2,2-Difluoroethyl Acetate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

FROM 19 AUGUST 2015 TO 21 SEPTEMBER 2016.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009.

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2-difluoroethyl acetate

EC Number:

801-773-4

Cas Number:

1550-44-3

Molecular formula:

C4H6F2O2

IUPAC Name:

2,2-difluoroethyl acetate

Test material form:

liquid

Remarks:

Clear and colorless.

Test animals

Species:

rat

Strain:

other: Crl:CD(SD).

Details on species / strain selection:

Rats have historically been used in safety evaluation studies for inhalation toxicity testing. The Crl:CD(SD) rat was selected based on consistently acceptable health status and on extensive experience with this strain at the testing facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories International, Inc., Raleigh, North Carolina, U.S.A.
- Females: nulliparous and non-pregnant.
- Age at study initiation: approximately 8 weeks old.
- Weight at study initiation: male rats weighed between 257 and 305 grams, female rats weighed between 173 and 233 grams. The weight variation of selected rats did not exceed ± 20% of the mean weight for each sex.
- Housing: except during exposure, animals were housed in pairs (or individually when necessary) in solid bottom caging with Enrich-o'Cobs™ bedding as enrichment.
- Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 was available ad libitum. During the urine collection period, animals were fasted overnight for 12 to 20 hours after approximately 1 to 3 hours of access to food following exposure.
- Water: except during exposure, tap water was available ad libitum, including during the urine collection period.
- Acclimation period: at least 6 days. The animals were released from quarantine based on normal observations for body weights and clinical signs.

DETAILS OF FOOD AND WATER QUALITY:
As specified in the test facility animal health and environmental monitoring program, the following procedures were performed periodically to ensure that contaminant levels were below those that would be expected to impact the scientific integrity of the study:
• Water samples were analysed for total bacterial counts, and the presence of coliforms, lead and other contaminants.
• Samples from freshly washed cages and cage racks were analysed to ensure adequate sanitation by the cage washers.
Certified animal feed was used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence of these contaminants below the maximum concentration stated by the manufacturer would not be expected to have impacted the integrity of the study.
The animal health and environmental monitoring program was administered by the attending laboratory animal veterinarian. Evaluation of these data did not indicate any conditions that affected the validity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26°C.
- Humidity (%): 30-70%.
- Photoperiod: Animal rooms were artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle.

IN-LIFE DATES: From 20 August 2015 to 22 December 2015.

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: the exposure chambers were constructed of stainless steel and glass (NYU style) with a nominal internal volume of 750 litres. Tangential turrets on top of the exposure chambers and conical shaped stainless steel baffles (suspended below the turrets) promoted uniform distribution of the test material vapour throughout the exposure chambers. The chamber volume was chosen so that the total volume of the test animals did not exceed 5% of the chamber volume.
- Method of holding animals in test chamber: during exposure, animals were individually placed in stainless steel, wire-mesh modules (sexes separate) and exposed, whole-body, inside the exposure chambers. The modules were placed on racks of stainless steel bars inside the exposure chambers so that the animals were elevated slightly from the bottom of the exposure chambers. Animals were exposed to the test substance during both the time it took for the chamber to reach concentration and the time it took for the test substance concentration to decrease below the acceptable exposure limit (AEL).
- Source and rate of air: Houseline generation air was metered to the round-bottom flasks with Brooks model 5851E mass flow controllers (MFCs) and carried the vapor and air mixtures into glass transfer tubes that led to turrets on top of the exposure chambers.
- System of generating test material vapour: chamber atmospheres were generated by flash evaporation of test material in air. The liquid test material was metered into heated round-bottom flasks with Harvard Apparatus model 22 Syringe Infusion Pumps. The round-bottom flasks were heated to approximately 150°C via Electrothermal Unimantle heaters to vaporize the test material. Houseline generation air was metered to the round-bottom flasks with Brooks model 5851E mass flow controllers (MFCs) and carried the vapour and air mixtures into glass transfer tubes that led to turrets on top of the exposure chambers. Conditioned high-efficiency particulate arrestance (HEPA) air was manually adjusted with iris valves (one per chamber) and entered through the chamber turret portals. The air control chamber was set up similarly except that there was no test material supply. The Unimantles and MFCs were monitored and controlled by the Camile Inhalation Toxicology Automated Data System (CITADS). Chamber concentrations of test material were controlled by varying the test material feed rates to the round-bottom flasks.
- Temperature, humidity in air chamber: chamber temperatures were targeted at 19-25°C and measured with type J Omega thermocouples. Chamber relative humidities were targeted at 30-70% and measured with Omega model HX71-V1 humidity sensors. Chamber temperatures, relative humidities, and airflows were automatically recorded by CITADS at approximately 15 minute intervals during the exposures.
- Air flow rate and air change rate: chamber airflows were set at the beginning of the exposure to achieve at least 10 air changes per hour and monitored using Omega Model FMA 1005 A-V1 thermoanemometers. Chamber oxygen concentrations were targeted to be at least 19%, measured with a Teledyne Analytical Instruments model GB-300 O2 monitor and recorded once from each chamber during the exposures.
- Treatment of exhaust air: the chamber atmospheres were vented into an exhaust stack via a dedicated variable speed fan controlled by an Allen-Bradley Powerflex 40 controller.

TEST ATMOSPHERE
- Brief description of analytical method used: the vapour concentration of the test material was determined by GC 4 times per day* in each test chamber and at least once per week in the control chamber. Samples of chamber atmosphere were drawn from the chambers through midget, fritted glass impingers containing acetone as a collection medium. Aliquots of the collection medium were injected into an Agilent Technologies model 6890N GC equipped with an Agilent Technologies model 7683B Series injection tower and an FID. All samples were chromatographed isothermally at 80°C on a 30 meter X 0.320 mm OD HP-5 fused silica glass column coated with a 0.25 μm film (5% Phenyl 95% dimethylpolysiloxane). The atmospheric concentration of the test material was determined from a standard curve derived from liquid standards. Standards were prepared by weighing small amounts (25-50 mg) of the liquid test material into a glass flask and filling the flask with acetone. Sample results (injection time, date, valve position, and measured concentration) were recorded by CITADS. Upon completion of the exposures, GC sample results were transferred to CIRAS, which collated sample calculations.
*: Four impinger samples were taken from each test chamber during all 6-hour exposures. Three samples were taken from each test chamber during one 5-hour exposure that was shortened to 5 hours to accommodate an eye exam.
- Samples taken from breathing zone: yes. Prior to the start of the exposure phase, the distribution of the test material was determined in the high-concentration chamber. Vapour samples were collected from the center of the chamber and 8 separate locations inside the exposure chamber and averaged. Individual samples from the 8 separate locations in the chamber were compared to the overall average for determination of homogeneous distribution of test material in the exposure chamber. The vapor concentration of the test material in the 100 ppm chamber was determined by GC to demonstrate uniform distribution of the chamber atmosphere. Samples of chamber atmosphere were continually drawn from the chamber (through ¼ inch outside diameter [OD] Teflon® sample lines) and were directly injected into an Agilent Technologies model 6890N GC equipped with a programmable pneumatic gas sample valve and a flame ionization detector (FID). All samples were chromatographed isothermally at 80°C on a 30 meter X 0.530 mm OD DB-5 fused silica glass column coated with a 3.0 μm film (5% Phenyl 95% dimethylpolysiloxane). The atmospheric concentration of the test material was determined from a standard curve derived from gas standards. Standards were prepared by injecting known volumes of the liquid test material into Tedlar® bags containing known volumes of air. Sample results (injection time, date, valve position, and measured concentration) were recorded by CITADS. Upon completion of the exposures, GC sample results were transferred to the Camile Inhalation Reporting and Analysis System (CIRAS), which collated sample calculations.

VEHICLE
- Justification for use and choice of vehicle: chamber atmospheres were generated by flash evaporation of the test material in air with a round-bottom evaporation flask.
- Concentration of test material in vehicle: chamber concentrations of the test material were controlled by varying the test material feed rate to the heated flasks.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

See in the field above named “Details on inhalation exposure / TEST ATMOSPHERE”.

Duration of treatment / exposure:

- Treatment / exposure: 90-days. To accommodate the laboratory facilities schedule, the initiation of exposures was staggered by one day. Due to the staggered start, animals received a partial week of exposures during the first and last weeks of the study. However, the total number of exposures was 65. The exposure period was defined as the period between initiation of the first exposure and completion of the last exposure.
- Recovery period: approximately 1 month.

Frequency of treatment:

Each group of animals was exposed for 6 hours/day, 5 days/week, over an approximate 90-day period (weekends and holidays excluded) for a total of 65 exposures.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Group 1 (control)

Doses / concentrations 2

Dose / conc.:

1 ppm

Remarks:

Group 2
Equivalent to 0.005 mg/L

Conversion from ppm to mg/L:
Test concentrations (mg/m3) = Test concentrations (ppm) x Molecular weight (g/mol) / 24.5 (L/mol)
Where:
Test concentrations (ppm) = 1
Molecular weight (g/mol) = 124.09
24.5 L/mol = gas constant at 25 °C and 1013.25 hPa
Test concentrations (mg/m3) = 1 x 124.09 / 24.5
Test concentrations (mg/m3) = 5
Test concentrations (mg/L) = 0.005

Doses / concentrations 3

Dose / conc.:

10 ppm

Remarks:

Group 3
Equivalent to 0.051 mg/L

Conversion from ppm to mg/L:
Test concentrations (mg/m3) = Test concentrations (ppm) x Molecular weight (g/mol) / 24.5 (L/mol)
Where:
Test concentrations (ppm) = 10
Molecular weight (g/mol) = 124.09
24.5 L/mol = gas constant at 25 °C and 1013.25 hPa
Test concentrations (mg/m3) = 10 x 124.09 / 24.5
Test concentrations (mg/m3) = 51
Test concentrations (mg/L) = 0.051

Doses / concentrations 4

Dose / conc.:

100 ppm

Remarks:

Group 4
Equivalent to 0.51 mg/L

Conversion from ppm to mg/L:
Test concentrations (mg/m3) = Test concentrations (ppm) x Molecular weight (g/mol) / 24.5 (L/mol)
Where:
Test concentrations (ppm) = 100
Molecular weight (g/mol) = 124.09
24.5 L/mol = gas constant at 25 °C and 1013.25 hPa
Test concentrations (mg/m3) = 100 x 124.09 / 24.5
Test concentrations (mg/m3) = 506
Test concentrations (mg/L) = 0.51

No. of animals per sex per dose:

15 animals/sex/group.
The first 10 animals in each group were designated for subchronic toxicity, and the remaining animals in each group were designated for recovery.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The design concentration of 100 ppm was selected as the high level for the current 90-day inhalation study based on a previous 4-week inhalation study in rats (see "Rep. dose Inh. tox 28d KS V1 2015SHEU" in the same IUCLID section) during which exposures to 100 ppm of the test material resulted in clinical pathology findings (decreased blood glucose and ketonuria) in both sexes and microscopic changes in the nose of male rats. The design concentration for the low level group was selected to be 1 ppm because this concentration was anticipated to cause no toxicologically significant effects. The design concentration for the middle level group was selected to be 10 ppm because this concentration was anticipated to be an appropriate level of magnitude to demonstrate a concentration-response between the low- and high-level groups.
- Animal assignment: Animals of each sex were selected for use on study based on adequate body weight gain and freedom from any ophthalmology abnormalities or clinical signs of disease or injury. They were distributed by computerized, stratified randomization into study groups so that there were no statistically significant differences among group body weight means within a sex. The weight variation of selected animals did not exceed ± 20% of the mean weight for each sex. The first 10 animals in each group were designated for subchronic toxicity, and the remaining animals in each group were designated for recovery. Animal #111, originally designated for recovery, was sacrificed at the end of exposure period. The change was recommended by the laboratory animal veterinarian based on occasional observations of slight bloody urine. This observation was not considered test substance-related because the animal was in the air-exposed control group. Animal #110, originally designated for subchronic toxicity, was sacrificed at the end of recovery period as a replacement.
- Fasting period before blood sampling for clinical biochemistry: at least 15 hours.
- Post-exposure recovery period: a recovery period of approximately 1 month was included to determine recovery from any potential effects.

Positive control:

No positive control was used in this specific study.

Examinations

Observations and examinations performed and frequency:

See also more details in the section "Any other information on materials and methods incl. tables".

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: cage-site examinations to detect moribund or dead animals, abnormal behavior and appearance among animals were conducted at the time of loading the animals into the chamber and unloading the animals from the chambers on exposure days and at least once daily on non-exposure days.
During the daily exposures, the response to an alerting stimulus was determined for the animals as a group within each exposure chamber. The alerting response was determined prior to the initiation of each exposure, 3 times during exposure, and after the conclusion of each exposure prior to animal removal from the exposure chamber. Study technicians judged whether the group of animals within a given exposure chamber displayed a normal, diminished, enhanced, or absent alerting behavior in response to a standardized auditory stimulus. In addition, study technicians observed the animals for clinical signs prior to the initiation of each exposure and 3 times during each exposure. Clinical signs observed were recorded for the animals collectively within a chamber, since individual animal identification was not visible to the observer.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: immediately following each exposure, each animal was individually handled and examined for abnormal behavior and appearance. Clinical signs of hair loss, fur/skin stains associated with restraint, urination and defecation during exposure were not recorded for the post-exposure observations. Detailed clinical observations, including (but not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic, tonic, stereotypical, or bizarre behavior were conducted prior to the start of the exposure period and then approximately once a week during both exposure and recovery periods. Any abnormal clinical signs noted were recorded.

BODY WEIGHT: Yes.
- Time schedule for examinations: all animals were weighed shortly before the first exposure, twice weekly during the exposure period, once weekly during the recovery period and at the time of euthanasia. At every weighing, each animal was individually handled and examined for abnormal behavior and appearance.

FOOD CONSUMPTION: Yes.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: The amount of food consumed by each animal over an approximately 7-day weighing interval was determined by weighing each feeder at the beginning and end of the interval and subtracting the final weight and the amount of spillage from the feeder during the interval from the initial weight divided by the number of animals in the cage. From these measurements, mean daily food consumption over the interval was determined.

FOOD EFFICIENCY: No.

WATER CONSUMPTION: No.

OPHTHALMOSCOPIC EXAMINATION: Yes.
Two ophthalmology examinations were conducted by a veterinary ophthalmologist.
1) The baseline examination was performed on all animals received for the study, prior to assignment to groups. Any animals with pre-existing ophthalmology abnormalities were eliminated from consideration for use in the study.
2) All surviving animals were examined prior to sacrifice at the end of the exposure period (i.e., on test day 86 for males and day 85 for females).
Both eyes of each animal were examined by focal illumination and indirect ophthalmoscopy. The eyes were examined in subdued light after mydriasis had been produced.

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: at the end of the treatment and recovery periods prior to the scheduled sacrifice.
- Anaesthetic used for blood collection: Yes (isofluorane anesthesia).
- Animals fasted: Yes (at least 15 hours).
- How many animals: 10 animals per group for the subchronic toxicity part of the study and 5 animals per group for the recovery part of the study.
- Parameters checked in Table 1 were examined.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: at the end of the treatment and recovery periods prior to the scheduled sacrifice.
- Animals fasted: Yes (at least 15 hours).
- How many animals: 10 animals per group for the subchronic toxicity part of the study and 5 animals per group for the recovery part of the study.
- Parameters checked in Table 2 were examined.

URINALYSIS: Yes.
- Time schedule for collection of urine: at the end of the treatment and recovery periods prior to the scheduled sacrifice.
- Metabolism cages used for collection of urine: Yes.
- Animals fasted: Yes (at least 15 hours).
- Parameters checked in Table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: No.

IMMUNOLOGY: No.

BRONCHOALVEOLAR LAVAGE FLUID (BALF): No.

LUNG BURDEN: No.

Sacrifice and pathology:

SACRIFICE
Rats were euthanized by exsanguination while under isoflurane anesthesia and a complete necropsy was performed on each rat.

GROSS PATHOLOGY: Yes.
- All study animals underwent a gross evaluation at the end of the exposure and recovery periods.

ANATOMIC PATHOLOGY: Yes.
- Organs checked in Table 4 were examined.

HISTOPATHOLOGY: Yes.
- Organs checked in Table 4 were examined.

Statistics:

See Table 5 in the section "Any other information on materials and methods incl. tables".

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- Observations during exposures: no abnormality was detected in any of the exposure groups during the exposures.
- Weekly detailed clinical observations: one male rat exposed at 100 ppm was hunched-over on test day 44. Other clinical signs observed in male and female rats included hair loss and superficial wounds. These effects were regarded as not test material-related or not adverse due to the nature of effect or lack of concentration-response relationship.
- Post-exposure clinical observations: no test material-related clinical sign of toxicity was observed in any of the exposure groups during the post-exposure clinical observations. Red discharge or slight bloody urine were observed in one control male rat from test day 61 until sacrificed.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no test material-related deaths. Two male rats (one exposed at 10 ppm and one exposed at 100 ppm) were found dead during the study (test days 85 and 84, respectively); a cause of death could not be determined. One male rat (exposed at 100 ppm) was sacrificed early due to fractured incisors. The other animals survived until the scheduled terminal sacrifice.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No statistical significant changes in body weight and body weight gain were observed in any male exposure groups when compared to the air-exposed control group. There were occasional variations in body weight gains and these changes were regarded as not test material-related because they were sporadic and did not follow any concentration-response or temporal relationship.
In females, statistically significant lower body weights were observed in female rats exposed at 100 ppm when compared to air-exposed control female rats during mid- to later stages of exposure period (test days 29-89) and early stage of recovery period (test days 95-102); the maximum reductions were 8.1% and 14%, respectively. At the later stage of recovery period (test days 109-124), reductions in body weights were continued at 9.4-12%. Although these changes during the late recovery period were not statistically significant, they were regarded as biologically relevant because of the magnitude. Overall, the lower body weights observed in female rats exposed at 100 ppm were regarded as test material-related and adverse based on the magnitude of change and inability to recover.
In contrast, reduction in body weight gain was only observed in female rats exposed at 100 ppm during test day 1-29 but not in any later period.
In conclusion, female rats exposed to 100 ppm of test material showed adverse, test material-related body weight reductions which were not correlated with daily food consumption or changes in body weight gains when compared to the air-exposed control groups. This effect was not reversible following the 1-month recovery period.
See Table 7 in the section "Any other information on results incl. tables".

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Male rats exposed at 100 ppm showed statistically significant (5.1%) lower daily food consumption when compared to air-exposed control male rats during the entire exposure period (test days 1-91). In contrast, the same group of exposed male rats consumed significantly (5.8-8.2%) more food during the early recovery phase (test days 96-110) and recovered from any food consumption effects by late recovery period (test days 110-123). There were occasional variations observed in exposed male and female rats. These changes were regarded as not test material-related because they were sporadic and did not follow any concentration-response or temporal relationship.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmological abnormalities were identified during the examination prior to sacrifice at the end of the exposure period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Hematology:
There were no adverse changes in hematology parameters in male or female animals. Hematology data from one male rat in the control group was excluded from statistical analysis due to marked alterations of hematology parameters resulting from the kidney tumor in this animal.
The following statistically significant changes in mean hematology parameters were not adverse or not related to exposure to the test material:
- Absolute neutrophil count (ANEU) was lower in males at the end of the exposure period in the 100 ppm group (47% below the control). The mild decrease in ANEU could potentially be treatment-related since females in the 100 ppm group also had decreased ANEU (41% below the control) although it was not statistically significant. The mild decrease in ANEU was not considered adverse since most individual values were within the laboratory’s 95% historical control range for similarly aged animals (0.67-4.05 x 10E3 /μL), the changes were not associated with clinical signs of neutropenia and values returned to normal following 1 month of recovery.
- Absolute reticulocyte count (ARET) was lower in males at the end of the exposure period in the 100 ppm group (18% below the control). The statistically significant differences in ARET were minimal and not considered adverse since all individual values in the 100 ppm group were within the laboratory’s 95% historical control range for similarly aged animals (114-214 x 10E3 /μL). Values returned to normal following 1 month of recovery.
- Mean corpuscular hemoglobin concentration (MCHC) was minimally decreased in males in the 100 ppm group (2% below control). Although statistically significant, the difference is not considered treatment related due to the minimal change and because all individual values were within the laboratory’s 95% historical control range for similarly aged animals (31.9-34.2 g/dL).
- Hemoglobin (HGB) and red blood cell indices (mean cell volume [MCV], mean cell hemoglobin [MCH] and mean corpuscular hemoglobin concentration [MCHC]) were lower in females at 100 ppm at the end of the exposure period. These differences were all minimal (<10%) compared to the control group. These changes were possibly test material-related but not considered adverse since there was no evidence of anemia and there were no changes in other hematology parameters including total red blood cell count (RBC), haematocrit (HCT), red cell distribution width (RDW), or ARET. Following 1 month of recovery, the 100 ppm group means for HGB, MCV, MCH and MCHC were similar to the control.
- Platelet count (PLT) was higher in females at the end of exposure in the 1 and 100 ppm groups (28% and 32%, respectively). These changes were minimal and there was no concentration response therefore it was not considered treatment-related. Following the 1-month recovery period, all group mean PLT counts were similar to control.
The following statistically significant changes in mean hematology parameters were considered to be unrelated to treatment because they did not occur in a concentration-related pattern or occurred only after 1 month of recovery:
- Platelet count was lower in males at 1 ppm after 1 month of recovery (18% below the control).
- Absolute basophil count (ABAS) was lower in males at 1, 10 and 100 ppm after 1 month of recovery (54%, 57% and 53% below the control).
- Absolute reticulocyte count (ARET) was higher in males at 1 and 10 ppm after 1 month of recovery (12% and 23% above the control).
- In females, red cell distribution width (RDW) was lower in the 1 and 10 ppm groups at the end of the exposure period (5% and 4% below the control, respectively) and higher in the 100 ppm group at the end of the recovery period (7% above the control).

- Coagulation:
There were no statistically significant changes in coagulation parameters in male or female animals at the end of exposure period or after 1 month of recovery.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Glucose (GLUC) was statistically significantly decreased in males and females at 100 ppm (29% and 49% below control, respectively). These decreases were associated with increases in urine ketones in these groups indicating a shift in energy metabolism in these animals from gluconeogenesis to incomplete oxidation of fatty acids. Additionally, glucose levels in most female individual animals in the 100 ppm group were below the 95% laboratory reference intervals for animals of the same age and sex (111 – 221 mg/dL). Therefore, the decreases in blood glucose in the 100 ppm male and female groups were considered to be test material related and adverse. Blood glucose levels were also statistically decreased in the 10 ppm female group (22% below control). However, these decreases in blood glucose were not associated with increases in urine ketones, and glucose values in individual animals in this group were within or only slightly below the 95% laboratory reference interval. Therefore, the changes in blood glucose in the 10 ppm female group were considered test material-related but non-adverse. The changes in blood glucose were reversible following 1 month of recovery and were similar to the respective control group.
See Table 8 in the section "Any other information on results incl. tables".
The following statistically significant changes in mean clinical chemistry parameters were not adverse or not related to exposure to the test material:
- Inorganic phosphorus (IPHS) was higher in the 100 ppm male group at the end of the exposure period (17% above the control). There were no similar changes in females. This change in IPHS was considered possibly related to treatment but non-adverse since it was not associated with relevant changes in other clinical pathology parameters. Following 1 month of recovery, the mean IPHS value was similar to the control.
- Bilirubin (BILI) was lower in the 100 ppm female group at the end of the exposure period (18% below the control). BILI was not statistically significantly lower in males at this same concentration and biologically relevant changes in BILI generally occur as increases rather than decreases. Therefore, lower BILI in the 100 ppm female groups was considered to be unrelated to treatment and non-adverse. Following 1 month of recovery, the mean BILI value was similar to the control.
The following statistically significant changes in mean clinical chemistry parameters were considered to be unrelated to treatment because they did not occur in a concentration-related pattern or occurred only after 1 month of recovery:
- Blood urea nitrogen (BUN) was 22% below the control following the exposure period in the 1 ppm male group. Following 1 month of recovery, BUN in this group was 16% above the control. BUN remained within the 95% historical control range (11 – 16 mg/dL) throughout the study.
- Aspartate aminotransferase (AST) was statistically significantly higher in the 1 ppm female group at the end of the recovery period (55% above the control).

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

As noted above, urine ketones were increased in the 100 ppm male and female groups at the end of the exposure period. These changes correlate with the decreases in blood glucose and were therefore considered to be exposure-related and adverse.
The following statistically significant changes in mean urinalysis parameters were not adverse or not related to exposure to the test material:
- Urine pH was lower in the 100 ppm female group at the end of the exposure period (6% below the control). Lower urine pH was likely exposure-related but was considered non adverse based on the lack of association with other changes indicative of adverse primary target organ toxicity. Following the recovery period, group mean values for urinary pH in females were similar to controls.
The following statistically significant changes in mean urinalysis parameters were considered to be unrelated to treatment because they did not occur in a concentration-related pattern, or occurred only after 1 month of recovery:
- Urine pH was higher in males in the 1 and 100 ppm treatment groups at the end of the recovery period.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related organ weight changes. There was statistically significant decrease in kidney weights in male rats at all exposure concentrations. However, the difference in weight compared to control was small and not concentration dependent (-8.4, -10.8, and -9.8%; groups 4, 3, and 2, respectively). There were no changes in organ weights in females and there was no microscopic correlate for lower kidney weights. Therefore, the decreased kidney weights were not considered treatment-related.
One male rat in the control group (animal 111) had a large nephroblastoma obscuring the right kidney. The kidney weights for this animal were excluded from all statistical analyses.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At necropsy, there were no test material-related gross observations. All gross observations were typical of background findings in rats of this age, sex, and strain.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test material-related microscopic findings. All microscopic observations were consistent with normal background lesions in rats of this age and stock. Microscopic findings for the 2 male rats that were found dead were consistent with agonal changes. No cause of death could be determined.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Urine and plasma fluoride:
Plasma fluoride concentrations at the end of the exposure period were statistically significantly higher in the 10 and 100 ppm female groups when compared to the control. At this same timepoint, the mean amounts of excreted fluoride (urine fluoride) were increased in males and females exposed to the test material (statistically significant at 10 and 100 ppm). Increased excreted fluoride in these animals was considered to be secondary to exposure to the test material, and indicates exposure to and metabolism of the fluoride-containing test material. Following 1 month of recovery, group mean plasma fluoride levels were similar to the respective control groups. Urine fluoride decreased following a 1 month recovery in the male recovery group at 100 ppm but remained slightly elevated and statistically significant when compared to control (85% above the control). Urine fluoride in the female recovery groups returned to control values.
There were no other changes in urine and plasma fluoride parameters in male or female animals.

Details on results:

- NOAEC determination: Females exposed to 100 ppm test material showed adverse, test material-related body weight reductions which were not correlated with daily food consumption or changes in body weight gains when compared to control groups. This effect was not reversible following the 1-month recovery period. No statistical significant changes in body weight and body weight gain were observed in any male exposure groups when compared to the control group. Statistically significant decreases in blood glucose were observed at exposure concentrations ≥ 10 ppm in females and at 100 ppm in males. The decreases in blood glucose at 100 ppm in males and females were associated with increases in urine ketones and were therefore considered to be test material-related and adverse. At 10 ppm in females, the decreases in blood glucose were not associated with any change in urine ketones and were therefore considered to be test material-related but non-adverse. The changes in blood glucose were reversible in all exposure groups following a 1-month recovery period. There were no other adverse changes in hematology, chemistry and urinalysis parameters in male or female animals. There were no test material-related changes in organ weights, gross observations or microscopic findings for either male or female rats at any exposure concentration. Consequently, the NOAEC was based on clinical pathology findings (decreased blood glucose and ketonuria) observed in both sexes at 100 ppm and adverse and unrecoverable body weight reduction in females exposed at 100 ppm and was determined to be 10 ppm* for male and female rats.
*NOAEC conversion from ppm to mg/L:
NOAEC (mg/m3) = NOAEC (ppm) x Molecular weight (g/mol) / 24.5 (L/mol)
Where:
NOAEC (ppm) = 10
Molecular weight (g/mol) = 124.09
24.5 L/mol = gas constant at 25 °C and 1013.25 hPa
NOAEC (mg/m3) = [10] x 124.09 / 24.5
NOAEC (mg/m3) (mg/m3) = 50,65
NOAEC (mg/L) = 0.051 mg/L.

- Discussion of the systemic effects observed: Albeit that a series of changes was observed, they did not translate in clear, organ-specific adverse observations, nor in adverse findings for apical endpoints. Moreover, some changes observed showed good reversibility in recovery groups.
---> On the above basis, a classification as STOT RE was not considered warranted and no target organ/system was identified in the table below named “Target system / organ toxicity”.

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

10 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Chamber concentrations of test material:

- Chamber distribution:
Samples taken from various locations in the chamber demonstrated differences that were less than 10% from the overall mean vapor concentration; therefore, the test material atmosphere was considered to be homogenously distributed in the locations where animals were exposed.

 

- Chamber concentrations:
Air-exposed control rats were exposed to an atmosphere containing 0.0 ± 0.0 ppm test material (mean ± standard error of the mean). Rats in the 1, 10, and 100 ppm target concentration groups were exposed to vapor concentrations of 1.0 ± 0.0049, 10 ± 0.044, and 100 ± 0.50 ppm test material, respectively.

Conversion from ppm to mg/L:
Test concentrations (mg/m3) = Test concentrations (ppm) x Molecular weight (g/mol) / 24.5 (L/mol)
Where:
Test concentrations (ppm) = 1 or 10 or 100
Molecular weight (g/mol) = 124.09
24.5 L/mol = gas constant at 25 °C and 1013.25 hPa
Test concentrations (mg/m3) = [1 or 10 or 100] x 124.09 / 24.5
Test concentrations (mg/m3) = 5 or 51 or 506
Test concentrations (mg/L) = 0.005 or 0.051 or 0.51

 

- Chamber environmental conditions:
The daily mean chamber temperatures for all exposure groups were 20-21°C and the daily mean relative humidity (RH) ranged from 60 to 68%. There were individual incidences where RH values deviated from the target range (i.e., 30-70%); however, they did not adversely affect the results or interpretation of this study. There was 131-132 L/min airflow through the chambers which provided 10-11 air changes per hour. The oxygen concentration in the chambers was 21%.

 

In conclusion, the chamber distribution, concentrations, and environmental conditions were considered adequate for the conduct of the study.

 

Table 6: Chamber concentrations of test material

Target Concentration (ppm)	Group	Measured concentration (ppm)^a
		Mean	S.E.M	Range	N
0	1 (Males)	0.0	0.0	0.0	13
	1 (Females)	0.0	0.0	0.0	13
	1 (Combined^b)	0.0	0.0	0.0	13
1	2 (Males)	1.0	0.0049	0.93-1.2	65
	2 (Females)	1.0	0.0049	0.93-1.2	65
	2 (Combined)	1.0	0.0049	0.93-1.2	66
10	3 (Males)	10	0.044	9.5-11	65
	3 (Females)	10	0.043	9.5-11	65
	3 (Combined)	10	0.043	9.5-11	66
100	4 (Males)	100	0.50	88-111	65
	4 (Females)	100	0.47	88-111	65
	4 (Combined)	100	0.49	88-111	66

^a Values represent the mean, standard error of the mean (S.E.M.), and range of the daily mean values obtained from n exposures.

^b Male and female exposure starts were staggered by 1 day. Each sex received 65 exposures.

N: number of values used in calculation.

 

 

Body weights:

Female rat body weights are detailed in Table 7.

Abbreviation:

SD: Standard Deviation

Table 7: Mean body weights of female rats

Dose	0 ppm	1 ppm	10 ppm	100 ppm
Day(s) relative to start date	Mean (SD)
1	201.6 (13.1)	202.8 (13.4)	200.8 (11.6)	197.9 (9.6)
5	214.5 (11.5)	216.1 (13.9)	214.9 (10.5)	207.9 (10.2)
8	225.1 (14.2)	227.2 (12.6)	223.6 (10.3)	217.9 (12.7)
12	235.1 (16.1)	235.7 (12.3)	234.8 (10.7)	225.1 (12.6)
15	242.0 (15.8)	244.8 (13.5)	241.5 (10.4)	232.9 (12.4)
19	254.6 (17.2)	256.2 (13.5)	253.2 (11.7)	242.8 (13.1)
22	257.0 (16.0)	257.9 (14.2)	255.4 (14.6)	245.7 (14.7)
26	264.4 (15.5)	266.4 (13.7)	262.4 (15.4)	252.9 (16.3)
29	270.9 (18.4)	271.6 (17.7)	265.5 (14.4)	256.3 (13.3)#^1
33	273.3 (21.4)	276.9 (17.0)	270.8 (16.0)	259.1 (14.6)
36	279.2 (20.0)	281.0 (16.8)	275.6 (14.4)	263.4 (14.7) #^1
40	282.2 (18.7)	288.5 (19.6)	278.2 (15.8)	265.4 (15.6) @^2
43	284.2 (17.4)	290.0 (19.7)	279.6 (15.9)	265.8 (15.1) #^1
47	289.4 (18.5)	295.1 (18.0)	283.3 (16.6)	272.0 (16.2) #^1
50	291.4 (17.2)	297.1 (19.8)	288.6 (21.4)	276.8 (17.9)
54	295.9 (16.6)	297.4 (19.7)	289.6 (20.8)	277.4 (18.5) #^1
57	298.5 (19.1)	301.5 (20.1)	293.4 (21.0)	279.2 (17.3) #^1
61	303.1 (20.3)	304.8 (21.9)	297.1 (23.0)	281.8 (18.8) #^1
64	307.2 (19.0)	305.9 (19.6)	301.3 (20.1)	285.5 (17.4) #^1
68	309.6 (18.7)	311.6 (22.7)	304.1 (21.2)	289.5 (19.1) #^1
71	310.6 (18.8)	314.2 (25.4)	304.1 (19.7)	289.6 (19.5) #^1
75	311.0 (18.3)	315.1 (23.4)	308.4 (24.0)	290.6 (21.0) #^1
78	315.8 (17.7)	317.4 (26.3)	310.2 (26.4)	293.8 (20.2) #^1
82	317.1 (17.9)	322.3 (25.1)	310.9 (26.5)	294.2 (19.4) #^1
85	319.6 (20.8)	319.1 (22.8)	312.7 (26.5)	293.6 (17.3) #^1
89	318.4 (20.7)	325.0 (24.4)	316.5 (28.1)	294.7 (17.8) #^1
91	312.4 (20.8)	332.2 (27.0)	315.5 (26.7)	298.0 (18.5)
92	303.4 (28.0)	308.5 (23.2)	299.8 (26.2)	275.4 (20.4) #^1
95	333.8 (15.7)	312.2 (21.0)	313.8 (23.4)	287.6 (27.6) #^1
102	340.2 (15.1)	312.6 (25.4)	320.0 (26.0)	298.8 (27.5) #^1
109	339.6 (15.0)	317.9 (27.4)	322.4 (27.9)	304.2 (28.4)
116	347.3 (18.4)	328.6 (30.1)	334.8 (28.2)	314.8 (37.1)
123	355.0 (23.4)	328.1 (28.9)	339.0 (31.2)	315.6 (40.2)
124	333.1 (23.2)	306.2 (28.6)	314.6 (30.7)	293.5 (37.9)

¹ #: Test Dunnet 2 Sided p <0.05.

²@: Test Dunnet Non Parametric 2 Sided p < 0.05.

 

Clinical chemistry:

The blood glucose results are presented in Table 8 below.

Table 8: Mean Glucose (GLUC) in male and female rats

	Days relative to start date	0 ppm	1 ppm	10 ppm	100 ppm
Males		Mean (SD)
GLUC (mg/dL)	92	193 (42)	184 (37)	178 (36)	136 (38) #^1
	124	211 (63)	191 (28)	200 (29)	199 (11)
Females		Mean (SD)
GLUC (mg/dL)	92	164 (22)	159 (24)	129 (25) #^1	83 (18) #^1
	124	182 (11)	162 (39)	171 (19)	185 (41)

¹#: Test Dunnet 2 Sided p < 0.05.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the NOAEC of 2,2-Difluoroethyl acetate was 10 ppm for male and female rats, based on clinical pathology findings (decreased blood glucose and ketonuria) observed in both sexes at exposure concentration of 100 ppm and adverse and unrecoverable body weight reduction in females exposed at 100 ppm.

Executive summary:

The repeated dose toxicity of 2,2-Difluoroethyl acetate was investigated in a 90-day study by inhalation route performed according to OECD test guideline 413 under GLP compliance.

Four groups of male and female Crl:CD(SD) albino rats (15 animals per sex per group) were exposed whole body, 6 hours per day, 5 days a week to vapour concentrations of 0 (air control), 1 ± 0.0049, 10 ± 0.044 and 100 ± 0.50 ppm test material (equivalent to 0, 0.005, 0.051 and 0.51 mg/L test material respectively) over a 90-day period for a total of 65 exposures. Test atmospheres were generated by flash evaporation of test material in air. Animals were observed for acute clinical signs of toxicity daily and detailed clinical observations were evaluated weekly. Body weight and food consumption parameters were evaluated twice a week and once a week, respectively. Ophthalmological examinations were performed on all rats during acclimation and the week before the end of the exposure period. Following the final exposure, blood and urine samples were collected for clinical pathology evaluations (hematology and coagulation, serum chemistry, urinalysis) and the main study animals (10 animals per sex per group) were sacrificed for anatomic pathology assessment (organs weight and organs gross and microscopic examination). Following an approximate 1-month recovery period, blood and urine samples were collected for clinical pathology evaluations and the animals from the recovery group (5 animals per sex per group) were sacrificed for evaluation of anatomic pathology endpoints.

There was no test material-related mortality, clinical signs of toxicity and ophthalmological abnormalities during the course of this study.

Females exposed to 100 ppm test material showed adverse, test material-related body weight reductions which were not correlated with daily food consumption or changes in body weight gains when compared to control groups. This effect was not reversible following the 1-month recovery period. No statistical significant changes in body weight and body weight gain were observed in any male exposure groups when compared to the control group.

Statistically significant decreases in blood glucose were observed at exposure concentrations ≥ 10 ppm in females and at 100 ppm in males. The decreases in blood glucose at 100 ppm in males and females were associated with increases in urine ketones and were therefore considered to be test material-related and adverse. At 10 ppm in females, the decreases in blood glucose were not associated with any change in urine ketones and were therefore considered to be test material-related but non-adverse. The changes in blood glucose were reversible in all exposure groups following a 1-month recovery period. There were no other adverse changes in hematology, chemistry and urinalysis parameters in male or female animals.

There were no test material-related changes in organ weights, gross observations or microscopic findings for either male or female rats at any exposure concentration.

Under the conditions of this study, the NOAEC of 2,2-Difluoroethyl acetate was 10 ppm for male and female rats based on clinical pathology findings (decreased blood glucose and ketonuria) observed in both sexes at 100 ppm and adverse and unrecoverable body weight reduction in females exposed at 100 ppm.