2,2-Difluoroethyl Acetate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Screening for reproductive / developmental toxicity

Data waiving (study scientifically not necessary / other information available):

The study does not need to be conducted because a pre-natal developmental toxicity study is available on 2,2-Difluoroethyl acetate.

 

Extended one-generation reproductive toxicity

Data waiving (study scientifically not necessary / other information available):

The extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because a pre-natal developmental toxicity study is available

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Additional information

The three available repeated dose toxicity studies in rats (28-day studies by oral and inhalation routes and 90-day study by inhalation route, see in IUCLID sections 7.5.1 and 7.5.2) covered investigations of reproductive organs that were helpful to screen potential effects on fertility. During these three studies, weight measurements, gross and microscopic examinations were performed at the time of necropsy for the following reproductive organs: ovaries (including oviducts), uterus (including cervix), mammary glands, vagina, testes, epididymides, prostate, seminal vesicles with coagulating glands (including fluids).
During the 28-day repeated dose toxicity study by oral route, there were no effects on organ weight, no gross observations and no microscopic findings considered to be test substance-related in any of the investigated reproductive organs at any dose tested (100, 300, and 1000 mg/kg/day of test material).
During the 28-day repeated dose toxicity study by inhalation route, organ weight changes were observed in testes (decrease of absolute weight at 1500 ppm and increase of testes-to-body weight ratio at 750 and 1500 ppm) and epididymides (decrease of absolute weight and epididymides-to-brain weight ratio at 1500 ppm). These changes were considered secondary to the decrements in male body weights at these same concentrations based on the absence of correlative microscopic findings. Indeed, there were no microscopic findings, as well as no gross observations, considered to be test material-related in any of the investigated reproductive organs. There were no test material-related organ weight change in other reproductive organs in males and females and no test material-related changes in any reproductive organ weights in males and females following the recovery period.
During the 90-day repeated dose toxicity study by inhalation route, there were no effects on organ weight, no gross observations and no microscopic findings considered to be test material-related in any of the investigated reproductive organs at any dose tested (1, 10 and 100 ppm of test material).
In conclusion the findings from the repeated-dose toxicity studies do not raise any alert regarding potential morphological and histological changes in reproductive organs that could be indicative of fertility impairment.

Effects on developmental toxicity

Description of key information

A lower maternal body weight gain at 500 ppm was considered to be related to exposure to 2,2-Difluoroethyl acetate and to be adverse. A concomitant lower mean fetal weight at 500 ppm was observed and was found the only fetal finding that was considered to be adverse, yet only in presence of clear maternal toxicity. Therefore, the NOAEC of 2,2-Difluoroethyl acetate for both maternal and developmental toxicity was 100 ppm.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

FROM 7 OCTOBER 2015 TO 17 JUNE 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

yes

Remarks:

Shorter acclimation period than the 5 days required in the guideline but it had no impact on the study as animals were monitored for weigh gain and freedom from adverse clinical signs prior to initiation of exposure.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD).

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories International, Inc., Raleigh, North Carolina.
- Strain: the rat was selected for this study because it is a preferred species for developmental toxicity testing as recommended by test guidelines. The Crl:CD(SD) strain was chosen because extensive background information is available from the literature, the supplier, and previous studies conducted at the testing facility. This strain is also considered suitable relative to hardiness and incidence of spontaneous disease.
- Sex: Female (nulliparous, time-mated, GD 0 = day mating confirmed).
- Age at study initiation: approximately 10 weeks.
- Weight at study initiation: approximately 201-225 grams.
- Fasting period before study: not specified.
- Housing: except during exposure, animals were housed in groups (2-3 per cage) in solid-bottom caging with bedding and Nestlets™ as enrichment.
- Diet: except during inhalation exposures, all animals were fed PMI® Nutrition International, LLC Certified Rodent LabDiet® ad libitum.
- Water: except during inhalation exposures, all animals were provided tap water ad libitum.
- Acclimation period: at least 1 day. The animals were released from quarantine by the animal resources supervisor or designee based on acceptable body weights and freedom from adverse clinical signs. The use of vendor supplied time-mated females combined with the breeding lot staggering necessary for scheduling of cesarean sections necessitated a shortened acclimation period relative to the guideline requirement of at least 5 days. There was no adverse impact on the study as a result of this deviation since the animals were monitored for weight gain and freedom from adverse clinical signs prior to initiation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26°C.
- Humidity (%): 30%-70%.
- Photoperiod (hrs dark / hrs light): animal rooms were artificially illuminated (fluorescent light) on an approximate 12 hour light/dark cycle.
Excursions outside of these ranges were of insufficient magnitude and/or duration to have adversely affected the validity of the study.

IN-LIFE DATES: From 11 October 2015 to 30 October 2015.

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on 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 liters. 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 vapor throughout the exposure chambers. 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 modules 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 the target 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 the 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 vapor 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.
- 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 vapor concentration of the test material was determined by gas chromatography (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.
- Samples taken from breathing zone: yes. Prior to the start of the exposure phase, the distribution of the test material vapor was determined in the 500 ppm exposure chamber using GC and the analytical method described above. Vapor 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. The vapor concentration of the test material in the 500 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.
- 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”.

Details on mating procedure:

No details on mating procedure were provided in the study report as the females were purchased already time-mated from the rat supplier.

Duration of treatment / exposure:

From gestation day (GD) 6 to 20. The exposure period was defined as the period between initiation of the first exposure and completion of the last exposure.

Frequency of treatment:

Each group of animals was exposed for 6 hours/day, 7 days/week during GD 6-20.

Duration of test:

Each group of dams was exposed daily from around the time of implantation to the end of gestation, i.e. during GD (gestation days) 6-20 and the dams were euthanized on GD 21.

Dose / conc.:

500 ppm

Remarks:

Group 5
Equivalent to 2.5 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) = 500
Molecular weight (g/mol) = 124.09
24.5 L/mol = gas constant at 25 °C and 1013.25 hPa
Test concentrations (mg/m3) = 500 x 124.09 / 24.5
Test concentrations (mg/m3) = 2532
Test concentrations (mg/L) = 2.5

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

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.:

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.:

0 ppm

Remarks:

Group 1 (control).

No. of animals per sex per dose:

22 time mated rat females per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: exposure concentration levels selected for this study were 0, 1, 10, 100 and 500 ppm. These levels were selected in consultation with the study sponsor based on results from a previously conducted 4-week inhalation study (see "Rep. dose Inh. tox 28d KS V1 2015SHEU" in IUCLID section 7.5.2), rats (20/sex/group) in which rats were exposed to 100, 750, or 1500 ppm of test material for 5 days/week (6 hrs/day). Clinical pathology findings (decreased blood glucose and ketonuria) were observed at all exposure concentrations in both male and female rats. Microscopic changes in the nose were observed in male rats at all exposure concentrations and in female rats exposed to 750 and 1500 ppm.
- Animal assignment: Before test substance exposures began, animals were randomly assigned to control or experimental groups using a computerized randomization procedure designed to produce a homogeneous distribution of body weights across groups within each breeding lot. The randomization resulted in a distribution in which the weight variation of selected animals did not exceed ± 20% of the mean weight for each sex..
- Fasting period before blood sampling: no (but no thyroid hormones measurements were done).
- Time of day for (rat) dam blood sampling: not specified.

Maternal examinations:

See also the Figure 2 in the section “Overall remarks, attachments”, detailing the schedule/frequency of In-life observations.

CAGE SIDE OBSERVATIONS: not specified.

MORTALITY / MORIBUNDITY: Yes.
- Time schedule: at least once daily during quarantine/pretest period, twice daily (AM and PM) during the testing period.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: on GD 4 during quarantine/pretest period, on three occasion during the testing period:
1) Twice daily on GD 6-20 (pre- and post-exposure); Clinical signs of fur/skin strains associated with restraint, urination, and defecation during exposure were not recorded for the post-exposure observations.
2) Three times during exposure; the observation(s) were only performed if the animals were visible to the observer through the chamber atmosphere. Observation(s) were recorded for the animals collectively within a chamber, since individual animal identification was not visible to the observer.
3) Once on GD 21.

BODY WEIGHT: Yes.
- Time schedule for examinations: on GD 4 (quarantine/pretest period) and daily on GD 6-21 (testing period).

FOOD CONSUMPTION: Yes.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: food consumption was noted on GD 4 (quarantine/pretest period) and on GD 6, 8, 10, 12, 14, 16, 18, 20, and 21 (testing period). During the testing period food spillage was not recorded. However, excess spillage was documented in the study records. Food consumption was measured as food consumed per cage, which was then divided by the number of animals in each cage for individual food consumption values.

WATER CONSUMPTION: No.

POST-MORTEM EXAMINATIONS: Yes.
- Sacrifice on gestation day 21.
- Organs examined: a gross external and a visceral examination was performed immediately after euthanasia. Gross lesions were retained for possible examination; lesions for which a microscopic diagnosis would not be additive (e.g., osteoarthritis, pododermatitis, tail chronic dermatitis, calculus, and deformities of the teeth, toe, tail, or ear pinnae) were not saved. Microscopic examination of gross lesions was not performed since it was not considered necessary to meet the objective of the study.

OTHER:
Alerting behavior was observed before exposure, 3 times during exposure and after each exposure. The observation(s) were only performed if the animals were visible to the observer through the chamber atmosphere. Observation(s) were recorded for the animals collectively within a chamber, since individual animal identification was not visible to the observer. 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.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: yes.
Examinations included:
- Gravid uterus weight: yes. The gravid uterus of each dam having at least one viable fetus was weighed to permit calculation of maternal body weight adjusted to exclude the products of conception.
- Number of corpora lutea: yes. The corpora lutea count for each ovary of females with visible implantation sites was recorded.
- Number of implantations, early and late resorptions: yes. For each female with visible implantation sites, the types of implantations (live and dead fetuses, early and late resorptions) and their relative positions in the uterus were recorded. The types of implantations were classified as follows:
* live fetus: fully formed and responds to stimuli.
* dead fetus: fully formed with little or no evidence of maceration.
* late resorption: identifiable structures (i.e. digital rays).
* early resorption: no visible fetal structures.
Uteri with no visible implantation sites were placed in a 10% aqueous solution of ammonium sulfide to detect very early resorptions.

Blood sampling:

Females were euthanized by isoflurane inhalation and exsanguination. Blood samples were collected from the abdominal vena cava, placed in serum separator tubes and kept on ice until serum was prepared. Serum was stored frozen for glucose concentration analysis.

Fetal examinations:

Those fetuses classified as dead were examined externally, viscerally and skeletally to the extent possible. Data for these fetuses were excluded from all calculations performed for live fetuses.
- External examinations: yes. All fetuses classified as live were examined externally for alterations. The external examination included inspection of all externally observable structures as well as the fetal palate. External sex was recorded for each live fetus.
- Soft tissue examinations: yes. For each litter, approximately half of the live fetuses were examined for visceral alterations by fresh tissue dissection. The visceral examination included inspection of all viscera for presence, size, shape, color, location and texture. In addition, all live fetuses with malformations visible at external examination were examined for soft tissue alterations; decapitation of these fetuses for head examination was performed at the discretion of the study director or designee.
- Skeletal examinations: yes. All fetuses classified as live were fixed in alcohol after sacrifice, processed and the skeletons stained with alizarin-red. The skeletal bodies of all the fetuses and the skulls of half the fetuses (fetuses that were not designated for head examination) were examined for alterations. The skeletal examination included inspection of the individual bones and cartilage of the axial and appendicular skeleton for overall structure, relationship, and stage of ossification.
- Head examinations: Yes. The frozen heads of decapitated fetuses (fetuses that were decapitated prior to visceral examination; approximately half of the fetuses) were examined by a serial sectioning technique. The fetal head examination included inspection of the eyes, nasal cavities and internal brain structures.
- Anogenital distance of all live rodent pups: no.

Statistics:

See Table 1 in the section "Any other information on materials and methods incl. tables".

Indices:

Not specified.

Historical control data:

Historical control data provided to allow comparison with concurrent controls:
- Historical control mean fetal weights.
- Historical control incidence of fetal short thoracic and cervical ribs.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test material-related clinical observations at any concentration tested; the observations that were recorded were unremarkable and occurred infrequently.

Mortality:

no mortality observed

Description (incidence):

There was no test material-related mortality at any concentration tested; all animals on study survived until scheduled euthanasia.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were test material-related, adverse effects observed on body weight gain at 500 ppm; mean weight gain was 46% lower than controls from days 6 to 8 of gestation. Mean weight gain was lower than control for all subsequent study intervals (except for the gestation day 8-10 interval), which ranged from 7% to 25% lower than control. Overall mean weight gain from gestation days 6 to 21 was 14% lower than control. Mean adjusted (minus products of conception) body weight gain was 23% lower than control (not statistically significant) at 500 ppm.
There were no test material-related effects on body weight parameters at 100 ppm or lower; mean body weights and weight gains were generally comparable to control group values at these concentrations.
See also Table 3 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):

Test material-related reductions in maternal food consumption were observed at 500 ppm; mean food consumption was 11% lower than controls from days 6 to 8 of gestation. This reduction was not considered to be adverse because it was transient and had no impact on cumulative food consumption or on mean body weights.
There were no test material-related effects on food consumption at 100 ppm or lower; the data were generally comparable to the control group data at these concentrations.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no exposure-related changes in serum glucose in pregnant animals. Decreased serum glucose was observed in previous studies in rats. However, in those studies, rats were fasted prior to blood collection, whereas rats in the current study were not fasted. Thus, it could not be determined whether the differences in glucose response between previous studies and the current study are due to differences in fasted state.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test material-related maternal gross postmortem observations at any exposure concentration tested. The observations that were recorded were unremarkable, infrequent, and not dose dependent.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEC

Effect level:

100 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The reduction in mean fetal weight (10% lower than the control group) at 500 ppm was considered to be adverse based on the magnitude of the change. Mean fetal weight at this concentration was 5.50 g which was below the test facility historical control data range of 5.56 to 6.10 g. Individual litter means at this concentration generally fell below the range of litter means in the concurrent control group.
In contrast, the fetal weight reduction at 100 ppm (3% lower than the control group) was not considered to be adverse since the mean fetal weight (5.87 g) at this concentration was well within the range reported for test facility historical control data. In addition, almost all of the individual litter means for the 100 ppm group fell within the range of means for the concurrent control group.
There was no test material-related effect on mean fetal weight at 1 or 10 ppm.

See also Table 4 in the section "Any other information on results incl. tables" and Figure 3 attached in the section Overall remarks, attachments.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

There were no test-substance related increases in malformations.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Test material related skeletal variations included short thoracic ribs at 500 ppm and short cervical ribs at 100 ppm and 500 ppm. Short thoracic ribs (short ribs at the 13th thoracic vertebra) were observed in 20 fetuses and 8 litters in the 500 ppm group compared to none in control. For comparison, the test facility historical control data range was 0 to 4 (fetuses) and 0 to 3 (litters). The incidence of short cervical ribs in the 500 ppm group was 19/11 (fetuses/litter) and 9/4 in the 100 ppm group, compared to none in the control. For comparison, the test facility historical control data range was 0 to 9 (fetuses) and 0 to 3 (litters). Both short thoracic ribs and short cervical ribs are classified as fetal variation and as such were not considered to be adverse findings.

Visceral malformations:

no effects observed

Key result

Dose descriptor:

NOAEC

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Remarks on result:

other: Concomittant with a lower maternal body weight gain at 500 ppm

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

500 ppm

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

presumably yes

Concentration verifications

 

- 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 of test material (mean ± standard error of the mean). Rats in the 1, 10, 100, and 500 ppm target concentration groups were exposed to vapor concentrations of 1.0 ± 0.0077, 10 ± 0.073, 100 ± 1.0, and 500 ± 3.6 ppm of 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, 10, 100 and 500.
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 or 500] x 124.09 / 24.5
Test concentrations (mg/m3) = 5 or 51 or 506 or 2532
Test concentrations (mg/L) = 0.005 or 0.051 or 0.51 or 2.5.

 

- 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 46 to 63%. 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 130-132 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 2: chamber concentrations of test material

Target concentration (ppm)	Group	Measured concentration (ppm) ^a
		Mean	S.E.M	Range	n
0	1	0.0	0.0	0.0	3
1	2	1.0	0.0077	0.97 – 1.1	19
10	3	10	0.073	9.5 – 11	19
100	4	102	1.0	88 – 111	19
500	5	501	3.6	466 - 539	19

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

 

Body weight changes

Table 3: mean maternal body weight changes

Female rats Days relative to mating	0 ppm	1 ppm	10 ppm	100 ppm	500 ppm
	Mean (Standard Deviation)
6 --> 8	10.0 (2.5)	10.5 (5.0)	9.3 (3.7)	8.2 (6.0)	5.4 (4.4) @@@^1
8 --> 10	12.5 (3.2)	14.1 (3.9)	13.1 (3.7)	13.7 (6.9)	13.8 (3.5)
10 --> 12	14.6 (3.5)	14.4 (4.7)	15.3 (4.5)	14.8 (4.2)	13.1 (3.8)
12 --> 14	13.4 (4.3)	15.0 (3.8)	14.4 (3.4)	13.8 (4.1)	11.8 (5.2)
14 --> 16	20.4 (3.3)	19.8 (4.6)	18.1 (4.1)	19.6 (3.8)	17.4 (4.5)
16 --> 18	30.8 (4.5)	31.4 (5.0)	30.0 (4.8)	30.1 (5.9)	25.7 (6.9) @^2
18 --> 20	31.0 (4.9)	34.5 (5.2)	32.9 (5.4)	33.1 (4.9)	28.7 (7.2)
20 --> 21	16.2 (7.0)	14.3 (6.5)	14.9 (6.8)	14.2 (5.5)	12.2 (6.7)
6 --> 21	148.8 (19.5)	153.9 (23.1)	148.1 (20.9)	147.6 (23.0)	127.9 (24.4) #^3
Adjusted	45.69 (15.88)	53.22 (16.40)	48.08 (16.48)	44.62 (15.91)	35.30 (14.77)

¹@@@: Test Dunnet Non Parametric 2 Sided p < 0.001.

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

³#: Test Dunnet 2 Sided p < 0.05.

 

Fetal body weight changes

Table 4: mean fetal weight

Dose group	0 ppm	1 ppm	10 ppm	100 ppm	500 ppm
	Mean (Standard Deviation)
Mean fetal weight (males) (g)	6.21 (0.26)	6.27 (0.32)	6.12 (0.36)	6.00 (0.28)	5.59 (0.48)
Mean fetal weight (females) (g)	5.95 (0.24)	5.96 (0.37)	5.75 (0.25)	5.71 (0.26)	5.29 (0.34)
Mean fetal weight (both sexes) (g)	6.07 (0.24)	6.13 (0.33)	5.92 (0.29)	5.87 (0.26) @^1	5.50 (0.49) @@^2

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

²@@ Test Dunnet Non parametric 2 Sided p < 0.001.

 

Conclusions:

Under the conditions of this study, the lower maternal body weight gain at 500 ppm was considered to be adverse. A concomitant lower mean fetal weight at 500 ppm was observed and was found the only fetal finding that was considered to be adverse, yet only in presence of clear maternal toxicity. Therefore, the NOAEC of 2,2-Difluoroethyl acetate for both maternal and developmental toxicity was 100 ppm.

Executive summary:

The prenatal developmental toxicity of 2,2-Difluoroethyl acetate was investigated in a study by inhalation performed according to OECD test guideline 414 under GLP compliance.

Groups of 22 time-mated female Crl:CD(SD) rats were exposed whole body, 6 hours per day, from gestation day (GD) 6 to GD 20 to vapour concentrations of 0, 1 ± 0.0077, 10 ± 0.073, 100 ± 1.0 and 500 ± 3.6 ppm test material (equivalent to 0, 0.005, 0.051, 0.51 and 2.5 mg/L test material, respectively). Test atmospheres were generated by flash evaporation of test material in air. During the in-life phase of the study, the following endpoints were investigated: mortality/moribundity (twice daily), clinical observations (three times during exposure, twice daily out of exposure from GD 6 to 20 and once daily on GD 21), behavior in response to a standardized auditory stimulus (before, 3 times during, and after each exposure), body weight (daily from GD 6 to 21) and food consumption (every other day from GD 6 to 20 and on GD 21). On GD 21, the dams were euthanized and each underwent a gross external and internal examination. Blood was collected from the vena cava for serum glucose concentration analysis. The gravid uteri were removed via caesarean section and weighed. The uterine contents were examined, counted and removed; counts were recorded for corpora lutea and implantations. Implantations were classified as either early or late resorptions, or live or dead fetuses. Live fetuses were individually identified. Fetal body weight and sex was recorded and the fetuses were subsequently examined for external, visceral, head, and skeletal alterations.

There was no test material-related effect in dams at any concentration tested for the following endpoints: mortality, clinical observations, behavior in response to a standardized auditory stimulus, gross post-mortem observations and serum glucose. A test material-related and adverse reduction in mean maternal body weight gain occurred at 500 ppm. This effect was more or less important depending on gestation day (- 7 % to - 46 % as compared to controls) and was found to be statistically significant from gestation day 6 to 8 (- 46 %), from gestation day 16 to 18 (- 17 %) and for the overall exposure period (from day 6 to 21: - 14 %). Adjusted (i.e. without products of conception) mean body weight gain was also reduced at 500 ppm, but this was not found to be statistically significant (- 23 % for the overall exposure period as compared to controls). When looking at mean maternal body weight itself, a reduction was also observed at 500 ppm, but this was never found to be statistically significant (- 0.4 % to - 3.8 % as compared to controls depending on gestation days). Finally, a test material-related reduction in maternal food consumption was observed at 500 ppm; this effect was only significant from gestation days 6 to 8 with a 11 % reduction as compared to controls. During the other study intervals, maternal food consumption was also reduced (from 0.9 to 4.6 % lower than in controls), but this was not statistically significant.

A test material-related and adverse reduction in mean fetal weight occurred at 500 ppm (10 % lower than control). This observed reduction in mean fetal weight was concomitant to reductions in maternal body weight gain and adjusted body weight gain. This effect was thus considered as indicative of maternal toxicity, and not developmental toxicity. Otherwise, there were no test material-related effects on reproductive outcome and quantitative litter data. The mean numbers of implantation sites, resorptions, live fetuses, as well as mean litter sex ratio values were comparable across all groups tested. There were no test material-related increases in malformations. Test material-related skeletal variations included short thoracic ribs at 500 ppm and short cervical ribs at 100 ppm and 500 ppm. Both short thoracic ribs and short cervical ribs are classified as fetal variation and as such are not considered to be adverse findings.

Under the conditions of this study, the lower maternal body weight gain at 500 ppm was considered to be adverse. A concomitant lower mean fetal weight at 500 ppm was observed and was found the only fetal finding that was considered to be adverse, yet only in presence of clear maternal toxicity. Therefore, the NOAEC of 2,2-Difluoroethyl acetate for both maternal and developmental toxicity was 100 ppm.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

100 ppm

Study duration:

subacute

Species:

rat

Quality of whole database:

A GPL-compliant study performed according to OECD test guideline 414 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

A prenatal developmental toxicity study is available and revealed as the only fetal finding a lower mean fetal weight at 500 ppm which is concomitant to maternal toxicity (i.e. lower maternal body weight gain at the same concentration), and which is thus not indicative of developmental toxicity. No dedicated fertility study is available, but the findings from the available repeated-dose toxicity studies do not raise any alert regarding potential morphological and histological changes in reproductive organs that could be indicative of fertility impairment.

---> O​n the abov​e basis, a​ classific​ation as toxic to reproduction in accordance with criteria of the CLP Regulation was​ not consi​dered needed.