 10 animals per sex per dose were used for the control and 2.0µg/L levels for assessment of recovery.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A 2-week range finding study (Huntingdon Life Sciences study number PXW0018) was conducted at achieved concenrations 0.0183, 0.0600 and 0.253 mg/L for 6 hours per day, 5 days per week. In this preliminary study treatment-related changes were observed in the nasal turbinates and larynx; it was not possible to establish a No Adverse Effect Level and it was recommended tha the high dose level of the subsequent 90-day study should be set at least 10-fold lower than the low dose level of the preliminary study.
On this basis the high level of the 90-day study was set at 2.0µg/L; the low dose level was set at 0.02 µg/L (100-fold lower than the high dose level) and the intermediate dose levels (0.6 and 0.06µg/L) were set to investigate any dose response which might be seen.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Inspected daily for signs of ill health or reaction to treatment. Animals were inspected for signs related to dosing before exposure, during exposure (note that observation was restricted due to tube restraint), when the animal was returned to its home cage, and as late as possible in the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed weekly physical examination was performed on each animal to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded twice weekly during the week before treatment commenced (Week -1), on the day that treatment commenced (Day 1), twice weekly from Week 1 to Week 4 and then weekly from Week 5 onwards and before necropsy.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: All animals examined pre-treatment; all animals of groups 1 and 5 (control and high dose level) assessed in week 13, and all recovery animals assessed in recovery week 4.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: All animals assessed in week 13; all recovery animals assessed in recovery week 4.
- Anaesthetic used for blood collection: Yes - Isoflurane
- Animals fasted: Yes
- Parameters checked in table [No. 1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: All animals examined in week 13; all recovery animals examined in recovery week 4
- Animals fasted: Yes
- Parameters checked in table [No.2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; refer to table no. 3
HISTOPATHOLOGY: Yes; refer to table 3
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Signs associated with the dosing procedure included wet fur and red stained fur and were evident on occasion for some animals from all groups, on return to their home cage. These signs were considered to be associated with the method of restraint and exposure used and not directly related to exposure to the test item.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Animal no. 36, a male exposed to 2.56 μg/L, was killed for welfare reasons on Day 53, due to clinical signs of decreased activity, piloerection, whole body pallor and general poor clinical condition. Macroscopic findings comprised enlargement of the liver and spleen, dark areas on the lungs, a small thymus and reduced skeletal muscle on the hind limbs with the animal thin.

The principal microscopic finding was a malignant lymphoma which correlated directly or indirectly with all the macroscopic findings and was the cause of death in this individual. This was considered to be incidental and unrelated to exposure to 1,3-bis(aminomethyl)cyclohexane.

Ulceration of the respiratory epithelium of the ventral larynx was seen in this animal and considered most likely to be due to treatment considering that the larynx of several other males at this exposure level were also affected.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
Differences from controls were inconsistent between the sexes and between exposure levels.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Differences from controls were inconsistent between the sexes and between exposure levels.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Differences from controls were inconsistent between the sexes and between exposure levels.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination performed after 13 weeks of treatment revealed no test item related lesions.
The macroscopic examination performed after 4 weeks of recovery revealed no test item related lesions.

The incidence and distribution of all findings were consistent with the background of macroscopic changes commonly seen in CD rats at this laboratory.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Animals killed after 13 weeks of treatment:

Changes related to exposure to 1,3-bis(aminomethyl)cyclohexane were seen in the larynx.
Findings were seen in the larynx of both sexes exposed to 0.710 or 2.56 μg/L.
In males these comprised of erosion or ulceration, inflammation and squamous metaplasia of the respiratory epithelium of the ventral larynx in the region of the epiglottis. In a few affected animals findings extended onto the lateral wall of the larynx and the arytenoids.
In females findings were confined to minimal squamous metaplasia of the respiratory epithelium of the ventral larynx in the region of the epiglottis.


Animals killed after 4 weeks of recovery:

Changes related to previous exposure to 1,3-bis(aminomethyl)cyclohexane were seen in the larynx.
Squamous cell metaplasia of the respiratory epithelium was seen two males previously exposed to 1,3-bis(aminomethyl) cyclohexane indicating partial recovery. There were no findings seen in the larynx of previously treated females indicating complete recovery.

Changes related to exposure to 1,3-bis(aminomethyl)cyclohexane which were considered to have an uncertain relationship to treatment were seen in the larynx.
Necrosis of the ventral cartilage was seen in one male and one female previously exposed to 1,3-bis(aminomethyl)cyclohexane . Although seen as a treatment related finding in a previous study (Envigo Study No. PXW0018) at higher doses of the test article it was considered to have an uncertain relationship to treatment in the current study due to its absence in any terminal animals.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
0.059 other: µg/L (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
0.71 other: µg/L (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
not specified
Conclusions:
Four groups of rats were exposed to the test article, 1, 3-bis(aminomethyl)cyclohexane, by snout-only inhalation administration, for 6 hours a day, 5 days a week, for 13-weeks at achieved aerosol concentrations of 0.0231, 0.0586, 0.710 or 2.56 μg/L. A control group of rats received air only at the same airflow as the high exposure group. Recovery from any effects was evaluated during a 4 week recovery period.
Histopathological changes were seen in the larynx of both sexes exposed to 0.710 or 2.56 μg/L, primarily affecting the respiratory epithelium of the ventral larynx in the region of the epiglottis. In males findings comprised of erosion or ulceration, inflammation and squamous metaplasia of the respiratory epithelium, whereas in females only squamous metaplasia was seen. After a 4 week recovery period the findings in the larynx had resolved apart from minimal squamous metaplasia of the respiratory epithelium in two males indicating partial recovery. The larynx of females was normal indicating complete recovery.
The No Adverse Effect Level (NOAEL) was considered to be 0.0586 μg/L.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed

Additional information

To assess the repeated dose oral toxicity and reproductive/developmental toxicity,1,3 -Cyclohexanedimethanaminewas dosed to both sexes of SD rats [Crl:CD(SD)] at 10, 60, and 300 mg/kg. In addition, the reversibility of the induced toxic changes was evaluated. The male rats were dosed from 14 days before mating to the day before necropsy (including the mating period; 42 days in total), and the female rats were dosed from 14 days before mating to Day 4 of lactation (including the mating period, gestation period, and delivery). Each test group consisted of 12 males (recovery animals included) and 12 females (5 females each were added for recovery test in the control and 300 mg/kg groups). The control group received the vehicle (purified water) only.

 

As major toxicological changes, the following changes were noted at 300 mg/kg: death of a male, suppressed body weight gain in males, salivation in both sexes. In the histological examination, focal squamous epithelial hyperplasia and keratosis, focal inflammatory cell infiltration, and ulcer were found in the forestomach in both sexes, and atrophy of the seminiferous tubule was found in males. Besides, the following changes were also noted at 300 mg/kg: increased reticulocyte and white blood cell counts and decreased total protein in males, increased ALAT (GPT) and ALP activities in females, increased absolute and relative adrenal weights in males, and increased relative kidney and adrenal weights in females. These changes disappeared in the 2-week recovery period or tended to recover. There were no test substance effects including neurologic symptoms in the detailed clinical observation, functional test, or motor activity observation.

In conclusion, the no observed effect level of 1,3-Cyclohexanedimethanaminefor repeated dose toxicity was considered to be 60 mg/kg/day in both sexes.

In accordance with ECHA Decision number CCH-D-0000003714 -74 -03 -F (Final decision on compliance check), a 90-day repeat dose inhaled toxicity study has been commissioned. The study will be conducted according to OECD test guideline 413.

This study was delayed due to difficulty in determining suitable dose levels which could be tolerated by the test animals for the duration of the dosing period; a 2 -week preliminary study was conducted at exposure levels 0.2, 0.06, and 0.02 mg/L, howevertreatment-related finding were observed at all levels and it was determined that these levels would not premit the determination of a threshold effect level during the main study.

Following extensive discussions with the toxicologists of the testing laboratory involved, it was decided that the main study would be performed using a high dose level 10 -fold lower than the low dose in the preliminary study and that a number of modification over the original study design would be employed including the addition of a 5th dose group to investigate dose-response relationship and the addition of a 4 -week recovery period to explore the reversibility of the pathology findings.

This study is expected to commence in November 2015 (animal arrival during October 2015) with the draft report available in May 2016.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: stomach

Justification for classification or non-classification

The NOEL for repeated dose toxicity (42 days' exposure) was concluded to be 60 mg/kg/day, with toxic effects observed at 300 mg/kg/day. In terms of classification for Specific Organ Toxicity according to the CLP regulation (EC regulation 1272/2008), the level at which classification as Category 2 for specific organ toxicity - repeated exposure is 100 mg/kg/day by the oral route. As damage to the stomach was seen in the repeated dose test, but only at 300 mg/kg/day, there is insufficient evidence to suggest that classification according to the CLP regulation is required.