Registration Dossier

Administrative data

Description of key information

No toxicity was observed in male or female rats exposed by inhalation to concentrations up to 50000 ppm (233000 mg/m3) for 6 hours a day for 2, 4, or 13 weeks.  In a 28-day inhalation toxicity study in rabbits, subacute/chronic cardiac inflammation was observed in males exposed to greater than 500 ppm (2330 mg/m3) and females exposed to greater than 1000 ppm (4660 mg/3).  

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2011 - 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study conducted under GLP. The study was selected as a key study because the information provided for the hazard is sufficient to evaluate the need for classification and labelling and/or risk assessment.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
except this study was only 4 weeks in duration, did not include ophthalmology or functional observational battery and motor activity evaluation, and was conducted in rabbits
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
study conducted in rabbits
GLP compliance:
yes
Remarks:
US laboratory - no certificate available
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Robinson Services, Inc.Mocksville, North Carolina 27028
- Age at study initiation: Approximately 4 to 5 months old
- Weight at study initiation: 2946 - 3551 kg males, 3073 - 3754 kg females
- Fasting period before study: none
- Housing:All animals were housed individually in suspended cages with grid bottoms. A stainless steel food hopper was attached to each cage
- Diet : ad libitum
- Water: ad libitum
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23C
- Humidity (%): 40 to 67%
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2010-10-05 To:2010-12-15
Route of administration:
inhalation: gas
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: MMAD = 3-6 um, concentrations no more than 0.015 mg/m3 with no substantial difference between the test substance chambers and the Air Control chamber indicating atmospheres were essentially gas only
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: chamber volume ~ 1500 liters
- Method of holding animals in test chamber: not applicable - whole body exposure
- Source and rate of air: room air, 300 liters/min
- Method of conditioning air: not reported
- System of generating particulates/aerosols: test substance a gas - no particulates or aerosols generated
- Temperature, humidity in air chamber: 17-23C, 49 - 62% humidity,
- Air flow rate: 300 liters/min
- Air change rate: 12/hour
- Method of particle size determination:TSI aerodynamic particle sizer
- Treatment of exhaust air: in house filtering system consisting of a coarse filter, a HEPA filter and activated charcoal

TEST ATMOSPHERE
- Brief description of analytical method used: MIRAN ambient air analyzer
- Samples taken from breathing zone: yes

VEHICLE: not applicable
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
6 hours a day
Frequency of treatment:
7 days/week
Remarks:
Doses / Concentrations:
0, 500, 1000/1500, 4500/5500 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
Phase 1 - 5/sex/group for 7 day exposure and 14 day exposure groups
Phase 2 - 10/sex/group for main study, 5/sex/group for recovery
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: In the rabbit developmental toxicity study, lethality was observed at 5500 ppm and higher and cardiac inflammation was observed in all group (lowest concentration tested 2500 ppm). In Phase 1 animals were exposed to 0, 500, 1500, and 5500 ppm. Due to mortality of one male and one female at 5500 ppm, the high concentration group was reduced to 4500 ppm.
- Rationale for animal assignment: none
- Rationale for selecting satellite groups: Phase 1 - groups exposed for 7 or 14 days for use as a range-finding study and also to monitor the time course for cardiac inflammation
- Post-exposure recovery period in satellite groups: yes 28 day recovery
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations : morbidity, mortality, and signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: pretest and weekly

BODY WEIGHT: Yes
- Time schedule for examinations: pretest and weekly and prior to necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: Phase 1 - up to 5 sex/group, Phase 2 - main study up to 10 sex/group, recovery up to 5/sex/group
- Parameters examined: Hemoglobin concentration, Hematocrit, Erythrocyte count, Platelet count, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Red cell distribution width, Total leukocyte count, Reticulocyte count, Differential leukocyte count, Prothrombin time, Activated partial thromboplastin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Phase 1 - at necropsy, Phase 2 - pretest, main test - selected parameters (see * below) evaluated weekly and at necropsy. All samples were collected in the morning
- Animals fasted: Yes
- Parameters examined: creatine kinase-Total *(UV, NAC-activated Kinetic), creatine kinase-MB* (CK-MB: electrophoretic isoenzyme separation), creatine kinase-MM * (CK-MM: electrophoretic isoenzyme separation), alanine aminotransferase* (Kinetic – Modified IFCC Technique), aspartate aminotransferase* (Kinetic – Modified IFCC Technique), alkaline phosphatase* (Kinetic – Modified AMP Buffer), urea nitrogen (Kinetic – Modified Urease), creatinine (Kinetic – Modified Jaffe Method), glucose (Hexokinase Method), total cholesterol (Enzymatic – Modified Trinder Method), triglycerides (GPO Triglyceride-lipase Method), total protein (Biuret Technique), albumin (Bromocresol Green Method), bilirubin (total) (Modified Wahlefield et al) , sodium (Ion Selective Electrode), potassium (Ion Selective Electrode), chloride (Ion Selective Electrode), calcium (Cresolphthalein Complexone Method), phosphorus (Phosphomolybdate – UV Method) , Globulin (calculated value; total protein - albumin), Albumin/globulin ratio (calculated value; albumin / globulin), Indirect bilirubin (calculated value; total bilirubin – direct bilirubin), heart-fatty acid binding protein (H-FABP) (ELISA), myoglobin (ELISA)

URINALYSIS: Yes
- Time schedule for collection of urine: 19 hours following last exposure
- Metabolism cages used for collection of urine: No
- Animals fasted: fed for first 6 hours and fasted for final 12 hours
- Parameters examined.: Glucose, Ketones, Occult blood, pH, Bilirubin, Urobilinogen, Appearance, specific gravity, volume

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: blood gases evaluated within 2 hours of final exposure in Phase 1 - 15 day exposure groups, as noted in metabolism section - urinary metabolites, liver cytochrome P450 2E1 and 3A4 activity, blood and urine inorganic fluoride concentration, cardiac monofluoroacetate concentration
Sacrifice and pathology:
GROSS PATHOLOGY: Yes - Complete macroscopic examinations were performed on all animals. The macroscopic examination included including examination of the external surface and all orifices; the external surfaces of the brain and spinal cord; the organs and tissues of the cranial, thoracic, abdominal and pelvic cavities and neck; and the remainder of the carcass for the presence of macroscopic morphologic abnormalities were performed on all animals.
HISTOPATHOLOGY: Yes (* - Microscopic examination following all scheduled interim necropsies)
adrenal glands, aorta (thoracic), bone (sternum, distal femur), bone marrow (sternum, distal femur), brain , cecum, colon, epididymides, esophagus, eyes, gallbladder , Harderian gland, heart*, kidneys*, lacrimal glands, larynx, liver*, lungs (with mainstem bronchi)*, lymph nodes (mesenteric, mediastinal), mammary gland, muscle (rectus femoris), nasal cavity and turbinates, nerve (sciatic), ovaries, pancreas, pituitary gland, prostate gland, salivary glands (submandibular), seminal vesicles, skeletal muscle (rectus femoris), skin (dorsal – base of tail), small intestine (duodenum, ileum, jejunum and Peyer’s patches/GALT), spinal cord (cervical), spleen, stomach, testes, thymus, thyroid/parathyroid glands, trachea, urinary bladder, uterus (body/horns) with cervix, vagina, tissues with macroscopic findings, including tissue masses
Statistics:
The following parameters were analyzed statistically: body weight, food consumption, hematology, coagulation, clinical chemistry, urinalysis, organ weights. The parameters to analyze were identified as continuous, discrete or binary. Test-article treated groups were then compared to the control using the following procedures as appropriate: Bartlett's test for variance homogeneity, F1 approximate test for monotonicity, Williams' test for a monotonic trend, Dunnett's test , H1 approximate test for monotonicity, Shirley's test for a monotonic trend, Steel's test , Kruskal-Wallis’ test, Wilcoxon rank sum tests. For discreet data the following were used as appropriate: Jonckheere-Terpstra test ,Kruskal-Wallis test , Wilcoxon rank sum tests. For binary parameters the following were used as appropriate: Cochran-Armitage test , ¿2 test , Fisher’s Exact tests , chi-squared test , Fisher’s Exact tests
For all parameters, significant differences between control and test article-treated groups were expressed at the 5% (p<0.05), 1% (p<0.01) or the 0.1% (p<0.001) level.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see below
Mortality:
mortality observed, treatment-related
Description (incidence):
see below
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
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see details below
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see details below
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see details below
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: Phase 1 - one female exposed to 5500 ppm euthanized in moribund condition on day 5, decreased activity, ano-genital staining, decreased fecal volume and unformed stool, one male exposed to 5500 ppm found dead on day 7 with no previous clinical signs. The exact cause of death for these animals could not be determined. Histopathologically, minimal (female) to slight (male) myocardial subacute/chronic inflammation was not identified as the probable cause of death based on its limited severity and distribution of the cardiac lesions
Phase 2- One female observed with decreased activity and trembling on Days 18 and 19 and pale and decreased fecal volume on Day 19 was euthanized in a moribund condition on Day 19. Inflammation was not present in the heart and an exact cause of death could not be determined.
With the exception of those described above, no other test substance -related clinical signs were noted in Phase 1 or Phase 2.

BODY WEIGHT AND WEIGHT GAIN No statistically significant changes observed between test substance and air control animals.

FOOD CONSUMPTION : There were no test substance-related effects on food consumption in Phases 1 and 2 for the exposed animals when compared to the Air Control animals. A few statistically significant decreases or increases in food consumption were seen at various intervals but were not seen in any consistent treatment-related pattern

FOOD EFFICIENCY: not measured

WATER CONSUMPTION: not measured

OPHTHALMOSCOPIC EXAMINATION: not evaluated

HAEMATOLOGY : No noteworthy test substance-related changes were identified in the hematology or coagulation parameters from either Phase 1 or Phase 2.

CLINICAL CHEMISTRY: Test substance-related clinical chemistry findings consisted of elevated total creatine kinase (CK), isoenzyme CK-MM, isoenzyme CK-MB, heart fatty acid-binding protein (H-FABP), and/or myoglobin in male and female rabbits exposed to the test susbstance at =1500 ppm during Phase 1 (after 7 or 14 days of exposures) and in male and female rabbits exposed to the test substance at =1000 ppm and =500 ppm, respectively, in Phase 2 (primarily at Termination Day 29).
The greatest elevations were noted for total CK and CK-MM, were consistent with an effect on skeletal muscle, and observed only in test substance exposed rabbits. At no time during the study were H-FABP or CK-MB elevated in an animal without concurrent pronounced elevations in total CK, CK-MM, and/or myoglobin, suggesting that the minimal elevations in H-FABP and CK-MB were also secondary to skeletal muscle injury rather than cardiotoxicity. A few rabbits at =1000 ppm also had slightly higher values for aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT) which were also considered to be of skeletal muscle origin. There were no abnormal clinical signs in the rabbits exposed to the test substance that would suggest a means for skeletal muscle injury or otherwise explain the aforementioned chemistry changes. The clinical pathology data were generally unremarkable and comparable between the groups at the end of the 30-day recovery period.
Several test substance exposed rabbits with elevated chemistry biomarkers had no microscopic observations in either cardiac or skeletal muscle tissue. The correlation between the chemistry changes and histomorphologic findings was not consistent with cardiotoxicity. Several rabbits (exposed at =1000 ppm) with elevated skeletal muscle biomarkers had minimal to slight microscopic findings in the heart only, in skeletal muscle only, or in both heart and skeletal muscle. It should also be noted that several males and females had microscopic findings in the heart but no accompanying elevations in chemistry parameters.
The elevations in H-FABP and CK-MB were attributed to skeletal muscle injury in this study and occurred only in rabbits with high values for total CK, CK-MM, and myoglobin; they did not correlate closely with the minimal to slight microscopic findings observed in the myocardium. The higher total CK, CK-MM, and myoglobin values were attributed to acute skeletal muscle activity/injury and were seen consistently in rabbits exposed to the test substance at =1000 ppm.


URINALYSIS: No noteworthy test substance-related changes were identified in theurinalysis parameters from either Phase 1 or Phase 2.

NEUROBEHAVIOUR : Not evaluated

ORGAN WEIGHTS :
Liver: Test substance-related organ weight changes consisted of statistically significant increases in liver weight in the 1500 and 5500 ppm males on Day 8. On Day 15, liver weight had a tendency to be higher (not statistically significant) than concurrent controls in =500 ppm males. There was no liver weight effect after 28 days of exposure to the test substance.
Testes: Day 8 : statistically significant testicular weight increases (absolute, % body and % brain) occurred at 500 and 1500 ppm but not at 5500 ppm. They did not exhibit an exposure level relationship and are not considered to be related to test substance exposure
Day 28: At the end of 28 days of exposure, statistically significant increases in absolute testis weight occurred at 4500 ppm along with non-significant increases at 500 and 1000 ppm. Increased testicular weight was not associated with microscopic changes. Sexual immaturity in microscopically normal testes (normal for animals of this age) was observed in animals in the Air Control group and was associated with notably lower individual testicular weights. Increased testicular weight in the test substance exposure groups is likely due to normal variation in sexual maturation and is not related to the test substance.
HEARTS- Day 28 - At the end of 28 days of exposure to the test substance, statistically significant increases in absolute and relative (% body weight) mean heart weights in males at 4500 ppm were not considered to be related to the test substance. The mean weight increase was 10.4% in magnitude (compared to concurrent Air Controls) and present in 1 sex (males only). Only 2 animals in the 4500 ppm exposure group had heart weights increases beyond the concurrent control range. Microscopically, these 2 animals had subacute/chronic myocardial inflammation but other animals with similar cardiac findings did not have an effect on heart weight. The inflammation was minimal to slight in severity and did not appear to be of sufficient magnitude to cause notable increases in cardiac weight.

BLOOD GASES : No noteworthy test substance-related changes were identified in the blood gas parameters from animals exposed for 14 days. Since no changes were observed, blood gases were not evaluated in the main study.

GROSS PATHOLOGY : No test related gross pathological changes were observed in Phase 1 or Phase 2 animals

HISTOPATHOLOGY: NON-NEOPLASTIC
HEART: Changes in the heart occurred at all time frames during the exposures period and consisted of subacute/chronic myocardial inflammation, characterized by small aggregates of lymphocytes, macrophages and variable numbers of heterophils, that were occasionally associated with foci of myocardial degeneration/necrosis. Degeneration/necrosis was characterized by sarcoplasmic basophilia, vacuolation and myofiber shrinkage and was considered part of the inflammatory process (subacute/chronic inflammation) and not recorded as a separate finding. Inflammation was present in the ventricular myocardium (right ventricle, left ventricle, papillary muscle and interventricular septum) and was most prominent in the left ventricle and the papillary muscle.
Day 8 : Multiple foci of minimal to slight subacute/chronic myocardial inflammation occurred in both sexes at 5500 ppm and in 1 male at 1500 ppm. The finding was more common in males than females and exhibited an exposure-related increase in incidence in males
Day 15 : Subacute/chronic myocardial inflammation was present in males (only) at 5500/4500 ppm and females (only) at 1500 ppm. Findings were similar in character, distribution and severity (minimal to slight) to Day 8. The incidence of inflammation in the 5500/4500 ppm exposure group was decreased in both sexes compared to Day 8 and likely reflects the exposure reduction (5500 ppm to 4500 ppm) and 1 day of non-exposure to
the test stubstance in this group
Day 29: Minimal to slight subacute/chronic myocardial inflammation occurred in both sexes at 4500 ppm and in 1 male at 1000 ppm. It was slightly more common in males than females and exhibited an exposure-related increase in incidence and severity in males (Text Table 3.8.3-3). Changes at final sacrifice, like at Day 15, were similar in character, distribution, incidence and severity to Day 8, suggesting that they did not progress with time.
RECOVERY : Following a 28-day recovery period, subacute/chronic inflammation was not present within the heart, at any exposure level. In addition, there was no evidence of residual inflammatory cells or fibrosis indicating complete recovery.

SKELETAL MUSCLE : Acute skeletal muscle (myofiber) necrosis was present at all 3 time frames (Days 8, 15 and 29) in both sexes in all or nearly all exposure groups including the air control but was increased in incidence and/or severity in polyhaloalkene exposed animals on Days 15 and 29.
Acute necrosis was characterized by myofiber shrinkage, sarcoplasmic hyalinization and loss of cross striation. It was not associated with inflammation, fibrosis or replacement of myofibers by adipose tissue, indicative of a chronic, ongoing change.
DAY 8 : Notable increases in the incidence or severity of acute necrosis were not present. Minimal to slight necrosis was observed with similar incidence in control, 500 and 1500 ppm groups.
Day 15: Acute necrosis was increased in incidence and/or severity, compared to the air control, in both sexes at =1500 ppm although an exposure level-related response was not observed and the incidence/severity was similar to what was observed in controls following 8 days of exposure.
Day 29: Acute necrosis was increased in incidence and/or severity in males at =1000 ppm and females at 4500 ppm. The incidence and severity in males at 1000 ppm was similar to what was observed in controls following 8 days of exposure.
RECOVERY: Acute myofiber necrosis was similar in incidence and severity in the air control and polyhaloalkene exposure groups, indicative of complete recovery.



HISTOPATHOLOGY: NEOPLASTIC - none observed

HISTORICAL CONTROL DATA (if applicable)

OTHER FINDINGS: The biotransformation results suggest that subchronic test substance exposure does not lead to the formation of potentially toxic metabolites or a change in biotransformation products or capacity compared to a single test substance exposure. Furthermore, no major difference between male and female polyhaloalkene biotransformation in New Zealand White rabbits could be observed. Regarding monofluoroacetate, no accumulation could be observed in heart tissue (limit of detection was 0.01 ppm) of any animal showing major clinical or pathological heart effects.
Dose descriptor:
NOEC
Effect level:
ca. 500 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: histological lesions
Dose descriptor:
NOEC
Effect level:
ca. 1 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: histological lesions
Critical effects observed:
not specified

Text Table 3.8.1-1. Liver Weight Changes in Male Rabbits Exposed to
the test substance

Interval

Exposure Level (ppm)

0

500

1500

5500

5500/4500

Day 8

Mean Wt (g)

59.3676

64.9646

69.6320*

70.5268*

NA

% change

-

+ 9.4%

+17.3%

+18.8%

NA

Day 15

Mean Wt (g)

63.0268

72.6486

71.2114

NA

71.2590

% change

-

+15.3%

+13.0%

NA

+13.1%

Day 29

Mean Wt (g)

67.9458

65.1227

64.7674

NA

65.8299

% change

-

-4.2%

-4.7%

NA

-3.1%

* Statistically Significant p < 0.05. Mean Wt: mean absolute liver weight in grams. % change: when compared to concurrent Air Controls. NA: not applicable

TextTable 3.8.3-1: Sacrifice Day 8 - Microscopic Heart Findings in Rabbits Exposed to the test substance

 

Males

Females

polyhalopalkene (ppm)

0

500 

1500 

5500 

0

 500

1500

5500 

No. of animals examined

5

5

5

5

3

5

5

6a

 Myocardium: Subacute/Chronic Inflammation

 

 

 

 

 

 

 

 

 minimal

0

0

0

2

0

0

0

1

 slight

0

0

1

3

0

0

0

1

 Total Incidence

(%)b

0

(0)

0

(0)

1

(20)

5

(100)

0

(0)

0

(0)

0

(0)

2

(33)

aIncludes unscheduled decedents on Days 5 & 7

b% = No. of animals with finding / No. of animals examined

TextTable 3.8.3-2: Sacrifice Day 15 - Microscopic Heart Findings in Rabbits Exposed to the test substance

 

Males

Females

polyhaloalkene (ppm)

0

500 

1500 

5500/4500a

0

 500

1500 

5500/4500a

No. of animals examined

5

5

5

5

5

5

5

4

 Myocardium: Subacute/Chronic Inflammation

 

 

 

 

 

 

 

 

 minimal

0

0

0

0

0

0

0

0

 slight

0

0

0

2

0

0

2

0

 Total Incidence

(%)b

0

(0)

0

(0)

0

(0)

2

(40)

0

(0)

0

(0)

2

(40)

0

(0)

aAnimals exposed at 5500 ppm x 6 days followed by 1 day of non-exposure then exposed at 4500 ppm x 7 days

b% = No. of animals with finding / No. of animals examined

TextTable 3.8.3-3: Sacrifice Day 29 - Microscopic Heart Findings in Rabbits Exposed to the test substance

 

Males

Females

polyhaloalkene (ppm)

0

500 

1000 

4500

0

 500

1000

4500 

No. of animals examined

10

10

10

10

10

10

10

10a

 Myocardium: Subacute/Chronic Inflammation

 

 

 

 

 

 

 

 

 minimal

0

0

1

3

0

0

0

3

 slight

0

0

0

3

0

0

0

1

 Total Incidence

(%)b

0

(0)

0

(0)

1

(10)

6

(60)

0

(0)

0

(0)

0

(0)

4

(40)

aIncludes unscheduled decedent on Day 19

b% = No. of animals with finding / No. of animals examined

TextTable 3.8.3-4: Sacrifice Day 8 - Microscopic Findings inRectus femorisofRabbits Exposed to the test substance

 

Males

Females

polyhaloalkene (ppm)

0

500 

1500 

5500 

0

 500

1500

5500 

No. of animals examined

5

5

5

4

5

5

5

5

Myofiber Necrosis

 

 

 

 

 

 

 

 

 minimal

1

3

2

0

3

1

1

0

 slight

2

0

1

0

0

1

1

0

 Total Incidence

(%)a

3

60

3

60

3

60

0

0

3

60

2

40

2

40

0

0

a% = No. of animals with finding / No. of animals examined

TextTable 3.8.3-5: Sacrifice Day 15 - Microscopic Findings inRectus femorisofRabbits Exposed to the test substance

 

Males

Females

polyhaloalkene (ppm)

0

500 

1500 

5500/4500a 

0

 500

1500 

5500/4500a 

No. of animals examined

5

5

5

5

5

5

5

4

Myofiber Necrosis

 

 

 

 

 

 

 

 

 minimal

2

2

3

2

1

2

3

1

 slight

0

0

1

1

0

0

1

1

 Total Incidence

(%)b

2

40

2

40

4

80

3

40

1

20

2

40

4

80

2

40

aAnimals exposed at 5500 ppm x 6 days followed by 1 day of non-exposure then exposed at 4500 ppm x 7 days

b% = No. of animals with finding / No. of animals examined

TextTable 3.8.3-6: Sacrifice Day 29 - Microscopic Findings inRectus femorisofRabbits Exposed to the test substance

 

Males

Females

polyhaloalkene (ppm)

0

500 

1000 

4500 

0

 500

1000 

4500 

No. of animals examined

10

10

10

10

10

10

10

10a

Myofiber Necrosis

 

 

 

 

 

 

 

 

 minimal

3

2

2

3

3

2

3

2

 slight

0

0

1

2

0

0

0

2

moderate

0

0

0

0

0

0

0

2

Total Incidence

(%)b

3

30

2

20

3

30

5

50

3

30

2

20

3

30

6

60

aIncludes unscheduled decedent on Day 19

b% = No. of animals with finding / No. of animals examined

Conclusions:
The inhalation exposures of the test substance to rabbits for up to 28 days resulted in unscheduled deaths/sacrifices at 4500 (1 female) and 5500 ppm (1 male, 1 female). Exposure to the test substance was associated with microscopic findings in the heart and skeletal muscle at =1000 ppm with males more frequently affected than females. Subacute/chronic myocardial inflammation was observed in one 1000 ppm male, 1 male/2 females at 1500 ppm, 6 males/4 females at 4500 ppm, 2 males at 5500/4500 ppm and 5 males/2 females at 5500 ppm on Days 8, 15 and/or 29. The lesions were minimal to slight, did not progress over time and recovered after 28 days withot test substance exposure.
When compared to Air Control animals, increased incidence and/or severity of acute skeletal muscle necrosis was noted in both sexes at =1500 ppm on Day 15 and in males at =1000 ppm and in females at 4500 ppm on Day 29. The acute nature of the change was inconsistent with the duration of exposure suggesting that the change was not a direct effect of the test substance. Minimal to moderate skeletal muscle necrosis was generally associated with elevated myoglobin, total creatine kinase (total CK), isoenzyme CK-MM, heart fatty acid-binding protein (H-FABP), isoenzyme CK-MB, aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT) in males at =1000 ppm and females at =500 ppm.
Test substance-related increases in liver weight in the 1500 and 5500 ppm males, on Day 8 only, had no microscopic correlates and were considered a non-adverse adaptive response.
The anatomic and clinical pathology changes fully resolved following 28 days of recovery.
Under the conditions of this study and based on mortality, exposure to the test substance was tolerated up to 1500 ppm. The NOEL (No-Observed-Effect-Level) based on anatomic pathology findings was 500 ppm in males and 1000 ppm in females.
Executive summary:

Polyhaloalkene at target concentrations of 500, 1500 or 5500/4500 ppm (daily for 6 hours per day) for 6 or 13 days (Phase 1) or 500, 1000 or 4500 ppm test substance for 28 consecutive days (Phase 2). Five (5) animals/sex/group were euthanized and necropsied at the end of up to 7 or 14 days of treatment (interim sacrifices 1 and 2, respectively), and 10 animals/sex/group were euthanized and necropsied after up to 28 days of treatment (terminal sacrifice). Recovery animals, 5/sex/group, were euthanized and necropsied following 28 days of recovery. Parameters evaluated during the study were: viability, clinical observations, body weights, food consumption, blood gas values (13 days interim sacrifice), clinical pathology (each interim sacrifice, termination and end of recovery), organ weights, macroscopic observations and microscopic pathology.

 

The target and mean analytical (IR) and nominal concentrations are summarized as follows:

 

Group

Target
Concentration

Analytical
Concentration

Nominal
Concentration

 

(ppm)

 

(ppm)

Phase 1

1

0

0

0

2

500

497

NA

3

1500

1508

NA

4

5500 (Days 1 - 6)a

5533

NA

4

4500 (Days 7 – 15)

4338

6914

Phase 2

1

0

0

0

2

500

478

NA

3

1000

1010

NA

4

4500

4378

5938

aGroup 4 animals scheduled for sacrifice after 7 days of exposure were not exposed on Day 7.

NA = not applicable since the nominal was measured for all 3 groups combined and not measured for each individual group.

These results indicated an acceptable agreement between the targeted and analytical andnominal concentrations. The combined IR concentrations for Phase 1 were 6848 ppm compared to the combined nominal concentration of 6914 ppm; this 1% difference was considered typical for this type of exposure. The combined IR concentrations for Phase 2 were 5866 ppm compared to the combined nominal concentration of 5938 ppm; this 1% difference was considered typical for this type of exposure. Mean particle size distribution measurements for the exposures indicated that the atmospheres were essentially gas only, as expected, since there was no substantial difference between the test substance chambers and theAirControl chamber.

The inhalation exposures of the test substance to rabbits for up to 28 days resulted in unscheduled deaths/sacrifices at 4500 ppm (1 female) and 5500 ppm (1 male, 1 female). Exposure to the test substance was associated with microscopic findings in the heart and skeletal muscle at =1000 ppm with males more frequently affected than females.

Subacute/chronic myocardial inflammation was observed in one 1000 ppm male,1 male/2 females at 1500 ppm, 6 males/4 females at 4500 ppm, 2 males at 5500/4500 ppm and 5 males/2 females at 5500 ppm on Days 8, 15 and/or 29. The lesions were minimal to slight, did not progress over time and recovered after 28 days without test substance exposure.

When compared to Air Control animals, increased incidence and/or severity of acute skeletal muscle necrosis was noted in both sexes at =1500 ppm on Day 15 and in males at =1000 ppm and in females at 4500 ppm on Day 29. Indications of a chronic, ongoing change such as inflammation, fibrosis or replacement of myofibers by adipose tissue were not observed. The acute nature of the change was inconsistent with the duration of exposure suggesting that the change was not a direct effect of the test substance.

Minimal to moderate skeletal muscle necrosis was generally associated with elevated myoglobin, total creatinine kinase (total CK), isoenzyme CK-MM, heart fatty acid-binding protein (H-FABP), isoenzyme CK-MB, aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT) in males at =1000 ppm and females at =500 ppm. The greatest elevations were noted for total CK and CK-MM, were consistent with an effect on skeletal muscle, and observed only in test substance exposed rabbits. At no time during the study were H-FABP or CK-MB elevated in an animal without concurrent pronounced elevations in total CK, CK-MM, and/or myoglobin, suggesting that the minimal elevations in H-FABP and CK-MB were also secondary to skeletal muscle injury rather than cardiotoxicity.

Test substance-related increases in liver weight in the 1500 and 5500 ppm males, on Day 8 only, had no microscopic correlates and were considered a non-adverse adaptive response.

The anatomic and clinical pathology changes fully resolved following 28 days of recovery.

Under the conditions of this study and based on mortality, exposure to the test substance was tolerated up to 1500 ppm. The NOEL (No-Observed-Effect-Level)based onanatomic pathology findings was 500 ppm in males and 1000 ppm in females.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
2 330 mg/m³
Study duration:
subacute
Species:
rabbit
Quality of whole database:
An extensive toxicology database is available including a 2, 4, and 13 week studies in rats and a 28-day study in rabbits with interim sacrifices following 7 and 14 days of exposure and 28 day recovery period.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The following are the key findings of the 28-day inhalation toxicity study in rabbits. In this study male and non-pregnant female rabbits were exposed by whole body to 0, 500, 1000, or 4500 ppm. The results observed were similar to what was seen in the rabbit developmental toxicity study. Lethality was observed following repeated exposures of 4500 ppm (1 female) and 5500 ppm (1 male and 1 female) with no cause of death determined. Subacute/chronic cardiac inflammation was observed in 6 males and 4 females at 4500 ppm, one male at 1000 ppm and not observed in animals exposed to 500 ppm for 28 days. The no-observed effect level (NOEL) based on histological lesions is 500 ppm in males and 1000 ppm in females. Additional groups included in the study were animals exposed for 7 or 14 days (0, 500, 1500 and 4500/5500 ppm), as well as, a 28 day recovery group. A progression in severity of incidence of cardiac inflammation was not observed when the 7, 14 and 28 day data were compared. In addition, a full recovery was observed in the recovery group. 

 

When compared to the air control animals, test subjects had increased incidence and/or severity of acute skeletal muscle necrosis in both sexes at 1500 ppm on Day 15 and in males at 1000 ppm and higher and in females at 4500 ppm on Day 29. The acute nature of the change was inconsistent with the duration of exposure suggesting that the change was not a direct effect of HFO-1234yf. 

Minimal to moderate skeletal muscle necrosis was generally associated with elevated myoglobin, total creatine kinase (total CK), isoenzyme CK-MM, heart fatty acid-binding protein (H-FABP), isoenzyme CK-MB, aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT) in males at =1000 ppm and females at 500 ppm. The greatest elevations were noted for total CK and CK-MM. These were consistent with an effect on skeletal muscle, and observed only in HFO-1234yf-exposed rabbits. At no time during the study were H-FABP or CK-MB elevated in an animal without concurrent pronounced elevations in total CK, CK-MM, and/or myoglobin, suggesting that the minimal elevations in H-FABP and CK-MB were also secondary to skeletal muscle injury rather than cardiotoxicity.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Subacute/chronic cardiac inflammation observed in rabbits repeatedly exposed to the test substance. Mortality observed at exposures of 4500 ppm and higher. The NOEC was 500 ppm in males and 1000 ppm in female rabbits based on subacute/chronic cardiac inflammation. The inflamation did not appear to progress over time and were no longer observed following a 28-day recovery period.

Repeated dose toxicity: inhalation - systemic effects (target organ) cardiovascular / hematological: heart

Justification for classification or non-classification

No adverse effects observed in repeated dose inhalation toxicity studies at concentrations up to 50000 ppm (233000 mg/m3) for up to 13 weeks in rats and the no observed effect concentration was 500 ppm in male and 1000 ppm in female rabbits following 28 days of repeated inhalation exposure. Therefore the substance does not need to be classified for repeated exposure toxicity according to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) N0. 1272/2008.