Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
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



Test animals

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)

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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

Results and discussion

Results of examinations

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

Effect levels

open allclose all
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

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

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.