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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2012 - March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Justification for study design:
Details on study design:
- Dose selection rationale: In a previous acute inhalation study the inhalation LC50 in male and female rats was greater than 502000 ppm. Based on these results, exposure concentrations of 0, 2500, 10000, and 50000 ppm were selected for the current study.

Test material

Constituent 1
Chemical structure
Reference substance name:
Perfluoroethane
EC Number:
200-939-8
EC Name:
Perfluoroethane
Cas Number:
76-16-4
Molecular formula:
C2F6
IUPAC Name:
hexafluoroethane
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material : perfluoroethane (CAS 76-16-4) named H-30188 in the study
- Lot/batch number of test material: 1011MT0161 ; 1110CW0033 and 0911MT0144
- Purity: 99.99% according to Certificate of Analysis (COA - appendix A of the study)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under storage conditions: Stable based on analyses conducted by the sponsor. The test substance appeared to be stable under the storage conditions.
- Stability under test conditions: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed.
- Solubility and stability of the test substance in the dispersant/vehicle/test medium: Test substance is a gas miscible with the houseline air used in the study.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No treatment prior to testing
- Preliminary purification step: No

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat was selected for this study because it is a preferred species for reproductive toxicity testing and recommended by regulatory guidelines. The Crl:CD(SD) strain was chosen because extensive background data are available from the literature, the supplier, and previous studies at DuPont Haskell. This strain is also considered suitable relative to longevity, hardiness, and incidence of spontaneous disease.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories International, Inc., Kingston, New York
- Age at Arrival: Approximately 51 days
- Age at Start of Inhalation: Approximately 70 days
- Weight on day after arrival: 198-240 grams (males); 148-183 grams (females)
- Weight at start of inhalation: 317-445 grams (males) and 201-269 grams (females)
- Assigned to test groups randomly: Yes, under following basis: Computerized, stratified randomization so that no statistically significant group mean body weight differences occur within a sex.
- Housing: Individually in solid bottom caging with bedding, and enrichment as appropriate; sexes on separate racks except during cohabitation (for reproduction group)
- Cohabitation: Mating pairs (females placed in males’ cages) on a 1:1 basis in solid bottom caging with bedding and enrichment. Fourteen days after the first day of cohabitation, females without evidence of copulation were housed singly.
- Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum except when fasted.
- Water: Tap water ad libitum
- Acclimation period: approximately 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC (68-79ºF)
- Humidity (%): Relative humidity of 30-70%
- Photoperiod (hrs dark / hrs light): Artificial (fluorescent light) on an approximate 12-hour light/dark cycle (except during eye examinations and motor activity).

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air and supplemental oxygen
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:
For exposure numbers 1 to 10, all exposure chambers were constructed of stainless steel and glass (NYU style) with a nominal internal volume of 350 L. For exposure numbers 11 to 71, control animals were exposed to air in the same chamber while H-30188 (test material) test exposures were performed in stainless steel and glass (NYU style) chamber with a nominal internal volume of 150 L, due to technical issues regarding exhaust emissions. A stainless steel baffle below the chamber turret inside the chambers promoted uniform chamber distribution of the test atmosphere.

- Method of holding animals in test chamber:
Exposure in a chamber. Whole body exposure.

- Exposure mode :
During exposure, animals were individually placed in stainless steel, wire-mesh modules and exposed whole-body inside the exposure chamber, when animals were housed as mating pairs and exposed in the same manner.

- Atmosphere Generation:
H-30188 (test material) vapor and supplemental oxygen were metered into a 1-liter 3-neck mixing flask by Brooks model 5850 E or 5850 I mass flow controllers. The mixture left the mixing flask and entered the glass transfer tube where chamber air supply was added to the mixture by Brooks model 5850 E or 5851 I mass flow controllers. The gas mixture then entered the top of the exposure chamber through a turret. The air-control atmosphere was similarly generated in a different room without the H-30188 vapor.
Test atmospheres were exhausted from the bottom of each chamber through MSA filters using vacuum pumps and discharged into the fume hood.

- Chamber Distribution of Test Substance:
Uniform distribution of the test substance in the 150-L and 350-L exposure chambers was determined.


TEST ATMOSPHERE
- Brief description of analytical method used: During each exposure, the vapor concentration of H-30188 was determined by gas chromatography at least once an hour from the test chambers.
- Samples taken from breathing zone: yes


VEHICLE
- Justification for use and choice of vehicle: The test material is a gas. The mode of exposition is via inhalation.
- Composition of vehicle: Chamber atmospheres were generated by dilution of the tested substance in houseline air and oxygen.


DETAILS ON EXPOSURE DURATION
The animals were exposed approximately 2 weeks for 6 hours/day, 5 days/week. Due to technical issue, exposures were suspended for 2 weeks and resumed on test day 29. Following resumption of exposures, animals were exposed 6 hours/day, 5 days/week for approximately a 2-week premating period, followed by a cohabitation period of approximately 2 weeks (6 hours/day, 7 days/week). This exposure schedule continued during a gestation period of approximately 3 weeks for females with evidence of mating. Gestation dams were not exposed after gestation day 19 or during the approximately 4-day lactation period. Females without evidence of mating continued to be exposed for 24 days (6 hours/day, 7 days/week) after the end of the cohabitation period. Exposures for parental males continued until the day prior to euthanasia on test days 77-78.
Details on mating procedure:
Cohabitation was started on test day 49 and lasted until evidence of copulation was observed or the cohabitation period (2 weeks) had ended, at which time the mating pairs were separated.
Each female was continuously housed on a 1:1 basis with a randomly selected, nonsibling male of the same exposure concentration level.
Analytical verification of doses or concentrations:
yes
Remarks:
gas chromatography
Duration of treatment / exposure:
Males: 78 days
Females: 63 days (with evidence of mating) or 66 days (without evidence of mating)
Frequency of treatment:
6 hours/day, 5 days/week (during the pre-mating period) and 7 days/week (at the commencement of cohabitation)
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Dose / conc.:
2 500 ppm
Dose / conc.:
10 000 ppm
Dose / conc.:
50 000 ppm
No. of animals per sex per dose:
10 rats/sex/dose
Control animals:
yes
Details on study design:
- Dose selection rationale: In a previous acute inhalation study the inhalation LC50 in male and female rats was greater than 502,000 ppm. Based on these results, exposure concentrations of 0, 2500, 10,000, and 50,000 ppm were selected for the current study.
- Quarantine Period: 7 days
- selection criteria: Animals with adequate body weight gain and free of any clinical signs of disease or injury. Animals that did not meet these criteria were eliminated from consideration for use in the study. Weight variation of individual rats did not exceed ± 20% of the mean weight for each sex.
- Rationale for animal assignment: Randomization. Computerized, stratified randomization so that no statistically significant group mean body weight differences occur within a sex.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
- Daily animal health observations and general clinical observations. Detailed clinical observations (evaluation of fur, skin, eyes etc.) were recorded once during pretest and at least weekly thereafter.
- Regular measurement of body weight (during premating: twice weekly for the first 2 weeks and then weekly; during cohabitation: weekly; during gestation: on 0, 7, 14 and 19 gestation day; during lactation: on 0 and 4 lactation day; during post-cohabitation: weekly and at terminal sacrifice) and food consumption & food efficiency (during premating: weekly; during cohabitation: none; during gestation: on 0, 7, 14 and 19 gestation day; during lactation: on 0 and 4 lactation day; during post-cohabitation: none)
- Neurobehavioral evaluation consisting of abbreviated functional observational battery (FOB) assessments and motor activity (MA) was conducted throughout the study (at baseline, on test days 44 and 45)
- Clinical pathology evaluation (hematology and coagulation, clinical chemistry, urinalysis) was performed on test day 48.
Oestrous cyclicity (parental animals):
During cohabitation (test day 49), daily examination of each female for an intravaginal copulation plug in the vaginal lavage sample
Sperm parameters (parental animals):
During cohabitation (test day 49), daily examination of each female for sperm in the vaginal lavage sample
Litter observations:
During gestation (gestation day 20), female rats were observed at least twice daily for signs of delivery and offspring.
During lactation (lactation day 0 and 4), offspring were individually handled and examined for abnormal behavior and appearance; any dead or abnormal pups were recorded.
If litters died prior to day 4 of lactation, the dam was sacrificed. If the dam died prior to day 4 of lactation, the litter was examined externally, sacrificed and discarded.
Postmortem examinations (parental animals):
The necropsy included examination of the external surface, all orifices and the cranial, thoracic, abdominal and pelvic cavities (including viscera).
The uteri from all reproduction subset females were examined for the presence and number of implantation sites and the ovaries were examined for the number of corpora lutea.
The following organs were weighed fresh at necropsy from all adult animals: adrenals, brain, heart, kidneys, liver, lungs, spleen, thymus, ovaries, uterus, testes, epididymides, prostate, seminal vesicles, accessory sex organs.
Microscopic evaluation of all tissues collected from adult rats (liver, lungs, trachea, thyroid gland etc.) as well as of the reproductive organs from all male and female P1 rats suspected of impaired reproductive performance was done.
Postmortem examinations (offspring):
Pups found dead or euthanized in moribund condition underwent a gross examination to the extent possible.
Statistics:
Yes
Reproductive indices:
mating index (%), fertility index (%), gestation index (%), pre-implantation loss (%), post-implantation loss (%)
Offspring viability indices:
Pups born alive (%), viability index (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No test substance-related clinical signs of toxicity were observed in males or females at any exposure concentration during the premating, gestation, or lactation periods.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Test substance-related mortality did not occur in the animals assigned to the Reproduction Subset.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No adverse, test substance-related effects on body weight or weight gain were observed for males or females exposed to any concentration of the test substance during the premating, gestation, or lactation periods.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related effects or statistically significant differences in food consumption were observed for males or females exposed to any concentration of the test substance during the premating, gestation, or lactation periods.

Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related effects or statistically significant differences in food efficiency were observed for males or females exposed to any concentration of the test substance during the premating, gestation, or lactation periods.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no exposure-related changes in group mean hematology parameters in male or female rats at any concentration tested.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no exposure-related changes in group mean clinical chemistry parameters in male or female rats at any concentration tested.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in group mean urinalyses parameters in male or female rats at any concentration tested.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Under the conditions of the study, no test substance-related or adverse effects were observed on neurobehavioral parameters for the reproduction subset.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test substance-related microscopic findings in any of the P1 adults. All microscopic findings in these animals were consistent with normal background lesions in rats of this age and strain.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no test substance-related microscopic findings in any of the P1 adults. All microscopic findings in these animals were consistent with normal background lesions in rats of this age and strain.
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related reproductive failures in this study.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related reproductive failures in this study.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related reproductive failures in this study.

Details on results (P0)

Only one parental generation in the study, corresponds to "P0" in IUCLID.

Effect levels (P0)

Key result
Dose descriptor:
NOAEC
Effect level:
ca. 50 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No test substance-related effects on the offspring clinical signs occurred at any exposure concentration.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No test substance-related effects on the number of offspring born, born alive, percentage born alive, number of offspring surviving to lactation day 4, percentage surviving to lactation day 4 occurred at any exposure concentration
The viability index for the 50,000 ppm group was 96.5% compared to 100% for the control group. The 96.5% value is within the historical control range of 91.1-100% (Appendix SSS). Therefore, the significantly lower viability index for the 50,000 ppm group was within the range of normal biological variation and not considered to be test substance-related. One litter in the control group was euthanized on lactation day 0 due to the death of dam 159, as a result of dystocia.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test substance-related effects on the offspring body weight on lactation days 0 and 4 occurred at any exposure concentration.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test substance-related gross observations in pups.
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
ca. 50 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
gross pathology

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, the no-observed-adverse-effect concentration (NOAEC) of Zyron 116 (perfluoroethane) for systemic toxicity in male and female rats was 50,000 ppm, the highest concentration tested, based on the absence of effects in all endpoints. The NOAEC for reproductive effects was 50,000 ppm based on the absence of effects on reproductive endpoints and on offspring at the highest concentration tested.
Executive summary:

The purpose of this study was to determine the potential subchronic and reproductive toxicity from repeated inhalation exposure of male and female rats to 0, 2500, 10,000, or 50,000 ppm of the test substance.    

Groups of 20 young adult male or nulliparous, non-pregnant female Crl:CD(SD) rats were divided into the following subsets:  Subchronic (including a micronucleus evaluation), Subchronic with Recovery (including a neurobehavioral evaluation), and Reproduction (including a neurobehavioral evaluation).  

Chamber atmospheres of test substance were generated by dilution of the test substance vapor in air and supplemental oxygen.  An air control group was also evaluated using a similar generation method.  Vapor concentrations of the test substance were measured by gas chromatography (GC).

Detailed clinical observations (evaluation of fur, skin, eyes etc.) were recorded once during pretest and at least weekly thereafter. Examinations also include regular measurement of body weight, food consumption & food efficiency. Neurobehavioral evaluation and clinical pathology evaluation (hematology and coagulation, clinical chemistry, urinalysis) was performed.

Litter examinations (live, dead, or missing pups, individual pup weights and clinical observations) were determined at birth and again on postnatal day 4. Subsequently, lactating females and offspring were euthanized. Selected organs were weighed, and selected tissues were evaluated microscopically from the parental rats. Offspring were evaluated for external abnormalities only.

No test substance-related or adverse effects were observed on reproductive performance;

offspring weight, survival, or clinical signs; micronucleus endpoints, neurobehavioral

parameters, or clinical pathology parameters. There were no test substance-related effects on gross or microscopic observations either.

The NOAEL for reproductive effects was 50,000 ppm based on the absence of effects on reproductive endpoints and on offspring at the highest concentration tested.