(E)-1,2-difluoroethylene

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Remarks:

A 13 Week Inhalation Toxicity with a 4-Week Recovery Period

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation: 18th June 2020, Experimental start date: 01 July 2020, Experimental completion date (terminal necropsy): 27 August 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

25 June 2018

Deviations:

yes

Remarks:

Minor deviations that were not considered to have compromised the validity or integrity of the study (see principals of method section for full details)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Version / remarks:

[EPA 712–C–98–204], August 1998

Deviations:

yes

Remarks:

Minor deviations that were not considered to have compromised the validity or integrity of the study (see principals of method section for full details)

Principles of method if other than guideline:

The following deviations from the study plan occurred during the study but were not considered to have compromised the validity or integrity of the study:

1. It is a Protocol requirement to examine each study animal at least once pretest and weekly (once every 7 days) during treatment (after exposure). Animals in Groups 1-4 were examined prior to exposure on Treatment Day 30 and again after exposure. As a result, there are a total of two physical examinations performed on Treatment Day 30 for all animals in Groups 1-4, one pre-exposure and one post-exposure.

2. Blood samples for hematology, coagulation and clinical chemistry as well as urine samples for
urinalysis were collected during Week 14 instead of during Week 13 as per protocol.

3. The 1 hour post dose observations were completed twice for Group 5: the first time at 1038, exactly one hour after the end of dosing and the second time at 1313.

4. On Treatment Day 22, all animals were restrained for up to 19 minutes longer than the protocol specified time.

5. On Treatment Day 18, animals in Groups 1-3 animals were observed 20, 16 and 18 minutes earlier than the protocol specified time, respectively. On Treatment Day 22, Groups 1 and 4 animals were observed 17 minutes later and 20 minutes earlier, respectively. On Treatment Day 31, animals in Group 3 were observed 19 minutes earlier than the protocol specified time. On Treatment Day 42, animals in Groups 3 and 4 were observed 18 and 21 minutes earlier than the protocol specified time, respectively. On Treatment Day 44, animals in Group 1 were observed 16 minutes later than the protocol specified time. On Treatment Day 60, animals in Group 4 were observed 17 minutes earlier than the protocol specified time. On Treatment Day 65, animal Group 4 were observed were observed 20 minutes earlier than the protocol specified time. On Treatment Day 73, animals in Group 3 were observed 26 minutes earlier than the protocol specified time.

6. On Treatment Day 43, the PM viability check was not performed in error. On Recovery Day 19,
the AM viability check was not performed in error.

7. On Treatment Day 44, Animal No. 3067 was placed on dose holiday due to a kinked tail.

8. On Recovery Day 13-15, the Pristima computer system was not available and data had to be collected offline. The incorrect offline form (Daily Room Condition Record) was used to record the viability checks during this period, in error. "Animals were “within normal limits” was manually written for the AM viability checks on these days; however, the same was not documented for the PM viability checks on the same days and therefore the protocol requirement for twice daily viability checks cannot be confirmed.

9. On Randomization Days 10 and 13, animals in Group 5 were restrained for 2 hours and 3 hours, respectively, along with all other animals. These Group 5 animals did not require habituation to the restraint tubes because they were the animals scheduled to receive the positive control single oral gavage dose.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(E)-1,2-difluoroethylene

EC Number:

216-629-0

EC Name:

(E)-1,2-difluoroethylene

Cas Number:

1630-78-0

Molecular formula:

C2H2F2

IUPAC Name:

(E)-1,2-difluoroethene

Test material form:

gas

Specific details on test material used for the study:

Test item: (E)-1,2-difluoroethylene (TKN1)
Description: Liquified gas
Storage conditions: Ambient conditions in solvent shed
Purtiy: ca 99.99% (GC area %)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Animal model: Albino Rats (Outbred) VAF/Plus CD (Sprague-Dawley derived) [Crl:CD(SD) IGS]

Details on species / strain selection:

The rat is an animal model commonly utilized in toxicity studies and will be the rodent species used in the toxicity testing program for this test item. In addition, an historical database of this species is available for comparative evaluation and prior studies have been conducted in rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

150 animals (75 males, 75 females) were placed on test. Animals approximately 8 weeks old at start of exposures. Weight range of the main study animals at start of treatment was 215 g to 341 g for males, and 150 g to 245 g for females.


Environmental Control:

Light/dark cycle: A twelve hour light/dark cycle was provided and controlled via an automatic timer. Note: Light cycles were interrupted for FOB/MA evaluations and fasting for necropsy, as required
.
Temperature and relative humidity: Monitored and maintained within the range of 20 to 26°C and 30 to 70%. There were no deviations from these ranges.

Animal Housing and Environmental Enrichment:

Cages: Polycarbonate cages with a stainless steel mesh lid.

Number of animals per cage: Nominally 2 or 3 of the same sex per cage. The number of animals in a cage may have been reduced due to mortality or isolation.

Bedding: Teklad 7070C Certified Diamond Dry Cellulose Bedding Envigo, Madison, Wisconsin.
Provided to each cage throughout the study and changed at appropriate intervals each week.
Analytical results of the bedding, provided by the manufacturer, are maintained on file at the Testing Facility. There were no known contaminants in the bedding that were expected to interfere with the objectives of this study.

Environmental enrichment: Provided to each cage throughout the study and replaced when necessary.

Feed:

Diet: Teklad Global 16% Protein Rodent Diet (Certified), 2016C Envigo, Madison, Wisconsin.

Availability: Without restriction (removed overnight before blood sampling for clinical pathology). Fresh feed was presented weekly.

Analysis: Analysis of each feed lot used during this study was performed by the manufacturer. Results were provided to the Testing Facility and are maintained on file at the Testing Facility. There were no known contaminants in the feed that were expected to interfere with the results of this study.

Water:

Supply: Potable water from the public supply via an automated watering system.

Availability: Without restriction.

Analysis: There were no known contaminants in the water which were expected to interfere with the results of this study.

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

Restraint was required for nose-only exposure for Groups 1-4 only. Animals were restrained (individual plastic exposure tubes) during the exposure procedures (up to 360 minutes). Animals were also restrained in tubes for up to 30 minutes each before and after exposure, during which time each group was loaded into and out of the tubes. The total time of restraint was expected to be up to 420 minutes, including the exposure time. The animals were habituated to the method of restraint with the duration incrementally increased over 3 days (up to 2/3 of the maximum anticipated exposure duration per day) preceding their initial test item exposure.


Administration of Test Item, Control Item and Positive Control Item:

Route (Groups 1 – 4): Inhalation - nose only exposure (see further details below)

Controls (Group 1): Air only.

Training for dosing: The animals on study were acclimated to the method of restraint, over a 3 day period immediately preceding the first test item exposure.

Duration of daily exposure: 360 minutes (6 h) per day, 5 days per week for 13 weeks followed by a 4-week exposure-free period.

Route (Group 5 and 6): Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.

Treated at: Constant doses in mg/kg.

Volume dose: 5 mL/kg body weight.

Individual dose volume: Calculated from the most recently recorded scheduled body weight.

Frequency and duration: Once, at approximately 24 (+1) hours prior to euthanasia

Formulation: A record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.


GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Components of the system:
- Exposure system:
-Directed flow nose-only exposure system
-Modular aluminium alloy construction comprising a base unit, 3 animal exposure levels (16 ports per level) and a top section
-Separate systems for control and treated exposures
-Unused ports sealed when not in use for sampling or animal exposure

-Animal Restraint:
Molded thermoplastic nose-only restraint tube

-Aerosol Generation:
-Sponsor cylinder
-Groups 2 - 4: Test item from cylinder directed into T-pieces where it was mixed with dilution airflow and then directed into chamber inlets
-Test item feed rate controlled by in-line flowmeters (real time measurement of atmosphere concentrations allowed for flow rate changes as needed to better target exposure levels)

-Inlet Airflow:
-Dry air from in-house compressed air system-breathing quality
-Dilution flow: 20.0 L/minute

-Extract Airflow:
-Drawn by in-house vacuum system
-Drawn from base of exposure system
-Filtered locally through a charcoal bed before discharge
-Extract flow: 20.0 L/minute

-Airflow monitoring:
-variable area flowmeters - calibrated before study, weekly and as necessary during study
-in-line flowmeters displayed continuously and monitored at regular intervals during each exposure

-System containment: Systems housed adjacent to HEPA filtered ventilated trolleys.

ADMINISTRATION:
Doses achieved with variable control and test item vapor concentrations and fixed exposure duration period.

Daily Procedure:
- Test item cylinder with gas vapor regulator connected to the exposure systems
- Rats restrained and loaded onto the system
- Inlet air supply and gas vapor regulator opened
- Animals exposed for 6 hours
- Exposure system operation parameters displayed continuously and monitored at regular intervals
- Chamber temperature, humidity and airflow rate documented approximately every 30 minutes during exposure
- Generation terminated, inlet air supply and vapor regulator switched off
- Animals remained on system for a minimum 1-minute chamber clear out period
- Animals returned to home cage at end of clear out period
- Exhaust airflow switched off
- Animal placement on exposure system altered weekly throughout duration of study

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Atmosphere Analysis:
Atmosphere concentration determined as follows:

-Atmosphere analysis: IR spectrophotometer
-Instrument conditions: Wilks Miran 1A-CVF ambient air analyzer
wavelength: 3.22 µm (Groups 1 to 4)
pathlength: 9.00 dial setting (Groups 1 to 4)
-Sample frequency: minimum of 3 samples/groups/exposure
-Sample location:
-Representative animal exposure position
-Alternated levels weekly throughout duration of study

Chamber Temperature and Relative Humidity:
Monitored using Extech/Springfield humidity and thermometer gauges as follows
-Frequency: continuously displayed, monitored regularly throughout exposure; documented at approximately 30 minute intervals
-Sample location: gauges located in restraint tubes on the exposure chambers
-Sample position remained constant throughout duration of study

Calculation of Concentration (IR):
The IR spectrophotometer was calibrated using test substance as supplied. Absorbance measurements acquired by the IR spectrophotometer were used to calculate analyzed concentration (ppm) using a 3rd order fit subjected to least squares regression analysis.

Duration of treatment / exposure:

6 hours/day, 5 days/week, for 13 consecutive weeks.

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/L air

Remarks:

(Air control)

Doses / concentrations 2

Dose / conc.:

3 000 mg/L air (nominal)

Remarks:

Mean Achieved: 3066 ppm

Doses / concentrations 3

Dose / conc.:

10 000 mg/L air (nominal)

Remarks:

Mean achieved: 10439 ppm

Doses / concentrations 4

Dose / conc.:

15 000 mg/L air (nominal)

Remarks:

Mean achieved: 14972 ppm

No. of animals per sex per dose:

20/sex/group in Groups 1 and 4
10/sex/group in Groups 2 and 3

For table detailing animal numbers and dose levels please refer to "Any other information on materials and methods section".

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Doses were selected based on the results of a 28-day (4 week) repeat dose study (Study NT27RG).
On the basis of these results including the irrelevance to humans for the degeneration of the neuronal cells of the vomeronasal organ, a high exposure level of 15,000 ppm was selected for this study with lower exposure levels of 3,000 and 10,000 ppm selected to provide a range of potential effects.


- Rationale for animal assignment (if not random): Animals were randomly allocated on arrival.
- Fasting period before blood sampling for clinical biochemistry: Animals were fasted overnight prior to each blood collection interval.

- Post-exposure recovery period: 4 weeks.

Positive control:

No positive control associated with the repeat dose toxicity study.

Examinations

Observations and examinations performed and frequency:

Daily Observations:
Animals were observed in their cages twice daily for mortality and general condition.

Dosing Observations:
On exposure days for Groups 1-4 and the day of dose administration for Group 5, all animals were observed for signs of toxic effects once prior to and at least once at approximately 1 hour after test item administration.

Animals in Group 1-4 were observed once as a group during each of the exposures.

Signs:

Animals in Groups 1-4 were removed from their cages and examined once pretest and weekly (once every 7 days) during the treatment (after exposure) and recovery periods.

Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as evaluations of respiration.

Body Weight:
Non-fasted body weights were recorded for animals in Groups 1-4 twice pretest and weekly during the treatment and recovery periods. Terminal, fasted body weights were obtained just prior to necropsy.


Food Consumption:
Food consumption was measured (weighed) weekly, beginning one week prior to treatment.

Ophthalmoscopy:
All animals were examined pretest and animals in Groups 1-4 were examined in Week 13.

Lids, lacrimal apparatus and conjunctiva were examined visually. The cornea, anterior chamber, lens, iris, vitreous humor, retina and optic disc were examined by indirect ophthalmoscopy.

The pupils of each animal were dilated prior to examination using tropicamide ophthalmic solution.

Neurobehavioral Studies:
Neurobehavioral evaluations were performed for 10 animals/sex/group from Groups 1-4 during Week 12, post-exposure. Testing at each interval was staggered over four sessions with approximately equal numbers of animals per sex per dose group in each session. Temperature, humidity, noise level and illumination of each room were measured and recorded to ensure that variations in environmental conditions were minimal during all evaluations.

Functional Observational Battery:

A functional observational battery was performed on all animals. With the exception of pretest, evaluations were performed “blind”, i.e., the observer did not know the identity of the animal’s dose group. Time of testing was counter-balanced across treatment groups.

Evaluations were performed according to the Testing Facility’s Standard Operating Procedures that include defined scales as listed below. Behaviors not adequately described by the defined scales were further characterized by descriptive comments. Testing proceeded from the least to the most interactive with the subject.

Home cage evaluations:

-Posture (Post)
-Palpebral Closure (Palp Close)
-Vocalizations (Vocal)
-Motor Movements (Motor)


Handling evaluations:

-Ease of Removal (Rem)
-Reactivity to Handling (Hand)
-Chromodacryorrhea (Chromo)
-Lacrimation (Lac)
-Salivation (Sal)
-Coat


Open field evaluations:
-Gait and Posture (Gait)
-Locomotion (Loco)
-Arousal (Arous)
-Piloerection (Pilo)
-Exophthalmia (Exo)
-Urine
-Fecal Pellets (Feces)

Motor movements:
-Fasciculations (Fasc)
-Tremors (Trem)
-Convulsions (Conv)


Reflex assessments
-Visual Approach (Visual Appr)
-Audition Assessment (Aud)
-Pinna Reflex (Pinna)
-Proprioception (Proprio)
-Pain Perception (Pain)
-Pupil Response (Pupil Resp)
-Air Righting (Air Right)

Other
-Grip Strength
-Landing Foot Splay:
-Body Temperature (Body Temp)

Motor Activity:
The locomotor activity of all animals was measured during a 60 minute session composed of 12 5-minute intervals using an automated motor monitor system. The total number of horizontal and vertical movements that occurred during each of the 5 minute intervals was recorded.

Clinical Pathology:

Blood obtained via the vena cava under isoflurane anesthesia was used to analyze hematology, coagulation and clinical chemistry parameters for 10 animals/sex/group in Groups 1-4 in Week 14 and for 10 animals/sex/group in Groups 1 and 4 for hematology and clinical chemistry only at the end of recovery. This was a terminal procedure and animals were not allowed to recover from anesthesia after blood collection. Animals were fasted overnight prior to each blood collection interval.

Urine obtained via a 12 to 16-hour overnight collection period was analyzed for 10 animals/sex/group in Groups 1-4 Week 14. Animals were fasted but not water-deprived during the collection period.

Hematology:

Blood samples (approximately 0.25 mL) were collected into tubes containing K2EDTA anticoagulant and analyzed for the following using a Siemens ADVIA 120 Hematology Analyzer:
• Hemoglobin (HGB)
• Hematocrit (HCT)
• Red blood cell count (RBC)
• Platelet count (PLT)
• Mean corpuscular volume (MCV)
• Mean corpuscular hemoglobin (MCH)
• Mean corpuscular hemoglobin concentration (MCHC)
• Red cell distribution width (RDW)
• Total white blood cell count (WBC)
• Reticulocyte count (RETIC)
• Differential white blood cell count
• Neutrophils (ANEU)
• Lymphocytes (ALYM)
• Eosinophils (AEOS)
• Basophils (ABASO)
• Monocytes (AMONO)
• Large unstained cells (ALUC)

1=Manual differential white blood cell counts were performed for verification and absolute values were calculated if necessary.

A peripheral blood smear was prepared for each animal at each blood collection interval and was available for confirmation of automated results and/or other evaluations deemed necessary by the Clinical Pathologist.

Coagulation:

Blood samples (approximately 1.0 mL) were collected into tubes containing sodium citrate anticoagulant and analyzed for the following using a Diagnostica Stago Products STA Compact MAX mechanical clot detection system:
• Prothrombin time (PT)
• Activated partial thromboplastin time (APTT)

Clinical chemistry:

Blood samples (approximately 1.0 mL) were collected into tubes with no anticoagulant, allowed to clot, centrifuged to obtain serum and analyzed for the following using a Siemens ADVIA 1800 Chemistry Analyzer:
• Aspartate aminotransferase (AST)
• Alanine aminotransferase (ALT)
• Alkaline phosphatase (ALKP)
• Blood urea nitrogen (BUN)
• Creatinine (CREAT)
• Glucose (GLU)
• Cholesterol (CHOL)
• Triglycerides (TRIG)
• Total protein (TP)
• Albumin (ALB)
• Total bilirubin (TBILI)
• Sodium (NA+)
• Potassium (K+)
• Chloride (Cl-)
• Calcium (Ca++)
• Inorganic phosphorus (PHOS)

Urinalysis:

Urine was collected into ice-chilled containers overnight (approximately 12 to 16 hours) from animals housed in metabolism cages.

Urine samples were analyzed for the following using Multistix reagent strips, interpreted using a Siemens Clinitek Advantus:

• pH
• Protein (PRO)
• Glucose (GLU)
• Ketones (KET)
• Bilirubin (BIL)
• Occult blood (BLD)

The samples were also analyzed for the following using manual methods:
• Appearance (APP)
• Specific gravity (Sp.G.) (Clinical Refractometer, Atago Uricon-N)
• Volume (VOL)

Sacrifice and pathology:

Necropsy:

Macroscopic Examinations:

Complete macroscopic examinations were performed on all main study animals. The necropsy included an external examination as well as a detailed internal examination.

Scheduled Necropsy:

Necropsy was performed on 10 animals/sex/group in Groups 1-4 after animals had been treated for 13 weeks and on 10 animals/sex/group in Groups 1 and 4 four weeks after termination of treatment. Animals were fasted overnight prior to necropsy. Necropsy schedules were established to ensure that approximately equal numbers of males and females from each group were examined at similar times of the day throughout the necropsy periods.

Method of Euthanasia:

Exsanguination following isoflurane inhalation

Organ Weights

Organs were weighed for all animals in Groups 1-4 at the scheduled necropsy intervals. Prior to weighing, the organs were carefully dissected and properly trimmed to remove adipose and other contiguous tissues in a uniform manner. Organs were weighed as soon as possible after dissection in order to avoid drying. Paired organs were weighed together.

Tissues Preserved:

Tissues were obtained from all animals in Groups 1-4 and preserved. Eyes and testes were placed in Modified Davidson’s solution initially and then retained in 10% neutral buffered formalin (NBF). Lungs and nasal turbinates were infused with 10% NBF prior to their immersion into a larger volume of the same fixative. All other tissues were preserved in 10% neutral buffered formalin (NBF).

Smear preparations of the marrow from the femur were air dried and fixed in absolute methanol.

In addition, approximately 0.03 grams were collected from the kidneys (right kidney adjacent to section for histology only), liver (left lobe only) and lungs (accessory lobe only) from each animal in Groups 1-5. The tissues were immediately submerged in liquid nitrogen. All other tissues from Group 5 animals were discarded.

Flash frozen tissues were placed in pre-labeled, chilled cryo vials and placed in a pre-labeled bag on dry ice. The samples were stored at approximately -80°C within 1 hour of collection.


Samples were analyzed using a validated microarray method (ThermoFisher/Affymetrix Clariom S Pico HT Rat) using the current version of the validated microarray procedure (including any and all updated versions).

The samples from the Groups 1, 4 and 5 animals at the Terminal Necropsy were analyzed.

Histopathology:

Slides of the tissues were prepared and examined microscopically for all animals in Groups 1 and 4 necropsied at the end of the treatment period. Any abnormalities not noted during macroscopic examinations which were seen during histology processing were recorded.

Processing:

After fixation, the tissues and organs from all animals in Groups 1 and 4 at the terminal necropsy were routinely processed, embedded in paraffin, sectioned at approximately 5 microns, mounted on glass slides, stained with hematoxylin and eosin and examined by light microscopy. The bones were decalcified in Formical-2000.

After decalcification the nasal turbinates were sectioned transversely following Histology Department guides. All sections were examined, post-fixation, for the presence of macroscopically visible morphologic abnormalities.

Two levels of the larynx were obtained following Histology Department guides.

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.

The following data types were analyzed at each timepoint separately:

body weight
body weight change from interval to interval
cumulative body weight change from baseline
food consumption
hematology
coagulation
clinical chemistry
urinalysis
organ weights
motor activity counts
forelimb and hind limb grip strength measurements
landing foot splay measurements

The following comparisons were made:
Group 1 vs 2, 3 and 4

The parameters to analyze were identified as either continuous (greater than 6 distinct values), discrete (between 3 and 6 distinct values) or binary (2 distinct values).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test item-related changes for clinical observations during or following the exposures.

Mortality:

no mortality observed

Description (incidence):

No TKN1-related mortality occurred.
All study animals survived to their scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no adverse test item-related changes for body weights during the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item-related changes for food consumption during the study.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no test item-related changes for ocular observations at end of 13 weeks of exposures. Thus, the examination was not done at end of recovery.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test item-related effects on hematology test results at any exposures at Week 14 or following a 4-week recovery period.
All differences in hematology test results, statistically significant or not, between control and test item-treated animals were consistent with normal variation and considered incidental. Most or all of the following characterized these differences: small magnitude, lack of dose relationship, and/or the absence of correlative findings.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test item-related effects on clinical chemistry test results at any exposures at Week 14 or following a 4-week recovery period.
All differences in clinical chemistry test results, statistically significant or not, between control and test item-treated animals were consistent with normal variation and considered incidental. Most or all of the following characterized these differences: small magnitude, lack of dose relationship, and/or the absence of correlative findings.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test item-related effects on urinalysis test results at any exposures at Week 14.
All differences in urinalysis test results, statistically significant or not, between control and test item-treated animals were consistent with normal variation and considered incidental. Most or all of the following characterized these differences: small magnitude, lack of dose relationship, and/or the absence of correlative findings.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were a few statistically significant differences between treated animals and control animals, listed below, however the differences were not consistent with any adverse test item-related changes at end of 13 weeks of exposures. Thus, the evaluation was not done at end of recovery.

Horizontal

Males

The 10000 and 15000 ppm TKN1 treated groups had significantly increased motor activity compared to control during interval 12 (p=0.034, Shirley’s test).

Females

There were no significant differences between the TKN1 treated groups and the control.

Vertical

Males

The 15000 ppm TKN1 treated group had significantly increased motor activity compared to control during interval 12 (p=0.011, Jonckheere-Terpstra test).

Females

There were no significant differences between the TKN1 treated groups and the control.

There were no test item-related changes for Functional Observational Battery at end of 13 weeks of exposures. Thus, the evaluation was not done at end of recovery.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No TKN1-related organ weight differences occurred at the terminal or recovery necropsy.
All differences in organ weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in relative (to body or brain weight) ratios but not both; lack of a exposure relationship or correlative findings; and/or the magnitude was considered small.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test TKN1-related macroscopic findings were present at terminal or recovery necropsy.

All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for Crl:CD(SD) rats of this age; therefore, they were considered not test article related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

TKN1-related microscopic findings occurred in the nose/turbinates in animals exposed to ≥ 3,066 ppm and persisted at end of the recovery period in animals exposed to 14,972 ppm.

At the terminal necropsy, degeneration (minimal to moderate) of the neuronal cells of the vomeronasal organ was present in the nose/turbinates in animals exposed to ≥ 3,066 ppm TKN-1. The finding exhibited an exposure-related increase in incidence and severity and severity was greater in males (minimal to moderate) than females (minimal to slight). Degeneration was characterized by cytoplasmic vacuolation of neuronal cells and was not associated with necrosis or inflammation in the vomeronasal organ or with changes in the main or accessory olfactory bulbs. Following a four-week recovery period, degeneration of the neuronal cells of the vomeronasal organ persisted in females exposed to 14,972 ppm TKN-1, but was decreased in incidence and severity (minimal) compared to the end of the dosing period (minimal to slight).

The vomeronasal organ is a sensory organ involved in detecting reproductive pheromones. It is well developed in rats and mice and experimental impairment has been shown to adversely affect mating behavior and reproduction (Doving 1998). Moderate neuronal cell degeneration observed at 14,972 ppm TKN-1 could potentially impair the vomero nasal organ’s function and may be adverse in this species. The vomeronasal organ is poorly developed or absent in humans and its functionality has not been definitively established (Meredith 2001). The significance of this finding to humans is unknown.

All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for Crl:CD(SD) rats of this age therefore, they were considered not test article related.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Coagulation:
There were no test item-related effects on coagulation test results at any exposures at Week 14.
All differences in coagulation test results, statistically significant or not, between control and test item-treated animals were consistent with normal variation and considered incidental. Most or all of the following characterized these differences: small magnitude, lack of dose relationship, and/or the absence of correlative findings.

Details on results:

No TKN1 was detected in Group 1 samples. Mean achieved concentrations for Groups 2 and 3 were 2% and 4% above target, respectively. Mean achieved concentration for Group 4 was below target by 0.2%.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

14 972 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no test item-related adverse effects

2

Key result

Dose descriptor:

NOAEL

Remarks:

nasal patholgy

Effect level:

10 439 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: nose/turbinates

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

10 439 ppm

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Incidence and Severity of TKN1-Related Microscopic Findings in the Nose/Turbinates – Terminal necropsy

Sex	TKN1
	Males				Females
Achieved Exposure (ppm)	0	3066	10439	14972	0	3066	10439	14972
Nose/Turbinates
Number Examined	10	10	8	10	10	10	10	10
Degeneration, Neuronal Cell, Vomeronasal Organ
Minimal	0	5	4	2	0	2	7	4
Slight	0	0	5	5	0	0	0	2
Moderate	0	0	0	2	0	0	0	0

 

Incidence and Severity of TKN1-Related Microscopic Findings in the Nose/Turbinates – Recovery necropsy

Sex	TKN1
	Males				Females
Achieved Exposure (ppm)	0		14972		0		14972
Nose/Turbinates
Number Examined	10		9		10		10
Degeneration, Neuronal Cell, Vomeronasal Organ
Minimal	0		0		0		2

Applicant's summary and conclusion

Conclusions:

Sprague-Dawley CD rats were exposed via nose-only inhalation with 0 (Air control), 3,066, 10,439, or 14,972 ppm (E)-1,2-difluoroethylene (TKN1) once daily, 5 days a week, for 13 consecutive weeks.

This study resulted in no test item-related adverse effects on mortality, clinical observations (including FOB/MA), body weights, food consumption, ophthalmology, hematology, coagulation, clinical chemistry, urinalysis, macroscopic necropsy observations or organ weights.

At the terminal necropsy, TKN1-associated degeneration (minimal to moderate) of the neuronal cells of the vomeronasal organ was present in the nose/turbinates in animals exposed to ≥ 3,066 ppm. The finding persisted following a 4-week recovery period in animals exposed to 14,972 ppm but was decreased in incidence and severity compared to the end of the exposures period. Moderate neuronal cell degeneration in animals exposed to 14,972 ppm TKN-1 may be adverse in this species.

Thus, the No-Observed-Adverse-Effect-Level (NOAEL) for nasal pathology was determined to be 10,439 ppm. The No-Observed-Adverse-Effect-Level (NOAEL) was otherwise determined to be 14,972 ppm.

Executive summary:

Purpose

The purpose of this study was to assess the toxicity of
(E)-1,2-difluoroethylene (TKN1) when administered via nose-only inhalation to rats for 6 hours per day, for 5 days per week, for 13 weeks followed by a 4-week exposure-free period.  This study allowed for selection of exposure levels for longer duration toxicity studies.

 

Methods

Sprague-Dawley CD^Ò rats (20/sex/group in Groups 1 and 4; 10/sex/group in Groups 2 and 3) were exposed via nose-only inhalation to 0 (Air control), 3,000, 10.000 or 15,000 ppm (E)‑1,2‑difluoroethylene (TKN1) gas once daily for 6 hours/day, 5 days/week, for 13 consecutive weeks. At the end of the treatment period, 10 animals/sex/group were euthanized and necropsied.  At the end of a 4-week recovery period, 10 animals/sex/group in Groups 1 and 4 were euthanized and necropsied. Parameters evaluated during the study were: viability, clinical observations, ophthalmology, body weights, food consumption, locomotor activity and functional observational battery (Week 12), clinical pathology (Week 14 and at end of recovery period), organ weights, macroscopic observations, and microscopic pathology.

 

Mean TKN1 atmosphere concentrations are summarized below:

Group	Target Atmosphere Concentration (ppm)	Miran (IR) Mean Achieved Atmosphere Concentration (ppm)
1 (Air)	0	0
2	3000	3066
3	10000	10439
4	15000	14972

 

Results

No TKN1 was detected in Group 1 samples. Mean achieved concentrations for Groups 2 and 3 were 2% and 4% above target, respectively. Mean achieved concentration for Group 4 was below target by 0.2%.

 

This study resulted in no test item-related adverse effects on mortality, clinical
observations (including FOB/MA), body weights, food consumption, ophthalmology, hematology, coagulation, clinical chemistry, urinalysis, macroscopic necropsy observations or organ weights.

 

At the terminal necropsy, TKN1-associated degeneration (minimal to moderate) of the neuronal cells of the vomeronasal organ was present in the nose/turbinates in animals exposed to ≥ 3,066 ppm. The finding persisted following a 4-week recovery period in animals exposed to 14,972 ppm but was decreased in incidence and severity compared to the end of the exposures period. Moderate neuronal cell degeneration in animals exposed to 14,972 ppm TKN-1 may be adverse in this species.

 

Conclusions

The No-Observed-Adverse-Effect-Level (NOAEL) for nasal pathology was
determined to be 10,439 ppm. The No-Observed-Adverse-Effect-Level (NOAEL) was otherwise determined to be 14,972 ppm.