Polyhaloalkene

Endpoint summary

Description of key information

No toxicity was observed in male or female rats exposed by inhalation to concentrations up to 50000 ppm (equivalent to 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 (equivalent to 2330 mg/m3) and females exposed to greater than 1000 ppm (equivalent to 4660 mg/m3). exposure 6 hours a day/ 7 days a week. However, the rabbit is not an appropriate model for human cardiotoxicity.

Therefore, a 28-day inhalation toxicity study in the minipig was conducted. No test substance-related effects were observed during the course of this study, in which animals were exposed to test concentrations up to 10200 ppm for 6 hours a day/ 7 days a week. For risk assessment purposes, the rat represents the species with a relevant, robust safety assessment database

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

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:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland
- Age at study initiation: 6 weeks
- Weight at study initiation: Mean weights 231 g (males) and 172 g (female)
- Fasting period before study: None
- Housing: Macrolon cages with wood shaving beding
- Diet: Ad libitum (overnight fast prior to necropsy)
- Water: Ad libitum
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 37-44
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: March 2006 To: June 2006

Route of administration:

inhalation: gas

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Cylindrical PVC column with a volume of ~ 70 liters surrounded by a transparent hook. The test atmosphere was introduced at the bottom and exhausted at the top
- Source and rate of air: At least 1 L/min
- Temperature, humidity in air chamber: 20-24C; 30-70%
- Air flow rate: At least 1 L/min

TEST ATMOSPHERE
- Brief description of analytical method used: Total carbon analysis
- Samples taken from breathing zone: Yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Total carbon analysis

Duration of treatment / exposure:

6 hours a day

Frequency of treatment:

5 days a week for 13 weeks

Dose / conc.:

5 000 ppm

Remarks:

Group 2: Low dose. Equivalent to 23300 mg/m3.

Dose / conc.:

15 000 ppm

Remarks:

Group 3: Mid dose. Equivalent to 69900 mg/m3.

Dose / conc.:

50 000 ppm

Remarks:

Group 4: High dose. Equivalent to 233000 mg/m3.

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: 50000 ppm (5%) was chosen as the high dose group to prevent secondary effects due to oxygen deprivation that can occur at higher concentrations
- Post-exposure recovery period in satellite groups: None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily

BODY WEIGHT:
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for per group determined and mean daily diet consumption was calculated as g food/kg body weight/day

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 was calculated as time-weighted averages from the consumption and body weight gain data

OPHTHALMOSCOPIC EXAMINATION: yes

HAEMATOLOGY:
- Time schedule for collection of blood: At scheduled necropsy
- Anaesthetic used for blood collection: Yes, Nembutal
- Animals fasted: Yes
- How many animals: All survivors
- Parameters listed in OECD guideline were examined.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: At scheduled necropsy
- Animals fasted: Yes
- How many animals: All survivors
- Parameters listed in OECD guideline were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes - those organs listed in the guideline plus nose (6-levels), larynx (3 levels), trachea (3 levels including bifurcation), and each lung lobe at 1 level.

Statistics:

Data were evaluated by the appropriate statistical test (one-way analysis of variance followed by Dunnett's multiple comparison test, one-way analyis of variance (ANOVA) followed by Dunn't multiole comparison testes, Krisckal-Wallis nonparametric Anova followed by Mann-Whitney U-tests, Fischers exact probability test.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEC

Effect level:

> 50 000 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Equivalent to 233000 mg/m3.

Key result

Critical effects observed:

no

Conclusions:

Exposure of rats to 5000, 15000 or 50000 ppm (23300, 69900, or 233000 mg/m3) test substance for 6 hours a day, 5 days a week, for 64 or 65 exposure days over a 98 day period did not result in adverse effects in any of the exposure groups. In the present sub-chronic toxicity study, the high concentration level of 50000 ppm was therefore considered the No-Observed-Adverse-Effect-Concentration for male and female rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

233 000 mg/m³

Study duration:

subchronic

Species:

rat

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

A number of repeated dose studies are available in which the toxicity of the test substance following inhalation exposure is assessed. For reasons of clarity, they will be described in chronological order in this endpoint summary.
In the period 2006-2007, 3 studies were performed assessing the effect of repeated dose inhalation exposure on rats. Test concentrations up to 50000 ppm (233000 mg/m3), and an exposure duration up to 13 weeks did not result in adverse findings in these studies.
In 2012-2013, a 28-day toxicity study following inhalation exposure of rabbits was conducted. The test substance was administered 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 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 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 at1000 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 on anatomic pathology findings was 500 ppm in males and 1000 ppm in females.
To help evaluate the relevance of the cardiac inflammation observed in rabbits following repeated exposure, consultation with several experts including cardiologists, toxicologists and pathologists indicated that the rabbit was not an appropriate model for human cardiotoxicity and that a more appropriate animal model was the minipig. The minipig is accepted by regulatory authorities as a non-rodent surrogate for humans in the detection of cardiotoxicity and is generally accepted to be a sensitive and representative model for human cardiac responses. A more detailed comparison between the rabbit and the minipig with regard to their role as toxicological models for predicting toxicity to humans is described in the toxicological endpoint summary / DNEL justification section.
In 2013-2014, a 28-day inhalation study was conducted in which 16 Göttingen minipigs were exposed to the test substance via whole-body exposure at target concentrations of 5000 or 10000 ppm (daily for 6 hours per day) for 7 days per week. At the end of the treatment period, all animals were euthanized and necropsied. Parameters evaluated during the study were: viability, clinical observations, body weights, food consumption, hematology and clinical chemistry, organ weights, macroscopic observations and microscopic pathology.
No test substance-related effects were observed during the course of this study. At Day 29, there were no HFO-1234yf-related microscopic findings involving the heart (left ventricle, right ventricle and septum) and skeletal muscle (rectus femoris, psoas and soleus muscles as well as the diaphragm). Therefore, there was no microscopic evidence of cardiotoxicity or skeletal muscle toxicity following whole-body inhalation of the test substance at up to 10,000 ppm for 28 days in male and female Gottingen minipigs. The results of this study demonstrate that minipigs respond very differently to HFO-1234yf inhalation exposure as compared to rabbits. Under the condition of this study, the No-Observed-Adverse-Effect-Level (NOAEL) for inhalation toxicity was determined to be the actual achieved exposure level of 10,200 ppm.
A detailed review of the cardiovascular pathology is provided as an attachment to the IUCLID dossier. The review was performed by an external expert upon request of the registrant.

Justification for classification or non-classification

No adverse effects observed in repeated dose inhalation toxicity studies at concentrations up to 50000 ppm (equivalent to 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. Furthermore, no adverse effects were observed in repeated dose inhalation toxicity studies at concentrations up to 10200 ppm in minipigs Therefore, the substance does not need to be classified for repeated exposure toxicity in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008 and its amendments.