2,2-Difluoroethyl Acetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

This repeated dose toxicity study was combined with a micronucleus evaluation. Only the information relevant for the repeated dose toxicity study were described here (for the micronucleus evaluation, see IU section 7.6.2).

Type of information:

experimental study

Adequacy of study:

key study

Study period:

FROM 29 AUGUST 2014 to 05 MAY 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Remarks:

The present 28-day repeated dose toxicity study by oral route included a micronucleus evaluation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008.

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 oral 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: 7 weeks old.
- Weight at study initiation: the males mean body weight varied between 224.9 and 228.4 g and the females mean body weight varied between 171.6 and 176.7 g within all dose groups. The weight variation of selected animals did not exceed ± 20% of the mean weight for each sex.
- Assigned to test groups randomly: yes, the animals were distributed by computerized, stratified randomization into study groups.
- Fasting period before study: not specified.
- Housing: animals were housed in groups in solid-bottom caging with bedding and appropriate species-specific enrichment.
- Diet: all animals were fed PMI® Nutrition International LLC Certified Rodent LabDiet® 5002 ad libitum, except during the neurobehavioral evaluations.
- Water: all animals were provided tap water ad libitum.
- Acclimation period: 6 days. The animals were released from quarantine based on normal observations for body weights and clinical signs.

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

IN-LIFE DATES: From 16 September 2014 to 14 October 2014.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Animals were dosed by intragastric intubation. The amount of test material each animal received was based on the most recently collected body weight and the formulation concentration.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was dissolved in corn oil. Neither the amount nor nature of any contaminants in the vehicle was expected to affect the integrity or validity of this study. Dose formulations were prepared without a correction for the sponsor reported purity. Dosing formulations of the test material were prepared and used within the established range of stability and stored at room temperature until used.

VEHICLE:
- Justification for use and choice of vehicle: corn oil was selected as solvent. The chosen solvent was among those recommended in the guideline and it was shown through sampling and analysis that the test material at 10 to 100 mg/mL in the vehicle and stored at room temperature for up to approximately 15 days was stable.
- Concentration in vehicle: 0, 10, 30 and 100 mg test material/mL of vehicle.
- Amount of vehicle: 10 mL/kg bw.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Sampling:
* Samples from each concentration were collected at the beginning and near the end of the study and were analysed to verify the concentration of the test material in the formulations.
* A sample of control was collected and analysed together with each set of samples to verify the absence of the test material in the vehicle.
* Stability of the test material in the concentration range from 10 to 100 mg/mL in the vehicle stored at room temperature for at least 5 hours after preparation and then for up to approximately 15 days was established concurrently.
- Storage until analysis: under refrigeration. On days samples were collected, back-up samples were taken and refrigerated for possible analysis. Whenever extra samples were taken, a sample of control was also collected. Remaining samples were discarded after the final report issued.
- Analysis: Analysis was performed by the DuPont Haskell Regulatory Analytical group. At the time of analysis, the samples were diluted with isopropyl alcohol and analysed by gas chromatography with mass spectrometry detection (GC/MS) or flame ionization detection (GC/FID).

Duration of treatment / exposure:

29 days.

Frequency of treatment:

Animals were dosed daily at approximately the same time (± 2 hours).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Test Group 4

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Test Group 3

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Test Group 2

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Test Group 1 / Vehicle alone

No. of animals per sex per dose:

- 10 animals/sex/dose in the control and 1000 mg/kg/day test groups.
N.B. The first 5 animals in each of these 2 groups were designated for repeated dose toxicity (Main Study) and micronucleus evaluation, and an additional 5 animals were designated for 1-month recovery (Recovery) evaluation.
- 5 animals/sex/dose in the 100 and 300 mg/kg/day test groups.

See also the Figure 1 on the study design and schedule attached in the section Overall remarks, attachments.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: 1000 mg/kg bw/day is the limit dose as recommended in OECD test guideline 407 and the other doses (300 and 100 mg/kg bw/day) were selected to assess a dose response for any observed effects and to establish a no-observed-effect level (NOAEL).
- Animal assignment: animals of each sex were selected for use on this study based on adequate body weight gain and freedom from any clinical signs of disease or injury. They were distributed by computerized, stratified randomization into study groups. The weight variation of selected animals did not exceed ± 20% of the mean weight for each sex.
- Fasting period before blood sampling for clinical biochemistry: 15 hours.
- Post-exposure recovery period: a recovery period of approximately 1 month was included to determine recovery from any potential effects (for the control group and high dose group).

Positive control:

No positive control was used in this repeated dose toxicity 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: at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: Day 1 and weekly thereafter.
At every weighing (excluding weights on days of neurobehavioral evaluations and necropsy), each animal was individually handled and examined for abnormal behavior and appearance. Detailed clinical observations in a standardized arena were also performed on all animals. The detailed clinical observations included (but were 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.

BODY WEIGHT: Yes.
- Time schedule for examinations: Day 1 and weekly thereafter. In addition, animals designated for neurobehavioral evaluation were weighed on the days of those observations. All animals were weighed on the day of sacrifice.

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

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: Yes.
From the food consumption and body weight data, the mean daily food efficiency was calculated.

OPHTHALMOSCOPIC EXAMINATION: not specified.

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 (15 hours).
- How many animals: all surviving main study and recovery animals.
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.
- Anaesthetic used for blood collection: Yes (isofluorane anesthesia).
- Animals fasted: Yes (15 hours).
- How many animals: all surviving main study and recovery animals.
- Parameters checked in Table 2 were examined.

PLASMA/SERUM HORMONES/LIPIDS: Not specified.

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 (15 hours).
- Parameters checked in Table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes.
- Time schedule for examinations: during acclimation (pre-test baseline), near the end of the treatment period (test day 28 or 29), and approximately 1 month after cessation of treatment (end of recovery period).
- Dose groups that were examined: all dose groups, all animals.
- Battery of functions tested:
1) Abbreviated functional observational battery (FOB): Righting reflex, approach and touch, sharp auditory stimulus, tail pinch, forelimb and hindlimb grip strength, pupillary constriction, polyuria, diarrhea.
2) Motor activity (MA): animals were individually tested in one of 32 identical, automated activity monitors (Kinder Scientific). Each monitor measured movement by detecting the interruption of photobeams by the animal. The monitoring device enabled the calculation of 2 dependent variables: duration of movements and number of ambulatory movements. All movements counted for the duration of movements, regardless of the position of the animal. A movement counted as an ambulatory movement only if the position of the animal changed along the X or Y axis. Each test session was 60 minutes in duration, with results expressed for the total session as well as for 6 successive 10-minute intervals.
The darkened MA room allowed the constriction of dilated pupils to be observed, in response to a beam of light. The MA cages were evaluated for the presence of polyuria or diarrhea.
Animals were counterbalanced by sex and treatment and tested in replicates over multiple days to minimize the influence of uncontrolled factors. The experimenter conducting the FOB was blind with respect to the group designation of the animal. The testing was performed by the same person each time. FOB and MA evaluations were conducted in a sound-attenuated room equipped with a white-noise-generation system to minimize variations in environmental test conditions. Animals were acclimated at least for 10 minutes in the FOB laboratory prior to initiation of evaluation. Body weights were collected during the FOB assessment, but were not compared statistically.
As regards motor activity,

IMMUNOLOGY: 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.
The gross examination included examination of external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.

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

HISTOPATHOLOGY: Yes.
- Organs checked in Table 5 were examined.

Optional endpoint(s):

Optional endpoints: Yes.
Micronucleus evaluation: Detailed in IUCLID section 7.6.2.

Statistics:

See more details in Table 6 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):

No adverse clinical signs were noted at any dose level. Salivation was noted in males (all treated groups) and females (low and high dose groups) at the time of and not post dosing. The reason was unknown, but likely due to test material odor. Other clinical signs observed were typical of this age and strain.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test material related mortality was noted at any dose level. One male rat in the control group and 2 male rats and 2 female rats in the 1000 mg/kg/day group were sacrificed moribund during the dosing period. Clinical signs (fast/labored breathing, cold to touch, decreased muscle tone, low posture, prostrate, etc) associated with moribundity were noted in all these animals. One female rat had a piece of bedding lodged in the duodenum opening at necropsy, and this animal was noted as ‘struggled during dosing’ multiple times. Another female rat died due to dosing error. Therefore, dosing errors/difficulties could be attributed as the reason for deaths of two female rats at 1000 mg/kg/day. Distinct gross or microscopic observations were not seen in two males that died in the 1000 mg/kg/day group. Since the clinical signs noted in these male rats were also observed in the control male and there were no gross or microscopic observations, the mortality in male rats is likely unrelated to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
Test material related, adverse effects in body weight parameters were observed at 1000 mg/kg/day. The body weight changes at 1000 mg/kg/day were not statistically significant for the first three weeks of the study, but the magnitude of change increased over time. On Day 29, the body weight at 1000 mg/kg/day was 14% lower (statistically significant) than control. The decrease in body weight persisted during recovery period (statistically significant in the first two weeks). Body weight gain at 1000 mg/kg/day was lower (statistically significant except week 1) compared to control throughout the study. Overall (Day 1-29) body weight gain was 31% lower than control. There were no statistically significant changes in body weight gains during recovery period. The changes noted in body weight parameters at 1000 mg/kg/day were considered test material related and adverse.
At 100 and 300 mg/kg/day, the body weight parameters were lower in a dose dependent manner throughout the dosing period compared to control and were not statistically significant except body weight gain at 300 mg/kg/day in the Day 15-22 interval. Final (Day 29) body weights at 100 and 300 mg/kg/day were 6 and 9%, respectively, lower compared to control. Overall (Day 1-29) body weight gains at 100 and 300 mg/kg/day were 12% and 21%, respectively, lower compared to control. Based on the dose dependency, the changes noted at 100 and 300 mg/kg/day were considered test material related, but not adverse because body weight decreases were <10% compared to control and the changes were not statistically significant.

Females:
There were no test material related changes in body weight parameters at any dose level. The final (Day 29) body weights at 100, 300, and 1000 mg/kg/day were 99%, 97%, and 103%, respectively, of control. Overall (Day 1-29) body weight gains at 100, 300, and 1000 mg/kg/day were 94%, 84%, and 100%, respectively, of control.

See Tables 9 and 10 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):

There were no test material related changes in food consumption at any dose level in males or females. Statistically significant changes at 1000 mg/kg/day – decreases in males and increases in females – were noted during recovery period. These changes were considered incidental.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Males:
Test material related, adverse decreases in food efficiency in males were observed at 1000 mg/kg/day. Statistically significant decreases were noted throughout the dosing period except first week. Overall (Day 1-29) food efficiency at 1000 mg/kg/day in males was 33% lower than control. While decreases in food efficiency were noted during the dosing period at 100 and 300 mg/kg/day, the changes were not statistically significant except at 300 mg/kg/day in the Day 15-22 weekly interval. Overall (Day 1-29) food efficiency at 100 and 300 mg/kg/day was 101% and 94%, respectively, of control. Therefore, the changes observed at 100 and 300 mg/kg/day were not considered test material related.
These data on males are detailed in Table 9.

Females:
There were no test material related changes in food efficiency in females at any dose level.

See Table 11 in the section "Any other information on results incl. tables".

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Mean total white blood cell count, as well as differential leukocyte counts, were decreased in male and female rats in all dose groups (variable statistical significance). Leukocyte parameters for most individual animals in these groups were within laboratory reference intervals. However, for a few animals across all treated groups, one or more leukocyte parameters were below laboratory reference intervals. Therefore, the decreases in leukocyte parameters in treated male and female groups were considered to be test material related and marginally adverse. The changes in leukocytes were reversible in the highest dose group following the recovery period.
There were no other adverse changes in hematology parameters in male or female animals. The following statistically significant changes in mean hematology parameters were not adverse or not related to exposure to the test material:
• Red blood cell mass parameters (red blood cell [RBC], hemoglobin [HGB] and hematocrit [HCT]) were minimally decreased (<10% compared to controls for all red cell mass parameters) in male and female in the 300 and 1000 mg/kg/day groups at the end of the dosing period (RBC and HCT were not statistically significant at 300 mg/kg/day in either sex). These changes were considered to be test material related but, based on their minimal nature, were considered to be non-adverse. There were no statistically significant differences in red cell mass parameters in the 1000 mg/kg/day recovery group.
• Statistical differences in red cell indices (red cell distribution width, mean corpuscular hemoglobin content) were observed in male and female rats in some treated groups at the end of exposure and/or after the recovery period. In absence of adverse changes in red cell mass parameters, these changes in red cell indices were not considered meaningful.
• Platelet count (PLT) was lower in the 1000 mg/kg/day male group at test day 30 (17% below the control). Although the group mean was minimally lower than the control group mean, all platelet values but one in the 1000 mg/kg/day male group were within the laboratory reference intervals for similarly aged animals (PLT 836–1460 x10E3/μL). Therefore, this difference was considered to be unrelated to treatment and non-adverse. Following the recovery period, there was no statistical significance noted in platelet count.
Additionally, there were no statistically significant changes in coagulation parameters in male or female animals.

See Table 12 in the section "Any other information on results incl. tables".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Glucose (GLUC) was lower in male and female rats in all dose groups at test day 30 (not statistically significant in the 1000 mg/kg/day male group; 19-64% below the control). These changes in blood glucose were associated with increased urine ketones (also at all dose groups) indicating a shift in energy metabolism in these animals from gluconeogenesis to incomplete oxidation of fatty acids. However, the decreases in GLUC were not associated with correlative clinical signs of hypoglycemia and thus were considered exposure related and marginally adverse. The changes in blood glucose and urine ketones were reversible in the highest dose group following the recovery period.
All other statistically significant changes in mean clinical chemistry parameters were considered spurious or were non-adverse based on the minimal degree of change, the direction of change, and/or lack of correlative changes in related parameters. These included the following:
• Higher alanine aminotransferase (ALT), in females at 1000 mg/kg/day at the end of the dosing period and in recovery animals, was not observed in males, was minimal and not associated with correlative changes in other liver related clinical chemistry parameters or with test material related microscopic changes. Therefore, this minimal change in ALT in females was considered of uncertain relationship to treatment but non-adverse.
• Lower sorbitol dehydrogenase (SDH) in males and females at all dose groups at test day 30 (not statistically significant in males at 300 mg/kg/day) was likely test material related based on the consistency of change in both males and females. However, these differences were considered non-adverse, as the direction of change—decreased rather than increased—has no known biological significance. Following the recovery period, there was no statistical significance noted in SDH.
• Higher blood urea nitrogen (BUN) in males and females at 1000 mg/kg/day at test day 30 was not associated to correlative changes in other kidney-related clinical chemistry or urinalysis parameters, or with microscopic changes in the kidneys. Therefore, these changes, while possibly test material related, were considered to be non-adverse. Following the recovery period, there was no statistical significance noted in BUN.
• Lower globulin (GLOB) in males at 300 and 1000 mg/kg/day at test day 30 did not occur in a dose-related manner, was minimal, and a similar difference was not observed in females at any dose level. Therefore, these differences were likely spurious but regardless, were considered non-adverse. Following the recovery period, there was no statistical significance noted in GLOB.
• Lower calcium (CALC) in females at 300 and 1000 mg/kg/day at test day 30 was minimal (<5% compared with controls) and similar differences were not present in males at any dose. Therefore, these differences were likely spurious and based on their minimal nature, were considered to be non-adverse. Following the recovery period, there was no statistical significance noted in CALC.
Other statistically significant changes in mean clinical chemistry parameters were considered to be unrelated to treatment and non-adverse because they did not occur in a dose-related pattern.

See Table 13 in the section "Any other information on results incl. tables".

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

As indicated in the "Clinical biochemistry findings" section above, urine ketones were increased in all test material exposed male and female groups at the end of the dosing period. These changes were correlative to the decreases in blood glucose and were thus also considered to be test material related. The increases in urine ketones were reversible following the recovery period.
There were no other adverse changes in urinalysis parameters in male or female animals. The following statistically significant changes in mean urinalysis parameters were not adverse or not related to exposure to the test material:
• Urine volume (UVOL) was higher, specific gravity (SG) and urine total protein (UMTP) were lower in all male dose groups at test day 30. In addition, UVOL was also higher in females at 1000 mg/kg/day at test day 30. These changes may indicate minimal diuresis; however, there were no changes in kidney-related clinical pathology parameters or kidney histopathology suggestive of renal injury. Therefore, these changes were considered to be possibly test material related, but non-adverse. Following the recovery period, there was no statistical significance noted in UVOL, SG or UMTP.
• Urinary pH was lower in females at 1000 mg/kg/day at test day 30. The difference relative to controls were minimal and there were no changes in urine pH in males. Therefore the lower pH in the 1000 mg/kg/day females was likely unrelated to treatment and was considered to be non-adverse. Following the recovery period, there was no statistical significance noted in pH.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Neurobehavioral evaluation:
1) Functional Observational Battery (FOB):
- Forelimb and Hindlimb Grip Strength:
There were no test material related effects on forelimb or hindlimb grip strength in males or females at any treatment level.
During the recovery period, forelimb grip strength was higher (p<0.05) in males in the 1000 mg/kg/day group compared with controls. This difference was considered spurious because it did not occur during the treatment period and because there were no corroborative differences in other grip strength parameters during the recovery period (hindlimb grip strength in males, forelimb or hindlimb grip strength in females).
- Manipulations and other FOB Endpoints:
There were no statistically significant differences or test material related effects on manipulations or other FOB parameters in males or females at any treatment level.
2) Motor activity
In males at 1000 mg/kg/day, the mean total (60-minute) duration of movement was 44% lower than control during the treatment period (statistically significant during the 3rd, 4th, and 5th 10-minute intervals as well as the 60-minute session total), and 27% lower than control during the recovery period (statistically significant during the 6th 10-minute interval but not the total session). Correspondingly, the mean total numbers of ambulatory movements were 57% and 33% lower than controls during the respective treatment (statistically significant) and recovery periods (not statistically significant). The decreases in duration of movement and number of ambulatory movements in males at 1000 mg/kg/day were considered adverse, but secondary to the systemic toxicity noted at this dose level during the treatment and recovery periods.
In females at 1000 mg/kg/day, the mean total (60-minute) duration of movement and total number of ambulatory movements were 19 and 32% lower than controls, respectively, during the treatment period. The differences were not statistically significant. The differences between female treatment groups were no longer present during the recovery evaluation. The lower motor activity values in females at 1000 mg/kg/day during the treatment period were considered to be possible test material related effects, but not adverse, due to the lack of statistically significance and low magnitude of difference in the context of individual animal data from the baseline evaluation.
There were no test material related effects on duration of movement or number of movements in males or females at 100 or 300 mg/kg/day. The mean total duration of movement and number of ambulatory movements were slightly lower among males at 100 and 300 mg/kg/day compared with controls during the baseline and treatment periods. The differences were not statistically significant during the treatment period, and the magnitudes of difference were similar before and during treatment (15-38% below control at baseline, 16-34% lower than control during treatment). Therefore, the differences were not considered test material related.
The following transient, statistically significant differences were observed during the baseline evaluation, which did not impact the interpretation of the study: during the 3rd and 4th 10-minute intervals, duration of movement was lower among males designated for 300 or 1000 mg/kg/day compared with the control group; these differences were not reflected in statistically significant differences in the 60-minute session total.
In conclusion as regards neurobehavioral evaluation, adverse, test material related decreases in duration of movement and number of ambulatory movements were observed in males at 1000 mg/kg/day during the treatment and recovery periods; the motor activity decreases were considered secondary to systemic toxicity. There were no adverse effects on motor activity in males at 300 mg/kg/day or below, or in females at any dose level. There were no test material related effects on any other neurobehavioral parameter evaluated in males or females at any dose level.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weights were collected for protocol designated organs in the 0, 100, 300 and 1000 mg/kg/day main phase groups; weights were collected from control and high dose recovery phase groups. Main phase scheduled sacrifice animals had 5/sex/group; recovery controls had 4 or 5 animals and recovery high dose had only 3 animals. Organ weight changes considered to be test material related were present in the thymus.
- Thymus:
Thymus weight parameters were statistically significantly decreased in the 1000 mg/kg/day males. Thymus weights were also lower in the 300 mg/kg/day males and the 1000 mg/kg/day females but these differences were not statistically significant except for absolute thymus weight in the 300 mg/kg/day male group. Thymic weight changes in the 1000 mg/kg/day male and female recovery groups were mostly similar to controls. However, thymic weight parameters in one 1000 mg/kg/day male appeared to be decreased relative to other individual animals in both the treated and control groups. Thymic weight change were not associated with test material related microscopic changes in the thymus (lymphoid necrosis and atrophy of the thymus were seen only in early death animals). Therefore, these changes were considered to be test material related but non-adverse.

- Other
In males, mean heart weight parameters were statistically higher in the 1000 mg/kg/day group. Mean heart weights in the 100 and 300 mg/kg/day group males were also higher, but the differences were not statistically significant. These heart weight differences in the male treated groups did not occur in a clear dose related manner and individual values in treated males were mostly within the range of concurrent controls. In addition, there were no correlative microscopic findings in the heart, and similar heart weight changes were not present in females. Therefore, these changes were of uncertain relationship to treatment and based on the absence of correlative microscopic findings, were considered to be non-adverse. Heart weights in the 1000 mg/kg/day male recovery group were similar to controls.
Mean kidney weight parameters were statistically higher in males administered 1000 mg/kg/day. These increases were not associated with test material related microscopic changes or with adverse changes in kidney related clinical chemistry parameters. Therefore, the higher kidney weights in the 1000 mg/kg/day male group were considered to be of uncertain relationship to treatment, but non-adverse. Kidney weight parameters in the 1000 mg/kg/day male recovery group were generally similar to or lower than controls.
Statistically significant differences in various kidney weight parameters were also observed in treated male and female groups. These included higher kidney weight relative to body weight in the 1000 mg/kg/day females and 100 mg/kg/day males; lower kidney weight relative to body weight in the 1000 mg/kg/day recovery group females; and decreased absolute in the 1000 mg/kg/day recovery group males. These kidney weight differences were not associated with changes in other kidney weight parameters at their respective doses or time points, or with correlative microscopic or clinical pathology changes. Therefore, these changes likely represent spurious findings.
All other organ weight differences, both statistically significant and non-significant, were considered to represent spurious findings complicated by a relatively small number of rats per group or, for the 1000 mg/kg/day males, to be secondary to body weight decrements observed in that group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was a wide variety of gross observations in this study with most being spontaneous background findings. One Group 2 male had a healed femur fracture and three rats had evidence of trauma due to gavage accidents.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Nose :
Microscopic findings in the nose (minimal to moderate reflux rhinitis), consistent with reflux/regurgitation of the gavage material into the nasal passages, were present in all treated male and female groups; incidence and severity were greater in males. Reflux rhinitis was also present in 3/3 males and 1/3 females in the 1000 mg/kg/day male and female recovery groups, respectively. In addition, secondary microscopic changes associated with decrements in body weight and food consumption and/or agonal stress were observed in some males and females administered 1000 mg/kg/day.
Reflux rhinitis was used as a summary diagnosis for a spectrum of microscopic changes in the nose that were consistent with effects due to reflux or regurgitation of the test material into the nasal passages. The reflux -related nasal lesions were generally characterized by the presence of any combination of the following: limited amount of amorphous grey material (assumed to be test material) within the nasal passages, especially the posterior nose; erosions/ulcers (often with fibrosis), atrophy and/or degeneration/regeneration of olfactory epithelium lining the nasal cavities; remodeling and/or fusion of the turbinate bones. There was infrequent and subtle inflammation associated with the reflux rhinitis. Although the caudal airways (levels 3 and 4) were most affected, lesions were occasionally noted in the more anterior nose (level 2). The dose-related increase in severity grades and the general increase in incidence of reflux rhinitis in both sexes suggest that the test material (in suspension with corn oil) was directly irritating to the nasal epithelium upon contact. The mechanism of reflux could not be determined.
See Table 14 in the section "Any other information on results incl. tables".

Other:
Other microscopic changes were observed in the 1000 mg/kg/day groups that were considered to be secondary to decrements in body weight and food consumption seen at this dose level (primarily in the 1000 mg/kg/day males) or secondary to agonal stress in early death animals.
Findings considered secondary to decrements in body weight and food consumption at 1000 mg/kg/day (main scheduled and unscheduled sacrifices) included depletion of bone marrow (3/7 males; 1/7 females) and depletion of zymogen granules (3/7 males; 2/7 females) in the pancreas. These changes were reversible as incidences of microscopic findings in the 1000 mg/kg/day recovery groups were similar to controls.
Microscopic findings typically seen in association with agonal stress were observed in two male and two female unscheduled deaths in the 1000 mg/kg/day groups. These changes included lymphoid necrosis and atrophy of the thymus, depletion of bone marrow and depletion of zymogen granules in the pancreas. Minimal lymphoid necrosis of the thymus was also seen in one scheduled sacrifice male at 300 mg/kg/day. However this finding was considered spurious, as lymphoid necrosis was not seen in scheduled sacrifice animals administered 1000 mg/kg/day and minimal lymphoid necrosis may be seen as a background finding.
All other microscopic observations were considered to be background lesions found in rats of this age and stock on gavage studies.
In conclusion as regards the microscopic findings, daily gavage of rats with the test material at 100, 300 or 1000 mg/kg/day for up to approximately 28 days resulted in reflux of the test material into the nasal cavity and subsequent rhinitis in male and female rats in all dosed groups. Reflux of the test material caused an array of lesions in the nose that were collectively termed reflux rhinitis; incidence and severity grades were higher in males. Although somewhat less severe than in the main phase, regurgitation rhinitis was present in most recovery phase animals. Tissue changes considered secondary to test material induced stress occurred in bone marrow (depletion), thymus (necrosis) and pancreas (decrease of zymogen granules); these changes were not observed in the recovery animals.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

- NOAEL determination: Clinical pathological adverse effects (decreases in leukocyte counts, decreased blood glucose, and increased urine ketones) related to the treatment and nasal lesions related to the treatment were observed in male and female rats at all dose levels. Consequently no NOAEL could be established. The NOAEL is < 100 mg/kg bw/day.

- Discussion of the effects observed:
* Local effects: Rather than primary target organ toxicity, the nasal lesions observed represent effects occurring secondary to regurgitation of the test substance which appeared as directly irritating to the nasal epithelium upon contact, at the concentrations applied. The fact that 2,2-Difluoroethyl acetate was concluded to be not irritating to skin and not irritating to eyes in GLP-compliant studies according to OECD test guidelines 404 and 405, respectively, is not contradictory and may reflect the fact that the nasal epithelium is more sensitive upon contact with 2,2-Difluoroethyl acetate.
* Systemic effects: 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, several 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:

NOAEL

Effect level:

< 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Dose formulation analysis:

Results of formulation analyses are presented in tables 7 and 8 below.

Table 7: Analysis results for samples formulated on September 16, 2014

Sample type	Test material (mg/mL)		Percent nominal
Formulation date: September 16, 2014	Nominal Average (SD)	Measured Average (SD)
Verification
Control	0	ND	-
10 mg/mL	10	9.04 (0.071)	90.4
30 mg/mL	30	29.3 (0.78)	97.7
100 mg/mL	100	97.5 (0.14)	97.5
Stability (at room temperature)
5 hours	10	9.18	91.8
	30	29.0	96.7
	100	94.2	94.2

ND: Not Detected

SD: Standard Deviation

 

Table 8: Analysis results for samples formulated on September 29, 2014

Sample type	Test material (mg/mL)		Percent nominal
Formulation date: September 29, 2014	Nominal Average (SD)	Measured Average (SD)
Verification Day 0
Control	0	ND	-
10 mg/mL	10	9.54 (0.15)	95.4
30 mg/mL	30	27.0 (1.3)	90.0
100 mg/mL	100	88.6 (6.0)	88.6
Stability (at room temperature)
Day 9	10	9.14	91.4
	100	92.0	92.0
Day 15	10	8.67	86.7
	100	91.5	91.5

ND: Not Detected

SD: Standard Deviation

 

 

Body weights:

Body weights are detailed in Tables 9 and 10 below.

Abbreviations:

SD: Standard Deviation

N: number of animals evaluated

%Diff: [(current group mean – control group mean) / control group mean] x 100  

Table 9: Mean body weights of male rats

Day(s) relative to start date	Dose	0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
1	Mean SD N %Diff	227.5 11.9 10 -	224.9 19.7 5 -1.1	228.0 5.9 5 0.2	228.4 17.1 10 0.4
8	Mean SD N %Diff	282.4 19.0 10 -	277.9 29.3 5 -1.6	280.1 8.2 5 -0.8	279.5 20.8 10 -1.1
15	Mean SD N %Diff	328.4 28.6 10 -	319.5 33.7 5 -2.7	316.1 9.4 5 -3.8	313.7 28.2 10 -4.5
22	Mean SD N %Diff	368.9 36.5 9 -	350.8 37.8 5 -4.9	341.0 9.1 5 -7.5	330.3 28.8 8 -10.4
29	Mean SD N %Diff	398.0 44.1 9 -	375.2 44.1 5 -5.7	363.0 10.7 5 -8.8	344.2 #^1 36.2 8 -13.5
36	Mean SD N %Diff	444.0 59.2 4 -	- - - -	- - - -	328.4 #^1 39.2 3 -26.0
43	Mean SD N %Diff	476.4 54.0 4 -	- - - -	- - - -	367.6 #^1 46.7 3 -22.8
50	Mean SD N %Diff	508.2 55.9 4 -	- - - -	- - - -	407.9 51.0 3 -19.7
57	Mean SD N %Diff	537.6 66.0 4 -	- - - -	- - - -	428.3 61.9 3 -20.3

¹ #: Test Dunnet 2 Sided p <0.05.

Table 10: Mean body weights of female rats

Day(s) relative to start date	Dose	0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
1	Mean SD N %Diff	171.6 10.2 10 -	173.8 10.0 5 1.3	174.6 9.7 5 1.8	176.7 12.3 10 3.0
8	Mean SD N %Diff	191.8 10.8 10 -	195.3 9.6 5 1.9	195.0 13.1 5 1.7	200.0 16.4 10 4.3
15	Mean SD N %Diff	207.2 12.2 10 -	210.9 13.8 5 1.8	207.6 15.2 5 0.2	216.1 22.5 9 4.3
22	Mean SD N %Diff	223.3 13.4 10 -	222.7 15.2 5 -0.3	221.7 15.9 5 -0.7	227.3 21.7 9 1.8
29	Mean SD N %Diff	236.7 14.8 10 -	234.9 17.0 5 -0.7	229.4 18.8 5 -3.1	243.0 21.9 8 2.7
36	Mean SD N %Diff	250.1 12.7 5 -	- - - -	- - - -	258.5 22.0 3 3.4
43	Mean SD N %Diff	260.5 8.9 5 -	- - - -	- - - -	282.3 #^1 16.1 3 8.4
50	Mean SD N %Diff	271.5 10.9 5 -	- - - -	- - - -	292.9 19.4 3 7.9
57	Mean SD N %Diff	280.4 11.6 5 -	- - - -	- - - -	304.0 27.4 3 8.4

¹ #: Test Dunnet 2 Sided p <0.05.

 

Food efficiency:

Food efficiency results for males rats are detailed in Table 11 below.

Table 11: Mean daily food efficiency of male rats

Day(s) relative to start date	Dose	0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
1 --> 8	Mean SD N %Diff	0.366 0.044 10 -	0.356 0.072 5 -3.0	0.367 0.023 5 0.0	0.361 0.059 10 -1.5
8 --> 15	Mean SD N %Diff	0.318 0.051 10 -	0.302 0.037 5 -5.0	0.264 0.033 5 -17.0	0.249 #^2 0.067 10 -21.6
15 --> 22	Mean SD N %Diff	0.252 0.105 10 -	0.235 0.042 5 -6.9	0.194 @^1 0.018 5 -22.9	0.139 @^1 0.087 10 -45.0
22 --> 29	Mean SD N %Diff	0.216 0.069 9 -	0.188 0.056 5 -12.9	0.175 0.043 5 -19.0	0.112 #^2 0.082 8 -48.1
1 --> 29	Mean SD N %Diff	0.270 0.104 10 -	0.273 0.049 5 1.4	0.254 0.013 5 -5.9	0.180 @^1 0.100 10 -33.2
29 --> 36	Mean SD N %Diff	0.113 0.059 4 -	- - - -	- - - -	0.096 0.045 3 -15.2
26 --> 43	Mean SD N %Diff	0.155 0.040 4 -	- - - -	- - - -	0.207 0.015 3 33.8
43 --> 50	Mean SD N %Diff	0.120 0.040 4 -	- - - -	- - - -	0.221 0.102 3 84.3
50 --> 57	Mean SD N %Diff	0.128 0.037 4 -	- - - -	- - - -	0.090 0.036 3 -29.5
29 --> 57	Mean SD N %Diff	0.129 0.015 4 -	- - - -	- - - -	0.148 0.025 3 15.2

Food efficiency = average weight gain / average consumed food

¹@: Test Dunnet Non-parametric 2 Sided p <0.05.

² #: Test Dunnet 2 Sided p <0.05.

Note: Interval 1-29 shows n=10 in the 0 and 1000 mg/kg/day groups; however, week 22-29 shows n=<10 due to animal deaths. Since the majority of values were calculated based on n=10, the database shows n=10 for interval 1-29.

 

Haematology:

Mean white blood cell count (WBC) are summarised in Table 12 below.

Table 12: Mean WBC for male and female rats

	Days relative to start date	0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Males		Mean (SD)
WBC (x 10^3/µL)	30	11.58 (1.80)	7.76 (2.70) #^1	7.08 (2.81) #^1	7.60 (2.93) #^1
	59	10.67 (2.25)	-	-	9.69 (2.28)
Females		Mean (SD)
WBC (x 10^3/µL)	30	9.99 (2.14)	7.05 (1.96)	6.69 (1.59) #^1	7.51 (2.81)
	59	6.05 (0.98)	-	-	8.15 (1.65)

¹# : Test Dunnet 2 Sided p < 0.05.

 

Clinical chemistry:

The blood glucose results are presented in Table 13 below.

Table 13: Mean Glucose (GLUC) in male and female rats

	Days relative to start date	0 mg/kg bw/day	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Males		Mean (SD)
GLUC (mg/dL)	30	134 (18)	49 (5)   @^1	61 (13) @^1	109 (49)
	59	126 (14)	-	-	127 (3)
Females		Mean (SD)
GLUC (mg/dL)	30	137  (18)	63 (10) @^1	60 (8) @^1	89 (40) @^1
	59	147 (11)	-	-	156 (13)

¹@: Test Dunnet Non Parametric 2 Sided p < 0.05.

 

Microscopic findings:

Incidence and severity of reflux rhinitis in rats are presented in Table 14 below.

Table 14: Microscopic Findings: Incidence and Severity of Reflux Rhinitis in Rats

Sex	Males				Females
Dose (mg/kg bw/day)	0	100	300	1000	0	100	300	1000
Nose (Main/unscheduled)
Number examined	(6)	(5)	(5)	(7)	(5)	(5)	(5)	(7)
Reflux rhinitis	0	2	4	4	0	2	1	1
Minimal	-	2	3	1	-	2	-	-
Mild	-	-	1	1	-	-	1	-
Moderate	-	-	-	2	-	-	-	1
Nose (recovery)
Number examined	(4)	(0)	(0)	(3)	(5)	(0)	(0)	(3)
Reflux rhinitis	0	-	-	3	0	-	-	1
Minimal	-	-	-	2	-	-	-	-
Mild	-	-	-	1	-	-	-	1

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the NOAEL of 2,2-Difluoroethyl acetate was undetermined (< 100 mg/kg/day) for male and female rats based on clinical pathology findings (decreases in leukocyte counts, decreased blood glucose, and increased urine ketones) and nasal lesions observed at all dose levels.

Executive summary:

The repeated dose toxicity of 2,2-Difluoroethyl acetate was investigated in a 28-day study by oral route performed according to OECD test guideline 407 under GLP compliance.

Four groups of male and female Crl:CD(SD) rats were administered 0, 100, 300, and 1000 mg/kg/day test material by gavage for 29 days consecutively (10 animals per sex in the control and 1000 mg/kg/day test groups and 5 animals per sex in the 100 and 300 mg/kg/day test groups). Main study animals (5 rats per sex per group) were sacrificed at the end of the 29-day dosing period. One-month recovery animals (3-5 per sex per group in control and high dose groups) were sacrificed approximately one month after the end of the treatment. The animals were examined daily after dosing for acute clinical signs of systemic toxicity. Body weights, food consumption and detailed clinical observations were evaluated weekly. Neurobehavioral evaluations were conducted during pretest and at the end of the dosing and recovery periods. Clinical and anatomic pathology endpoints (haematology and coagulation, serum chemistry, urinalysis, organs weight and organs gross and microscopic examinations) were evaluated at the end of the dosing and recovery periods.

No adverse clinical signs were observed in males or females at any dose level. One male in the control group and 2 males and 2 females in the 1000 mg/kg/day group were sacrificed moribund during the dosing period. Dosing errors/difficulties were attributed as the reason for deaths of the two females at 1000 mg/kg/day. Since distinct gross or microscopic observations were not seen in the two males that died in the 1000 mg/kg/day group and since the clinical signs noted in these males were also observed in the control male, the mortality is likely unrelated to treatment. It was thus concluded that there was no test material related deaths in males or females at any dose level.

Test material related adverse decreases in body weight parameters and food efficiency (= average weight gain / average consumed food) were noted in males at 1000 mg/kg/day (- 14 %, - 31 % and - 33 % in body weight, body weight gain and food efficiency, respectively, as compared to controls). The decrease in body weight persisted during the recovery period (statistically significant in the first two recovery weeks). Test material related yet limited changes in body weight parameters were noted in males at 100 mg/kg/day (- 6 % and - 12 % in body weight and body weight gain, respectively, as compared to controls) and 300 mg/kg/day (- 9 % and - 21 % in body weight and body weight gain, respectively, as compared to controls). Food efficiency during the overall exposure period was not significantly affected in those two groups. In females, there were no test material related changes in body weight or nutritional parameters at any dose level.

Adverse, test material related decreases in duration of movement and number of ambulatory movements were observed in males at 1000 mg/kg/day during the treatment (- 44 % and - 57 % in duration of movement and number of ambulatory movements, respectively, as compared to controls) and recovery (- 27 % and - 33 % in duration of movement and number of ambulatory movements, respectively, as compared to controls) periods. The decreases in motor activity parameters were considered secondary to systemic toxicity and not a primary neurotoxic effect. There were no effects on motor activity considered to be adverse in males at 300 mg/kg/day or below, or in females at any dose level. There were no test material related effects on any other neurobehavioral parameter evaluated in males or females at any dose level.

Decreased leukocyte counts (- 25 % to - 39 % as compared to controls), decreased blood glucose (- 19 % to – 64 % as compared to controls) and increased urine ketones (no percentage values available in the report) were observed in all male and female dosed groups with variable levels of statistical significance. These changes were reversible following the recovery period. There were no other adverse changes in haematology, chemistry and urinalysis parameters in male or female animals.

Statistically significant changes in some organ weight parameters (thymus, heart, and kidney) were noted but they were considered to be uncertainly related to treatment, and in any case non-adverse, because they were not associated with microscopic and/or clinical chemistry changes. Tissue changes considered to be secondary to decrements in body weight and food consumption occurred in bone marrow (depletion) and pancreas (decrease of zymogen granules) at 1000 mg/kg/day in the main study animals. These changes were reversible following the recovery period. Tissue changes occurred in bone marrow (depletion), thymus (necrosis) and pancreas (decrease of zymogen granules) at 1000 mg/kg/day in the rats sacrificed moribund during the dosing period. These findings were considered to be secondary to agonal stress in animals dead from non-treatment related reasons (see above) and spurious because not seen in scheduled sacrifice animals administered 1000 mg/kg/day.

Treatment-related adverse nasal lesions consistent with reflux of the test material into the nasal cavity and subsequent rhinitis in male and female rats were observed in all dosed groups. The incidence and severity grades were higher in males. Although somewhat less severe than in the main phase, regurgitation rhinitis was present in most recovery phase animals. The nasal lesions likely represent effects occurring secondary to regurgitation rather than primary target organ toxicity.

Under the conditions of this study, the NOAEL of 2,2-Difluoroethyl acetate was undetermined (< 100 mg/kg/day) for male and female rats based on clinical pathology findings (decreases in leukocyte counts, decreased blood glucose, and increased urine ketones) and nasal lesions observed at all dose levels.