2,2-Difluoroethyl Acetate

Endpoint summary

Description of key information

Repeated dose toxicity via oral route – 28-day 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.

Repeated dose toxicity via inhalation route – 28-day study: the NOAEC of 2,2-Difluoroethyl acetate was undetermined (<100 ppm) for male and female rats, based on clinical pathology findings (decreased blood glucose and ketonuria) observed at all exposure levels, and microscopic changes in the nose of male rats in all exposed groups and female rats in the 750 and 1500 ppm groups.

Repeated dose toxicity via inhalation route – 90-day 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.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

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.

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

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.

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).

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Test Group 4

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Test Group 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Test Group 2

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.

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".

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".

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

Critical effects observed:

no

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

A GPL-compliant study performed according to OECD test guideline 407 is available. It is considered as fully reliable (Klimisch score of 1) and the result is retained as key data.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

FROM 19 AUGUST 2015 TO 21 SEPTEMBER 2016.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009.

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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.

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.

Dose / conc.:

0 ppm

Remarks:

Group 1 (control)

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

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

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.

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".

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”.

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

Critical effects observed:

no

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.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

10 ppm

Study duration:

subchronic

Species:

rat

Quality of whole database:

A GPL-compliant study performed according to OECD test guideline 413 is available. It is considered as fully reliable (Klimisch score of 1) and the result is retained as key data.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

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 28 APRIL 2014 TO 01 SEPTEMBER 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Remarks:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

2009.

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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: the males mean body weight varied between 248 and 308 g and the
females mean body weight varied between 176 and 226 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, so that there were no statistically significant differences among group body weight means within a sex.
- Fasting period before study: not specified.
- Housing: except during exposure, animals were housed in pairs (sexes separated) in solid-bottom caging with Enrich-o'Cobs™ as bedding and enrichment.
- Diet: all animals were fed PMI® Nutrition International LLC Certified Rodent LabDiet® 5002 ad libitum, except during exposure. During the urine collection period, rats were fasted overnight for approximately 12 to 20 hours.
- Water: except during exposure, all animals were provided tap water ad libitum (even during the urine collection period).
- 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 20 May 2014 to 15 July 2015.

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

TYPE OF INHALATION EXPOSURE
Whole body.

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: exposure chambers were constructed of stainless steel and glass (NYU style) with a nominal internal volume of 350 L. A tangential feed inside the chamber promoted uniform chamber distribution of the test atmosphere. The chamber volume was chosen so that the total body 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 cages and exposed, whole-body, inside the exposure chamber. Animals were exposed to the test material during both the time it took for the chamber to reach concentration and the time it took for the test material to be purged from the chamber.
- Source and rate of air: Houseline generation air, metered into the flask by a Brooks model 5850E mass flow controller, carried the vaporized test substance into a supply air stream leading to the exposure chamber.
- System of generating test material vapour: chamber atmospheres were generated by flash evaporation of the test material in air with a round-bottom evaporation flask. The test material was metered into the evaporation flask with a Harvard Apparatus model 22 syringe infusion pump. The evaporation flasks were placed in Unimantle heaters that were heated up to 175ºC for the 100 and 750 ppm chambers and 195 ºC for the 1500 ppm chamber to vaporize the test material. Heat tapes set at approximately 95ºC were used to wrap the glass connection tubes from the evaporation flasks to the chamber inlets for the 1500 ppm chamber. Houseline generation air, metered into the flask by a Brooks model 5850E mass flow controller, carried the vaporized test material into a supply air stream leading to the exposure chamber. Chamber concentrations of test material were controlled by varying the test material feed rate to the heated flasks.
- Temperature, humidity in air chamber: chamber temperature was targeted at 19-25°C and recorded approximately once/hour during each exposure. Chamber relative humidity was targeted at 30-70% and recorded approximately once/hour during each exposure. Temperature and humidity were measured with a VWR dial-type thermometer/hygrometer.
- Air flow rate and air change rate: Chamber airflow was set at the beginning of each exposure to achieve at least 10 air changes per hour and monitored continually with a Brooks model 5850E mass flow controller. Airflows were recorded initially, approximately once/hour, and whenever changes were made during each exposure. Chamber oxygen concentration was targeted to be at least 19%, measured with a Teledyne Analytical Instruments model GB300 oxygen analyser, and recorded once during each exposure.
- Treatment of exhaust air: test atmospheres were exhausted into the laboratory’s exhaust stack.

TEST ATMOSPHERE
- Brief description of analytical method used: during each exposure, the atmospheric concentration of the test material was determined by gas chromatography (GC) at approximately 12 times per day in the test chambers. The control chamber was monitored at least once per day. Samples of chamber atmosphere were directly injected into an Agilent Technologies model 6890N GC equipped with a pneumatically operated gas sample valve and a flame ionization detector. All samples were chromatographed using an oven temperature rate of 80°C on a 30-meter J&W Scientific Inc. DB-5 fused silica glass column. The atmospheric concentration of the test material was determined from a standard curve derived from vapour standards. Standards were prepared prior to each exposure 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.
- 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 homogenous distribution of test material in the exposure chamber.

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: 4 weeks. 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 20 (see in the field below "Frequency of treatment). 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 a 4-week period (weekends and holidays excluded) for a total of 20 exposures.

Dose / conc.:

0 ppm

Remarks:

Group 1 (control)

Dose / conc.:

100 ppm

Remarks:

Group 2
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

Dose / conc.:

750 ppm

Remarks:

Group 3
Equivalent to 3.8 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) = 750
Molecular weight (g/mol) = 124.09
24.5 L/mol = gas constant at 25 °C and 1013.25 hPa
Test concentrations (mg/m3) = 750 x 124.09 / 24.5
Test concentrations (mg/m3) = 3799
Test concentrations (mg/L) = 3.8

Dose / conc.:

1 500 ppm

Remarks:

Group 4
Equivalent to 7.6 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) = 1500
Molecular weight (g/mol) = 124.09
24.5 L/mol = gas constant at 25 °C and 1013.25 hPa
Test concentrations (mg/m3) = 1500 x 124.09 / 24.5
Test concentrations (mg/m3) = 7597
Test concentrations (mg/L) = 7.6

No. of animals per sex per dose:

20 animals/sex/dose.
The first 10 rats in each group were designated for subchronic toxicity, and the remaining rats in each group were designated for neurobehavioral evaluation and recovery.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The previously performed acute inhalation toxicity study demonstrated that the 4-hour median lethal concentration (LC50) in male and female rats for this test material is > 5600 ppm (>28 mg/L). Animals exposed to 5600 ppm lost weight and displayed decreased activity, labored breathing, gasping, ataxia, lethargy, and hunched posture during the exposure and/or the 14-day recovery period. Another group of rats exposed to 1500 ppm showed decreased activity during the exposure and only one male displayed lethargy and hunched posture after the exposure. All animals survived and no gross lesions were observed in this study. During pilot studies for this 28-day repeated dose toxicity study by inhalation, 3 male and 3 female rats were exposed (6 hours/day) to 3000 ppm of test material for 3 days and lethality (1/3) was observed in males 2 days after the last exposure. Therefore, the high concentration of the test material for this 4-week inhalation toxicity study was selected based on previous inhalation studies, the above-mentioned pilot studies and knowledge about the physical/chemical properties of the test material.
- Animal assignment: Rats 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 rats did not exceed ± 20% of the mean weight for each sex. The first 10 rats in each group were designated for subchronic toxicity, and the remaining rats in each group were designated for neurobehavioral evaluation and recovery.
- 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.

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-side examinations to detect moribund or dead animals, abnormal behaviour and appearance among rats were conducted at the time of loading the animals into the chamber on exposure days and at least once daily on non-exposure days and during the recovery period.
- Observations during exposure: Additionally, 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 and 3 times during exposure. Study technicians judged whether the group of animals within a given exposure chamber displayed a normal, diminished, enhanced, or absent alerting behaviour 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 rat was individually handled and examined for abnormal behaviour and appearance. Clinical signs of hair loss and 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 behaviour, were conducted prior to the start of the exposure phase and approximately once a week during both exposure and recovery periods. Any abnormal clinical sign noted was recorded.

BODY WEIGHT: Yes.
- Time schedule for examinations: individual animal weights were recorded shortly before the first exposure, twice weekly thereafter and at the time of euthanasia. Animals designated for neurobehavioral evaluation and recovery were weighed weekly throughout the recovery period. At every weighing, each rat was individually handled and examined for abnormal behaviour 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 rats received for the study, after assignment to groups. Any rats with pre-existing ophthalmology abnormalities were eliminated from consideration for use in the study.
2) All surviving rats were examined prior to the sacrifice at the end of the exposure period (i.e., on test day 25 for males and day 24 for females).
Both eyes of each rat 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: 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.
- Animals fasted: Yes (at least 15 hours).
- How many animals: all surviving main study and recovery animals.
- 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: Yes.
- Time schedule for examinations: during acclimation (baseline), during week 4 of exposure, and at approximately one month after cessation of exposure (prior to the end of the recovery period).
- Dose groups that were examined: all dose groups, all animals designated for neurobehavioral evaluation and recovery (10 male and 10 female rats per dose group). For the baseline evaluation, FOB and MA assessments (see below) were also conducted on spare animals in the event that replacements were needed during acclimation or on test day 1.
- Battery of functions tested:
1) Abbreviated functional observational battery (FOB):
Righting reflex, approach and touch, sharp auditory stimulus (e.g., clicker), 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.

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.

Other examinations:

Micronucleus evaluation: Detailed in IUCLID section 7.6.2.

Statistics:

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

Clinical signs:

no effects observed

Description (incidence and severity):

- Observations During Exposures: no abnormality was detected in any of the exposure groups during the 6-hour exposures.
- Weekly Detailed Clinical Observations: no test material related clinical sign of toxicity was observed in any of the exposure groups during the weekly detailed clinical observations.
- Post-Exposure Clinical Observations: no clinical sign of toxicity was observed in any of the exposure groups during the post-exposure clinical observations.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no test substance-related deaths. All 40 main phase animals survived until the scheduled terminal sacrifice. Thirty-nine of the 40 recovery animals survived until the scheduled terminal sacrifice. One female rat from the 1500 ppm exposure group was found dead during the recovery period (test day 52); a cause of death could not be determined.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the exposure period, statistically significant reductions (6.4-14%) in body weight were observed in male rats exposed to 1500 ppm of test material (test days 3-28) when compared to air-exposed control male rats. Significant lower body weights (5.2-11%) were also seen in the 750 ppm male exposure group during the latter part of the exposure period (test days 10-28) while exposure to 100 ppm did not cause any significant body weight effect in male rats. During the entire exposure period (test days 1-28), male rats exposed to 100, 750, and 1500 ppm gained 13, 29, and 41% less weight than the air controls, respectively. These changes in mean body weight and weight gain of male rats were considered to be test material related and the body weight reduction observed in the 750 and 1500 ppm exposure groups were considered adverse because of the magnitude and consistency of the alterations.
At the beginning of the recovery period, male rats in the 1500 ppm exposure group weighed 9.9% less than the air controls (test day 35) and no significant effect was observed for the remaining portion of the study (test days 42-56). No significant body weight effect was observed for the 150 and 750 ppm exposure groups. By the end of the recovery period (test day 53), male rats in all exposure groups showed ≤5.1% body weight difference from the air controls, indicating that the body weight reductions observed during the exposure period were reversible. Body weight gains of male rats in the 100, 750, and 1500 ppm exposure groups were 30, 43, and 54% higher than the air control male rats, respectively, during the entire recovery period (test days 35-53).
Similar effects were observed in female rats. Female rats exposed to 750 ppm and 1500 ppm of test material had statistically significant lower body weights (4.1-8.8% during test days 10-28 and 4.3-11% during test days 8-28, respectively) than the air-exposed control female rats during the exposure period. They gained 39 and 52% less weight, respectively, than the air controls during the entire exposure period. Exposure to 100 ppm did not cause any significant effect on body weight and body weight gain of female rats. These changes in mean body weight and weight gain of female rats were considered to be test material related and the body weight reduction observed in the 750 and 1500 ppm exposure groups were considered adverse because of the magnitude and consistency of the alterations.
Female rats in the 750 and 1500 ppm exposure groups continued to weigh 7.7-8.5% less than air controls at the beginning of the recovery period (test days 34-41). By the end of the recovery period (test day 52), female rats in all exposure groups showed ≤5.3% body weight difference from the air controls, indicating that the body weight reductions observed during the exposure period were reversible. Body weight gains of female rats in the 100, 750, and 1500 ppm exposure groups were 23, 35, and 40% higher than the air control female rats, respectively, during the entire recovery period (test days 34-52).
See also Tables 7 and 8 in the section "Any other information on results incl. tables".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Male rats exposed to 750 and 1500 ppm of test material consumed significantly less food than air control male rats in every week during the exposure period and averaged 9.9 and 17% reduction, respectively, for the entire exposure period (test days 1-28). Female rats demonstrated the same trend and averaged 11 and 14% reduction in food consumption, respectively, for the same period of time. These changes in male and female rats were considered to be test material related and correlated with the body weight reductions observed in the 750 and 1500 ppm exposure groups.
During the recovery period, significant increase (≤9.8%) or decrease (≤8.0%) in food consumption was observed in male rats in the 100 ppm (test days 35-53), 750 ppm (test days 28-35 and 49-53), and 1500 ppm (test days 42-53) exposure groups. Female rats in the 100 ppm and 1500 ppm exposure groups demonstrated sporadic (but significant) increases (11-26%) in food consumption. These changes did not demonstrate concentration response relationships or correlate well with the changes in body weights.
See also Tables 9 and 10 in the section "Any other information on results incl. tables".

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test material related ophthalmological abnormalities were identified during week 4 of exposures. One (of 10) control male, 2 (of 10) males exposed to 100 ppm, 1 (of 10) female exposed to 100 ppm, and 1 (of 10) female exposed to 1500 ppm of test material were identified with multifocal retinal degeneration. The etiology of these findings was not known, but was not considered to be test material related or adverse due to the lack of a concentration-response relationship and the sporadic nature of the findings. All other animals examined had no visible ophthalmoscopic lesions.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- Hematology:
Absolute counts for reticulocytes [ARET] and white cells (total white cell count, as well differential white cells—neutrophils, lymphocytes, monocytes, eosinophils, and basophils) were decreased in the 750 and 1500 ppm male and female groups. Changes in red cell indices (mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], mean corpuscular hemoglobin concentration [MCHC], and red cell distribution width [RDW]) correlative to the decrease in reticulocytes were also present in these groups. In addition, red cell mass parameters (red blood cell count [RBC], hemoglobin [HGB], and hematocrit [HCT]) were decreased in the 750 and 1500 ppm female groups. These changes in hematology parameters were generally statistically significant at 1500 ppm, with more variable statistical significance at 750 ppm. The pattern of change in hematology parameters in these groups is indicative of decreased hematopoiesis of both the white and red cell lineages and is consistent with effects reported to occur in association with reduced food consumption and decrements in body weight.
Therefore, these hematology changes in the 750 and 1500 groups were likely secondary to the test material related decrements in food consumption and body weights observed at these exposure concentrations.
Statistically significant but minimal (less than 5% above controls) increases in RBC and HCT were present in males at 1500 ppm. These minimal changes were likely the result of slight hemoconcentration (dehydration) associated with the more severe effects on food consumption and body weight observed in the 1500 ppm male group compared with those seen in females at this exposure concentration. These more severe effects on food consumption likely resulted in associated decreases in water intake in this group.
These secondary hematology changes observed in the 750 and 1500 ppm groups at the end of the 4-week exposure period were reversible following the 4-week recovery period, during which time effects on food consumption and body weight were also reversible.
The following statistically significant changes were considered to be non-adverse and/or unrelated to exposure of the test material.
- A minimally higher ARET was present in the 1500 ppm male recovery group. This may reflect an appropriate residual hematopoietic response to the decreased erythropoiesis observed at the end of the exposure period at this concentration. However, based on its minimal nature and lack of association with changes in red cell mass parameters, this difference was considered to be non-adverse.
- RDW was minimally higher in the 750 and 1500 ppm male recovery groups and in all previously exposed female recovery groups. These differences were of uncertain relationship to exposure but were not associated with changes in red cell mass parameters, as all changes in red cell mass parameters were reversible following the recovery period. Therefore, these differences in RDW were considered to be non-adverse.
- Minimally lower HCT, MCV, and MCH were present in the 100 ppm female group at the end of the exposure period. These differences were of uncertain relationship to exposure but were not associated with statistically significant changes in RBC or HGB, and statistically significant differences in these parameters were not observed in males at this concentration. Therefore these differences were considered to be non-adverse.
- Absolute monocyte count was higher in 100 ppm recovery group males, and absolute eosinophil count was higher in females the 1500 ppm recovery group females. These changes were considered spurious and unrelated to exposure as similar changes were not observed at these concentrations at the end of the exposure period.
- Mean corpuscular hemoglobin content was lower in the 100 and 1500 ppm female recovery groups. These minimally lower group means were considered to be unrelated to exposure and non-adverse because they did not occur in a concentration-related manner, and occurred only after approximately 4 weeks of recovery.

- Coagulation:
There were no statistically significant or exposure-related changes in any coagulation parameter in male or female animals following the exposure period.

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

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Glucose (GLUC) was statistically significantly decreased (by approximately 34-44% relative to respective controls) in all male and female exposure groups. Individual GLUC values in most animals in these groups were below the laboratory reference interval (GLUC reference intervals: 86-134 mg/dL and 88-138 mg/dL for males and females, respectively). These changes in blood glucose were associated with increased urine ketones (also at all exposure concentration) 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 were reversible in all exposure groups following the recovery period.
All other statistically significant changes in clinical chemistry parameters were considered unrelated to exposure and/or non-adverse:
- Statistically significant but minimal changes in some serum protein and lipid parameters were observed in the 1500 ppm males or females at the end of the exposure period. These included statistically decreased triglycerides and albumin in males and decreased total protein and increased globulin in females. These serum protein and lipid changes were likely secondary to the decrements in food consumption and body weight seen at this concentration and, based on their minimal nature, were considered to be non-adverse.
- Cholesterol was lower in all female exposure groups. These differences were minimal, did not occur in a concentration-related manner, and similar changes were not observed in males, which were generally more severely affected than females for other parameters. Therefore, these changes were of questionable relationship to exposure but regardless, were considered non-adverse based on their minimal nature.
- Aspartate aminotransferase was higher in all male exposure groups and alanine aminotransferase was higher in the 750 or 1500 ppm male and female groups at the end of the exposure period. Such minimal increases in serum transaminases may occur secondary to decrements in food consumption and body weight as seen in these groups. However, based on their minimal nature (increased < 2-fold relative to control) and lack of correlative changes in related parameters, these changes were considered non-adverse.
- Sorbitol dehydrogenase was lower in all male and female exposure groups (not statistically significant in females exposed to 100 ppm) at the end of the exposure period. These changes may have been related to exposure to the test material but were considered non-adverse based on the direction of change (decreased, rather than increased), as decreases in SDH are not biologically relevant.
- Blood urea nitrogen (BUN) was minimally but statistically higher in all male exposure groups and in 100 and 750 ppm female groups (but not the 1500 ppm female group). These differences did not occur in a concentration-related manner and were not associated with changes in creatinine or with microscopic changes in the kidneys. Therefore, these differences were likely unrelated to exposure and based on their minimal nature, were considered to be non-adverse.
- Inorganic phosphorus was statistically higher in the 750 and 1500 ppm males and in the 100 and 1500 ppm females. These differences, though possibly related to exposure, did not occur in a concentration-related manner, were not associated with relevant changes in other clinical pathology parameters and were thus considered to be non-adverse.
- Calcium was lower in females exposed to 1500 ppm at the end of the exposure period. The degree of change was not biologically relevant (4% below the control) and a similar change was not observed in males. Therefore, the minimal difference in calcium was considered spurious and non-adverse.
The following statistically significant changes in mean clinical chemistry parameters were considered to be unrelated to exposure and non-adverse because they did not occur in a concentration-related manner, or occurred only after approximately 4 weeks of recovery:
- lower BUN in the 1500 ppm male recovery group and higher BUN in the 100 ppm female recovery group,
- higher inorganic phosphorus in the 100 ppm male recovery group,
- and higher sodium in the 100 and 750 ppm male exposure groups.

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 exposure period. These changes were correlative to the decreases in blood glucose and were thus also considered to be exposure-related. The increases in urine ketones were reversible following the recovery period.
The following statistically significant changes in mean urinalysis parameters were considered to be unrelated to exposure and/or non-adverse:
- Urine volume (UVOL) was increased in males and females exposed to 750 or 1500 ppm at the end of the exposure period (statistically significant except in the 1500 ppm male group). These increases in urine volume may have been exposure-related, however, they were not associated with changes in urine specific gravity—indicating no effects on the urine concentrating function of renal tubules—or with changes in other kidney-related parameters. Therefore, these changes were considered to be non-adverse. Following the recovery period, group mean values for UVOL were similar to controls.
- The concentration of urine total protein (UMTP) was decreased in males and females exposed to 750 or 1500 ppm (not statistically significant in the 1500 ppm males). These changes may have been secondary to the increases in urine volume (i.e., dilution) in these groups and were considered non-adverse as decreases in UMTP have no biological relevance. Following the recovery period, group mean values for UMTP were similar to controls.
- At the end of the exposure period, urine pH was lower in males and females at 100, 750, and 1500 ppm (statistically significant except in 100 ppm males). 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 were similar to controls for both sexes.

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 exposure level.
During the week 4 evaluation, hindlimb grip strength was significantly lower (p<0.05) among males at 750 and 1500 ppm compared with controls. The difference was considered spurious for the following reasons: the differences were not concentration-dependent (i.e. the magnitude of difference was lower at 1500 ppm than at 750 ppm); individual values for hindlimb grip strength (mean of 3 trials) at 7500 ppm were all within the range of values from the control group; the mean values for all exposure levels were within the range of historical control means (0.72-1.12 kg); and there were no corroborative differences in forelimb grip strength for males, or forelimb or hindlimb grip strength for 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 exposure level. Any observations noted by exception were unremarkable and occurred with low incidence across exposure levels.

2) Motor activity
There were no adverse effects on any motor activity parameter (duration of movement or number of ambulatory movements during any 10-minute interval or 60-minute total session duration) in males or females at any exposure level.
During the week 4 evaluation, males at 1500 ppm exhibited significantly lower (p<0.05) total duration of movements and total number of ambulatory movements, compared with controls. A similar difference was observed in baseline values collected for these animals prior to test material exposure, but the differences were not statistically significant at baseline, and they were slightly more severe during the week 4 evaluation (18% and 20% differences at baseline compared with 24% and 30% differences at week 4, for duration and number of movements, respectively). The magnitudes of these differences were not substantial; means were decreased up to 30%, with coefficients of variation up to 43%. The individual motor activity values generally overlapped with the values observed in the control group. In addition, there were no signs of low arousal or other signs of neurotoxicity during the FOB or clinical evaluations. Females at 1500 ppm also exhibited a slightly lower total duration of movements and total number of ambulatory movements during the week 4 evaluation compared with controls, but these differences were of lower magnitude (means were decreased up to 17%, with coefficients of variation up to 56%) compared with males, and were not statistically significant. Locomotor reductions have been previously attributed to general malaise or systemic toxicity in rats, resulting from varied conditions, without any associated neurochemical or cognitive/behavioural impairments. The lower mean motor activity values at 1500 ppm in males and females during the week 4 evaluation were considered possible effects of the test material but not primary neurotoxic effects, due to the evidence of systemic toxicity at this exposure level, the low magnitudes of difference in motor activity (especially considering the pre-existing differences in baseline values in the case of males), the lack of statistical significance in females, and the lack of other corroborative evidence of neurotoxicity. As such, any differences in motor activity values at week 4 were considered secondary to systemic toxicity, and therefore non-adverse.
There were no statistically significant differences or test material-related effects on duration of movements or number of ambulatory movements during the week 4 evaluation in males or females at 750 ppm and below. Slight differences between males at 750 ppm and control males corresponded to the pre-existing differences observed during the baseline evaluation, and were not statistically significant at either time point.
During the recovery evaluation, there were no statistically significant differences or test material-related effects on any motor activity parameter during the recovery evaluation in males or females at any exposure level.
In conclusion, under the conditions of the study, there were no adverse effects on any neurobehavioral parameter evaluated in males or females at any exposure level.

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no primary test material-related organ weight changes at any of the exposure concentrations tested. All statistically significant organ weight differences from control were considered to be secondary findings associated with body weight decrements or to represent spurious findings unrelated to exposure to the test material.
- Main Phase Organ Weights:
Test material-related decreases in terminal (fasting) body weights were present in males at all exposure concentrations and in females at 750 and 1500 ppm. Males were more severely affected than females, with terminal body weight decreases of 15.4 and 22.9% at 750 and 1500 ppm, respectively, compared to decreases of 7.6 and 12.6%, respectively, in females at these concentrations. A number of statistically significant organ weight changes that were present at these same concentrations were considered secondary to the decrements in body weights based on the absence of correlative microscopic findings and the pattern of change in the respective organ weight parameters. Thus, weight changes in organs typically unaffected by modest changes in body weight (brain, testes, thyroid gland) generally followed a pattern of increased organ-to-body weight ratio, without changes in absolute organ weight or organ-to-brain weight ratio; organs that typically decrease with decreased body weights (adrenal glands, epididymides, heart, kidneys, liver, lungs, spleen, thymus) generally followed a pattern of decreased absolute organ and organ-to-brain weight ratio, with variable effects on organ-to-body weight ratio depending on the organ. Minimal, statistically significant decreases in absolute brain and testes weights (which are typically stable with modest decreases in body weight) in the 1500 ppm males were consider secondary to the more severe decreases in body weight (approximately 23%) observed in this group.(33)
- Recovery Phase Organ Weights:
There were no test material-related changes in terminal body weights or in organ weights in male or female rats following the recovery period.
In males, statistically significant changes were limited to increased thymic weight parameters in the 100 ppm group. These changes were considered spurious as they did not occur in a concentration-related manner. In females, organ-to-body and organ-to-brain weight ratios were higher for thyroid gland at 1500 ppm and for adrenal gland at 750 and 1500 ppm. Liver-to-body weight ratio was also higher in the 1500 ppm females. These differences were not associated with statistically significant differences in the absolute weight of these organs and there were no statistically significant changes in these parameters at the end of the exposure phase of the study. Therefore, these statistically differences were also considered to be spurious findings.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All gross observations were consistent with normal background lesions in rats of this age and, therefore, were not test material-related.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related, minimal to moderate, degeneration/necrosis of the nasal olfactory epithelium lining the dorsal meatus was observed in males at all exposure concentrations and in females at 750 and 1500 ppm in the main phase animals.
The degeneration/necrosis was characterized by multifocal segments of disorganization, degeneration, and rarely individual cell necrosis of the olfactory epithelium in the dorsal meatus primarily at Young’s level II section; it was occasionally noted in level III. This microscopic finding was present in most males at ≥ 100 ppm and all females at ≥ 750 ppm.
In the recovery phase, test substance-related, minimal to moderate, regeneration was the predominate finding in the nasal olfactory epithelium (dorsal meatus) of most males and females at ≥ 750 ppm. It was also present in 1 female at 100 ppm but was considered equivocal as this lesion has been occasionally observed in control animals from other studies. Additional test substance-related changes present in the nasal olfactory epithelium (dorsal meatus) of recovery phase rats, but not observed in the main phase rats, were atrophy and intraepithelial cysts in the olfactory epithelium.
All other microscopic findings were typical of background lesions in rats of this age and strain and, therefore, not test substance-related.

See the details and Tables 13 and 14 provided in the section "Any other information on results incl. tables".

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Urine and plasma fluoride:
- There were no statistically significant increases in plasma fluoride at any concentration in male or female rats following the exposure or recovery periods.
- As expected following exposure to a fluoride-containing compound, urine total fluoride was increased in all exposure groups (not statistically significant in females exposed to 100 ppm). Following the recovery period, group mean urine fluoride values in male and female rats in all exposure groups were similar to the control group.

Details on results:

- NOAEC determination: Clinical pathological adverse effects (decreased blood glucose and ketonuria) related to the treatment were seen at all test material dose levels and microscopic treatment related adverse effects were seen in the nose of male rats in all exposed groups and in the nose of female rats in the 750 and 1500 ppm groups. Consequently no NOAEC could be established. The NOAEC is < 100 ppm*.
*NOAEC conversion from ppm to mg/L:
NOAEC (mg/m3) = NOAEC (ppm) x Molecular weight (g/mol) / 24.5 (L/mol)
Where:
NOAEC (ppm) < 100
Molecular weight (g/mol) = 124.09
24.5 L/mol = gas constant at 25 °C and 1013.25 hPa
NOAEC (mg/m3) < [100] x 124.09 / 24.5
NOAEC (mg/m3) < 506,5
NOAEC (mg/L) < 0.51 mg/L.

- Discussion of the effects observed:
* Local effects: Rather than primary target organ toxicity, the nasal lesions observed represent effects occurring secondary to inhalation 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”.

Key result

Dose descriptor:

NOAEC

Effect level:

< 100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable

Critical effects observed:

no

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 substance atmosphere was considered to be homogeneously 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 (of test material). Rats in the 100, 750, and 1500 ppm target concentration groups were exposed to vapor concentrations of 100 ± 0.63, 750 ± 1.3, and 1500 ± 2.9 ppm of test material (mean ± standard error of the mean), 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) = 100 or 750 or 1500
Molecular weight (g/mol) = 124.09
24.5 L/mol = gas constant at 25 °C and 1013.25 hPa
Test concentrations (mg/m3) = [100 or 750 or 1500] x 124.09 / 24.5
Test concentrations (mg/m3) = 506 or 3799 or 7597
Test concentrations (mg/L) = 0.51 or 3.8 or 7.6

- Chamber Environmental Conditions:
The daily mean chamber temperatures for all exposure groups ranged from 21-24°C and the daily mean relative humidity (RH) ranged from 43-66%. There were individual incidences where RH values deviated from the target range (i.e., 30-70%); however, they do not adversely affect the results or interpretation of this study. There was 60 L/min airflow through the chambers which provided 10 air changes per hour. The oxygen concentration in the chambers was 21%.

 

In conclusion, the chamber 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	20
	1 (Females)	0.0	0.0	0.0	20
	1 (Combined^b)	0.0	0.0	0.0	21
100	2 (Males)	100	0.66	94-110	20
	2 (Females)	100	0.62	94-110	20
	2 (Combined)	100	0.63	94-110	21
750	3 (Males)	750	1.4	740-760	20
	3 (Females)	750	1.2	740-760	20
	3 (Combined)	750	1.3	740-760	21
1500	4 (Males)	1500	2.9	1500	20
	4 (Females)	1500	2.3	1500	20
	4 (Combined)	1500	2.9	1500	21

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

^b Male and female exposure starts were staggered by 1 day. Each sex received 20 exposures.

N: number of values used in calculation.

 

Body weights:

Body weights are detailed in Tables 7 and 8 below.

Abbreviations:

SD: Standard Deviation

N: number of animals evaluated

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

 

Table 7: Mean body weights of male rats

Day(s) relative to start date	Dose	0 ppm	100 ppm	750 ppm	1500 ppm
1	Mean SD N %Diff	274.1 13.8 20 -	275.5 14.4 20 0.5	275.4 17.4 20 0.5	279.1 14.9 20 1.8
3	Mean SD N %Diff	290.2 16.7 20 -	290.3 16.1 20 0.0	281.0 18.4 20 -3.2	262.6 #^1 20.4 20 -9.5
8	Mean SD N %Diff	328.0 20.4 20 -	324.4 18.8 20 -1.1	317.4 22.1 20 -3.2	307.0 #^1 20.1 20 -6.4
10	Mean SD N %Diff	339.6 22.7 20 -	333.9 22.4 20 -1.7	321.9 #^1 22.6 20 -5.2	306.0 #^1 21.4 20 -9.9
14	Mean SD N %Diff	366.0 26.4 20 -	360.4 26.2 20 -1.5	346.6 26.9 20 -5.3	337.8 #^1 22.5 20 -7.7
17	Mean SD N %Diff	383.3 28.0 20 -	373.6 31.1 20 -2.5	354.8 #^1 28.5 20 -7.4	338.8 #^1 23.1 20 -11.6
21	Mean SD N %Diff	405.9 31.3 20 -	393.2 33.0 20 -3.1	371.9 #^1 32.9 20 -8.4	359.5 25.7 20 -11.4
24	Mean SD N %Diff	415.8 34.1 20 -	400.0 35.6 20 -3.8	373.9 #^1 32.4 20 -10.1	356.3 #^1 26.7 20 -14.3
28	Mean SD N %Diff	439.8 36.1 20 -	419.8 35.1 20 -4.5	392.5 #^1 36.7 20 -10.8	377.7 #^1 29.1 20 -14.1
30	Mean SD N %Diff	421.9 26.2 10 -	382.7 #^1 40.7 10 -9.3	357.1 #^1 26.7 10 -15.4	325.4 #^1 26.5 10 -22.9
35	Mean SD N %Diff	435.9 38.8 10 -	441.3 29.9 10 1.2	399.0 47.6 10 -8.5	392.8 #^1 30.2 10 -9.9
42	Mean SD N %Diff	469.0 38.4 10 -	484.0 34.2 10 3.2	442.5 52.7 10 -5.7	437.2 33.8 10 -6.8
49	Mean SD N %Diff	496.8 40.2 10 -	517.7 33.8 10 4.2	478.4 56.0 10 -3.7	478.5 40.2 10 -3.7
53	Mean SD N %Diff	504.4 35.8 10 -	530.1 40.2 10 5.1	497.2 59.1 10 -1.4	498.3 43.6 10 -1.2
56	Mean SD N %Diff	481.1 40.2 10 -	503.4 37.8 10 4.6	468.7 53.6 10 -2.6	467.2 41.4 10 -2.9

¹ #: Test Dunnet 2 Sided p <0.05.

 

Table 8: Mean body weights of female rats

Day(s) relative to start date	Dose	0 ppm	100 ppm	750 ppm	1500 ppm
1	Mean SD N %Diff	200.3 9.8 20 -	201.3 11.3 20 0.5	199.3 12.0 20 -0.5	202.1 12.1 20 0.9
3	Mean SD N %Diff	208.5 10.9 20 -	211.3 10.2 20 1.3	205.5 11.0 20 -1.4	203.9 11.6 20 -2.2
8	Mean SD N %Diff	219.4 12.3 20 -	216.9 11.8 20 -1.1	211.1 11.7 20 -3.7	209.9 #^1 12.1 20 -4.3
10	Mean SD N %Diff	225.5 12.1 20 -	224.5 10.3 20 -0.4	215.0 #^1 12.1 20 -4.7	212.9 #^1 12.4 20 -5.6
14	Mean SD N %Diff	232.8 11.8 20 -	232.3 9.8 20 -0.2	223.2 #^1 12.9 20 -4.1	220.1 #^1 13.1 20 -5.5
17	Mean SD N %Diff	237.5 13.8 20 -	238.9 11.4 20 0.6	224.3 #^1 14.4 20 -5.6	222.2 #^1 12.7 20 -6.4
21	Mean SD N %Diff	245.5 14.7 20 -	242.9 12.2 20 -1.1	227.2 15.6 20 -7.5	224.8 #^1 13.5 20 -8.4
24	Mean SD N %Diff	250.0 13.5 20 -	244.3 13.3 20 -2.3	230.3 #^1 14.6 20 -7.9	224.5 #^1 14.2 20 -10.2
28	Mean SD N %Diff	256.1 13.8 20 -	250.6 14.6 20 -2.1	233.5 #^1 14.3 20 -8.8	228.7 #^1 15.6 20 -10.7
30	Mean SD N %Diff	231.2 7.5 10 -	227.5 13.3 10 -1.6	213.6 #^1 14.0 10 -7.6	202.0 #^1 13.7 10 -12.6
34	Mean SD N %Diff	260.0 18.4 10 -	252.1 12.1 10 -3.0	237.9 #^1 11.1 10 -8.5	239.0 #^1 15.6 10 -8.1
41	Mean SD N %Diff	269.5 18.2 10 -	266.1 13.8 10 -1.3	248.7 #^1 16.1 10 -7.7	253.9 17.4 10 -5.8
48	Mean SD N %Diff	279.9 18.8 10 -	275.3 13.0 10 -1.7	262.9 14.6 10 -6.1	264.5 15.6 10 -5.5
52	Mean SD N %Diff	281.1 19.0 10 -	278.1 14.9 10 -1.1	266.3 14.5 10 -5.3	268.5 18.3 10 -4.5
56	Mean SD N %Diff	262.8 19.9 10 -	258.8 13.4 10 -1.5	249.3 12.7 10 -5.1	249.6 19.1 9 -5.0

¹ #: Test Dunnet 2 Sided p <0.05.

 

Food consumption:

 

Table 9: Mean daily food consumption by male rats

Day(s) relative to start date	Dose	0 ppm	100 ppm	750 ppm	1500 ppm
1 --> 8	Mean SD N %Diff	28.9 1.8 20 -	28.1 2.3 20 -2.8	26.4 #^1 1.9 20 -8.6	23.0 #^1 1.7 20 -20.5
8 --> 14	Mean SD N %Diff	28.2 1.9 20 -	27.6 2.7 20 -1.9	25.5 @^2 1.5 20 -9.7	23.0 @^2 5.0 20 -18.3
14 --> 21	Mean SD N %Diff	28.6 1.9 20 -	28.0 2.8 20 -2.0	25.5 #^1 1.6 20 -10.8	24.5 #^1 1.2 20 -14.5
21 --> 28	Mean SD N %Diff	28.6 2.0 18 -	28.1 2.2 20 -1.7	25.6 #^1 1.8 20 -10.3	24.1 #^1 1.6 20 -15.6
14 --> 28	Mean SD N %Diff	28.7 1.9 20 -	28.1 2.5 20 -2.2	25.6 @^2 1.6 20 -10.8	24.3 @^2 1.4 20 -15.3
1 --> 28	Mean SD N %Diff	28.6 1.7 20 -	28.0 2.5 20 -2.2	25.8 @^2 1.6 20 -9.9	23.7 @^2 0.7 20 -17.3
28 --> 35	Mean SD N %Diff	26.2 2.1 10 -	27.0 1.5 10 3.0	24.1 @^2 1.8 10 -8.0	24.6 1.4 10 -6.2
35 --> 42	Mean SD N %Diff	30.7 1.8 10 -	33.1 #^1 1.6 10 8.0	30.9 1.8 10 0.6	32.0 1.0 10 4.2
42 --> 49	Mean SD N %Diff	31.5 1.8 10 -	33.8 #^1 1.9 10 7.4	31.3 2.0 10 -0.4	33.5 #^1 0.8 10 6.4
49 --> 53	Mean SD N %Diff	28.6 1.3 10 -	31.2 @^2 2.1 10 9.0	30.5 @^2 2.5 10 6.7	31.4 @^2 0.9 10 9.8
28 --> 53	Mean SD N %Diff	29.3 1.7 10 -	31.3 #^1 1.7 10 6.7	29.0 1.9 10 -0.9	30.2 0.9 10 3.2

¹ #: Test Dunnet 2 Sided p <0.05.

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

 

Table 10: Mean daily food consumption by female rats

Day(s) relative to start date	Dose	0 ppm	100 ppm	750 ppm	1500 ppm
1 --> 8	Mean SD N %Diff	18.8 1.3 20 -	18.7 1.4 20 -0.3	16.7 @^1 0.7 20 -11.1	16.7 @^1 0.7 20 -10.9
8 --> 14	Mean SD N %Diff	19.2 1.2 20 -	19.2 0.6 20 0.0	17.2 @^1 0.8 20 -10.5	17.0 @^1 0.9 20 -11.6
14 --> 21	Mean SD N %Diff	20.1 1.2 20 -	19.7 1.0 20 -1.6	18.1 @^1 1.2 20 -9.6	16.7 @^1 1.0 20 -16.6
21 --> 28	Mean SD N %Diff	19.5 1.7 20 -	19.1 0.6 20 -2.3	16.8 @^1 0.7 20 -14.2	16.4 @^1 1.6 20 -15.9
1 --> 28	Mean SD N %Diff	19.4 1.3 20 -	19.2 0.8 20 -1.1	17.2 @^1 0.4 20 -11.4	16.7 @^1 0.9 20 -13.9
28 --> 34	Mean SD N %Diff	17.6 1.9 10 -	18.1 0.7 10 3.1	17.5 1.3 10 -0.1	18.2 2.8 10 3.4
34 --> 41	Mean SD N %Diff	20.9 1.0 10 -	23.4 @^1 3.2 10 12.0	20.9 1.1 10 0.1	23.7 @^1 4.1 10 13.4
41 --> 48	Mean SD N %Diff	20.9 1.4 10 -	22.1 1.4 10 5.8	20.9 1.0 10 0.1	21.4 5.8 10 2.7
48 --> 52	Mean SD N %Diff	18.5 1.7 10 -	23.0 @^1 5.5 8 24.3	18.8 0.8 10 1.8	23.4 @^1 5.9 10 26.2
28 --> 52	Mean SD N %Diff	20.0 1.5 10 -	22.3 2.7 10 11.2	20.1 0.9 10 0.7	22.1 @^1 2.0 10 10.6

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

 

Haematology:

Haematology parameters that showed changes considered to be related to treatment exposure are detailed in Table 11 below.

Table 11: Extract of haematology results for male and female rats

	Days relative to start date	0 ppm	100 ppm	750 ppm	1500 ppm
Males		Mean (SD)
RBC (x 10^6/ µL)	30	7.95 (0.32)	8.03 (0.25)	8.20 (0.32)	8.31 (0.47) #^1
	56	8.19 (0.37)	8.23 (0.33)	8.42 (0.27)	8.06 (0.35)
HGB (g/dL)	30	15.4 (0.5)	15.3 (0.5)	15.5 (0.6)	15.8 (0.7)
	56	15.1 (0.6)	15.1 (0.5)	15.5 (0.5)	15.1 (0.4)
HCT (%)	30	45.2 (1.5)	44.9 (1.3)	45.7 (1.8)	47.1 (2.1) #^1
	56	45.1 (1.7)	45.0 (1.5)	46.7 (1.3)	45.0 (1.7)
MCV (fL)	30	56.8 (1.4)	55.9 (1.0)	55.7 (1.2) #^1	56.7 (1.7)
	56	55.1 (1.1)	54.7 (0.9)	55.5 (1.0)	55.9 (1.0)
MCH (pg)	30	19.4 (0.4)	19.1 (0.5)	18.9 (0.4) #^1	19.0 (0.6) #^1
	56	18.5 (0.4)	18.4 (0.4)	18.4 (0.5)	18.8 (0.5)
MCHC (g/dL)	30	34.1 (0.3)	34.1 (0.4)	33.9 (0.4)	33.4 (0.6) #^1
	56	33.6 (0.2)	33.6 (0.3)	33.2 (0.4)	33.6 (0.7)
RDW (%)	30	11.3 (0.4)	11.1 (0.4)	11.4 (0.3)	11.9 (0.5) #^1
	56	12.5 (0.5)	13.0 (0.5)	13.7 (0.5) #^1	14.3 (0.6) #^1
WBC (x 10^3/µL)	30	11.56 (4.27)	10.60 (2.90)	9.97 (2.30)	8.43 (2.60) #^1
	56	10.46 (3.40)	10.64 (2.22)	11.79 (2.31)	10.40 (1.54)
ARET (x 10^3/µL)	30	195.1 (32.5)	177.2 (35.3)	144.1 (23.7) @^2	134.0 (23.5) @^2
	56	197.4 (36.9)	209.9 (33.7)	241.0 (20.9)	228.0 (11.6) @^2
Females		Mean (SD)
RBC (x 10^6/ µL)	30	7.81 (0.39)	7.80 (0.37)	7.71 (0.34)	7.69 (0.39)
	56	8.08 (0.44)	7.93 (1.34)	8.20 (0.32)	8.09 (0.30)
HGB (g/dL)	30	15.1 (0.7)	14.7 (0.6)	14.1 (0.6) #^1	14.0 (0.7) #^1
	56	15.4 (0.7)	14.7 (2.3)	15.3 (0.6)	15.2 (0.7)
HCT (%)	30	43.4 (1.8)	42.0 (1.9) #^1	40.9 (1.7) #^1	40.8 (2.0) #^1
	56	44.9 (1.8)	43.8 (7.0)	45.2 (1.7)	45.2 (2.0)
MCV (fL)	30	55.7 (1.2)	53.9 (1.1) @^2	53.1 (1.4) @^2	53.1 (0.9) @^2
	56	55.6 (1.1)	55.4 (1.5)	55.1 (1.3)	55.9 (1.9)
MCH (pg)	30	19.4 (0.5)	18.8 (0.4) #^1	18.3 (0.7) #^1	18.2 (0.4) #^1
	56	19.1 (0.4)	18.6 (0.3)	18.7 (0.5)	18.8 (0.7)
MCHC (g/dL)	30	34.8 (0.4)	34.9 (0.5)	34.6 (0.6)	34.2 (0.5) #^1
	56	34.4 (0.4)	33.6 (0.7) #^1	33.9 (0.5)	33.6 (0.3) #^1
RDW (%)	30	10.7 (0.3)	10.5 (0.3)	10.8 (0.3)	11.0 (0.4) #^1
	56	11.5 (0.3)	13.3 (2.4) @^2	13.4 (0.4) @^2	13.6 (0.6) @^2
WBC (x 10^3/µL)	30	8.98 (2.26)	9.49 (2.79)	7.14 (1.75)	7.26 (2.84)
	56	5.84 (1.35)	6.41 (2.22)	6.08 (1.28)	7.83 (5.07)
ARET (x 10^3/µL)	30	157.5 (36.4)	137.5 (27.2)	127.6 (36.4) #^1	108.2 (35.2) #^1
	56	142.6 (42.7)	169.3 (100.5)	149.3 (30.2)	157.6 (23.9)

¹#: Test Dunnet 2 Sided p < 0.05.

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

RBC: red blood cell count.

HGB: haemoglobin.

HCT: hematocrit

MCV: mean corpuscular volume.

MCH: mean corpuscular haemoglobin.

MCHC: mean corpuscular haemoglobin concentration.

RDW: red cell distribution width.

WBC: Mean white blood cell count.

ARET: Absolute counts for reticulocytes.

 

Clinical chemistry:

The blood glucose results are presented in Table 12 below.

Table 12: Mean Glucose (GLUC) in male and female rats

	Days relative to start date	0 ppm	100 ppm	750 ppm	1500 ppm
Males		Mean (SD)
GLUC (mg/dL)	30	123 (17)	79 (9)   #^1	76 (11) #^1	74 (11) #^1
	56	120 (12)	124 (15)	120 (16)	113 (7)
Females		Mean (SD)
GLUC (mg/dL)	30	117 (15)	78 (10) #^1	71 (7) #^1	65 (10) #^1
	56	124 (17)	123 (16)	120 (10)	129 (10)

¹#: Test Dunnet 2 Sided p < 0.05.

 

Microscopic findings:

Table 13: Incidence of Test Substance-Related Findings in Main Phase Rats

Sex	Males				Females
Group (exposure concentration ppm)	1 (0)	2 (100)	3 (750)	4 (1500)	1 (0)	2 (100)	3 (750)	4 (1500)
Nose degeneration/necrosis, olfactory epithelium (dorsal meatus)	0/10	6/10	10/10	8/10	0/10	0/10	10/10	10/10

 

Table 14: Incidence of Test Substance-Related Findings in Recovery Phase Rats

Sex	Males				Females
Group (exposure concentration ppm)	1 (0)	2 (100)	3 (750)	4 (1500)	1 (0)	2 (100)	3 (750)	4 (1500)
Nose: degeneration/necrosis, olfactory epithelium (dorsal meatus)	0/10	0/10	9/10	9/10	0/10	1/10^*	9/10	10/10
Nose: atrophy, olfactory epithelium (dorsal meatus)	0/10	0/10	2/10	4/10	0/10	0/10	1/10	6/10
Nose: cyst, olfactory epithelium (dorsal meatus)	0/10	0/10	6/10	2/10	0/10	0/10	4/10	1/10

* Findings considered equivocal.

 

 

Conclusions:

Under the conditions of this study, the NOAEC of 2,2-Difluoroethyl acetate was undetermined (<100 ppm) for male and female rats, based on clinical pathology findings (decreased blood glucose and ketonuria) observed at all exposure levels, and microscopic changes in the nose of male rats in all exposed groups and female rats in the 750 and 1500 ppm groups.

Executive summary:

The repeated dose toxicity of 2,2-Difluoroethyl acetate was investigated in a 28-day study by inhalation route performed according to OECD test guideline 412 under GLP compliance.

Four groups of male and female Crl:CD(SD) albino rats (20 animals per sex per group) were exposed whole body, 6 hours per day, 5 days a week to vapour concentrations of 0 (air control), 100 ± 0.63, 750 ± 1.3 and 1500 ± 2.9 ppm test material (equivalent to 0, 0.51, 3.8 and 7.6 mg/L test material, respectively) over a 4-week period for a total of 20 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. Neurobehavioral evaluations were performed during acclimation, during week 4 of exposures, and at approximately one month after cessation of exposures in recovery animals. 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 4-week recovery period, blood and urine samples were collected for clinical pathology evaluations, and the animals from the recovery group (10 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.

Male and female rats exposed to 750 and 1500 ppm showed adverse, test material-related body weight reductions which correlated with decreased daily food consumption and changes in body weight gains when compared to the control groups. These effects were reversible following the 4-week recovery period.

There were no adverse effects on any neurobehavioral parameter evaluated in males or females at any exposure level.

Statistically significant decreases in blood glucose and associated increases in urine ketone bodies were observed at all exposure concentrations in both males and females. There were changes in other clinical chemistry and hematology parameters in the 750 and 1500 ppm groups that were considered to be secondary to the decrements in food consumption and body weight observed at these concentrations. These changes were generally minimal and were considered to be non-adverse. All exposure-related clinical pathology changes were reversible following the 4-week recovery period.

There were no primary test material-related organ weight changes at any of the exposure concentrations tested. All organ weight changes were considered secondary to the body weight decreases and none were considered indicative of organ-specific toxicity based on the absence of correlative microscopic findings and the pattern of change in the respective organ weight parameters. All gross observations were consistent with normal background lesions in rats of this age and, therefore, were not test material-related.

Olfactory epithelium in the dorsal meatus of the nose was the only tissue found to have test material-related microscopic changes. Degeneration/necrosis was present after 4 weeks of exposure in most males at all exposure concentrations and all females at 750 and 1500 ppm. This change had a gradient preference for the anterior nasal olfactory epithelium lining the dorsal meatus at level II and sometimes extending into level III. In the recovery phase, three test material-associated tissue changes were present in the nasal olfactory epithelium of most rats at 750 and 1500 ppm. These changes consisted of regeneration, atrophy and intraepithelial cysts. Test material-related microscopic changes in the nose of main and recovery phase rats were considered adverse.

Under the conditions of this study, the NOAEC of 2,2-Difluoroethyl acetate was undetermined (< 100 ppm) for male and female rats based on clinical pathology findings (decreased blood glucose and ketonuria) observed at all exposure levels and microscopic changes in the nose of male rats in all exposed groups and female rats in the 750 and 1500 ppm groups.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

A GPL-compliant study performed according to OECD test guideline 412 is available. It is considered as fully reliable (Klimisch score of 1) and the result is retained as key data.

Justification for classification or non-classification

Discu​ssion of t​he effects​ observed:​
         ​ * Local e​ffects: Ra​ther than ​primary ta​rget organ​ toxicity,​ the nasal​ lesions observed in 28-day studies by oral and inhalation routes likely represent effects occurring secondary to regurgitation or inhalation of the tes​t substanc​e which ap​peared as ​directly i​rritating ​to the nas​al epithel​ium upon c​ontact, at​ the conce​ntrations ​applied. T​he fact th​at 2,2-Dif​luoroethyl​ acetate w​as conclud​ed to be n​ot irritat​ing to ski​n and not ​irritating​ to eyes i​n GLP-comp​liant stud​ies accord​ing to OEC​D test gui​delines 40​4 and 405,​ respectiv​ely, is no​t contradi​ctory and ​may reflec​t the fact​ that the ​nasal epit​helium is ​more sensi​tive upon ​contact wi​th 2,2-Dif​luoroethyl​ acetate.
​          ​* Systemic​ effects: ​Albeit tha​t a series​ of change​s was obse​rved, they​ did not t​ranslate i​n clear, o​rgan-speci​fic advers​e observat​ions, nor ​in adverse​ findings ​for apical​ endpoints​. Moreover​, several ​changes ob​served sho​wed good r​eversibili​ty in reco​very group​s.
---> O​n the abov​e basis, a​ classific​ation as S​TOT RE in accordance with criteria of the CLP Regulation was​ not consi​dered warr​anted and ​no target ​organ/syst​em has been ide​ntified.