Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Remarks:
No deviations that impacted the overall integrity of the study results or conclusions occurred.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Perfluorpropylvinylether (PPVE), MTDID 16437
- Substance type: pure active substance
- Physical state: liquid
- Analytical purity: 98.5%
- Storage condition of test material: At room temperature in the dark under nitrogen
- Other: Flush container with nitrogen after handling
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M Company, Lot 4000102238
- Expiration date of the lot/batch: No data
- Purity test date: 20th of July, 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from light.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague Dawley Crl:CD(SD) rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratoris, Inc. (Raleigh, N.C.)
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Approximately 51 days old
- Weight at study initiation: Males:
- Fasting period before study: All males and recovery phase females were fasted prior to clinical pathology blood collection when food, but not water, was withheld.
- Housing: Following receipt and until pairing, all F0 animals were housed 2–3/cage by sex in clean, solid-bottom cages with bedding material (Bed-O’Cobs; The Andersons, Cob Products Division, Maumee, OH). During cohabitation, breeding phase rats (10/sex/group) were paired in a solid-bottom cage with bedding material. Following the breeding period, the males were individually housed in solid-bottom cages until the scheduled necropsy. Following positive evidence of mating, the females were individually housed in solid-bottom cages with bedding material. The dams and their litters were housed in these cages until euthanasia on Lactation Day 13. Females that failed to deliver were housed in solid-bottom cages with bedding material until post-mating day 25. The 5 rats/sex in the control and high-exposure groups that were assigned to the recovery phase were not paired for mating and remained housed in groups of 2–3 in clean solid-bottom cages until euthanasia.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet 5002 ad libitum.
- Water (e.g. ad libitum): Reverse osmosis-purified tap water ad libitum.
- Acclimation period: 21 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5-24.3 C
- Humidity (%): 30.3-62.6
- Air changes (per hr): At least 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 22 August, 2016 To: 15 November, 2016

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: Air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposures were initially conducted using four 1000-L glass and stainless steel whole-body exposure chambers. However, from 19 Oct 2016 through the end of the study, the exposures were conducted using a 1000-L glass and stainless steel whole-body chamber for Group 1 and three 500-L glass and stainless steel whole-body chambers for Groups 2 to 4 in order to preserve test substance for study completion. The change in chamber size had no impact on the study because the environmental parameters and test substance concentrations were monitored and within specifications.
- Method of holding animals in test chamber:
- Source and rate of air: Air supplied to the exposure chambers was provided from the Charles River Laboratories Inhalation Department, breathing quality, in-house compressed air source and a HEPA- and charcoal-filtered, temperature- and humidity-controlled supply air source.
- Method of conditioning air: HEPA and charcoal filtered, temperature and humidity controlled.
- Temperature, humidity, pressure in air chamber: 19-25 degrees C, 30-70% humidity. Negative pressure was maintained in the chamber.
- Treatment of exhaust air: All exposure chamber exhaust passed through the facility
exhaust system, which consists of redundant exhaust blowers preceded by activated-charcoal and HEPA-filtration units.
- Atmosphere generation: various sized glass beads and heated to approximately 80°C using heat tape controlled using a temperature controller and a J-type thermocouple. A pump equipped with a piston controlled using a stroke rate controller was used for metering test substance from a glass reservoir to the top of the bead column. Test substance was delivered to the top of the bead column using Teflon tubing.

TEST ATMOSPHERE
- Brief description of analytical method used: Analyzed exposure concentrations were determined at approximately 45-minute intervals using a gas chromatograph. Samples were collected from the appropriate animal-breathing zone of the exposure chamber via one-eighth inch Teflon tubing. Gas sampling injection onto the chromatography column occurred via an internal gas-sampling valve with a sample loop, the chromatograph was displayed, the area under the sample peak was calculated and stored, and the concentration in parts per million (ppm) was calculated.
- Samples taken from breathing zone: yes
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing, the pair was separated.
- After successful mating each pregnant female was caged (how): Individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyzed exposure concentrations were determined at approximately 45-minute intervals using a gas chromatograph. Samples were collected from the appropriate animal-breathing zone of the exposure chamber via one-eighth inch Teflon tubing. Gas sampling injection onto the chromatography column occurred via an internal gas-sampling valve with a sample loop, the chromatograph was displayed, the area under the sample peak was calculated and stored, and the concentration in parts per million (ppm) was calculated.
Duration of treatment / exposure:
Animals were exposed for 6 hours/day.
Frequency of treatment:
28 days consecutively for male rats and 14 days prior to mating and continuing until Gestation Day 20 for female rats. No female exposures were conducted from Gestation Day 21 through Lactation Day 4. On Lactation Day 5, exposure began again and continued until 1 day prior to euthanasia.
Details on study schedule:
Test article or humidified, filtered air was administered as 6-hour whole-body exposures once daily. The males selected for pairing were exposed during Study Days 0–27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 days. The females selected for pairing were exposed during Study Days 0 (14 days prior to pairing) through Gestation Day 20. No exposures were conducted from Gestation Day 21 through Lactation Day 4. On Lactation Day 5, exposure began again and continued until Lactation Day 13 (the day prior to scheduled euthanasia) for a total of 50–53 days. Females that failed to deliver were exposed through the day prior to euthanasia (Post-Mating Day 25) for a total of 35–38 days. The extra 5 rats/sex in the control and high-exposure groups were not used for mating, but were treated on a comparable regimen beginning on Study Day 0; following 28 days of exposure for males and 49 days of exposure for females, these animals remained on study for a 15-day recovery period.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/L air (analytical)
Remarks:
Control
Dose / conc.:
3.26 mg/L air (analytical)
Remarks:
300 ppm
Dose / conc.:
10.92 mg/L air (analytical)
Remarks:
1004 ppm
Dose / conc.:
43.89 mg/L air (analytical)
Remarks:
4034 ppm
No. of animals per sex per dose:
10 (main group) with an additional 5/group in the control and high dose group for recovery animals.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a 14 day range-finding study.
- Rationale for animal assignment (if not random): Random
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (morning, afternoon)
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations were recorded daily and individual detailed physical examinations were recorded weekly (prior to exposure during the exposure period). Each male and female was also observed for signs of toxicity approximately 1 hour following completion of exposure. The absence or presence of findings was recorded for all animals. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual male body weights were recorded weekly throughout the study and prior to the scheduled euthanasia (see Appendix 1 - Study Protocol and Deviations). Individual female body weights were recorded weekly until evidence of copulation was observed or until euthanasia (for females assigned to recovery phase).

FOOD CONSUMPTION AND COMPOUND INTAKE: Food consumption was recorded on the corresponding weekly body weight days until pairing (for animals paired for breeding) or euthanasia (for animals assigned to the recovery period). Food consumption was measured on a per cage basis for the corresponding body weight intervals. Food consumption was normalized to the number of animals/cage and was reported as g/animal/day. Food intake was not recorded during the breeding period for animals selected for pairing. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 10, and 13; food consumption was reported as g/anima/day during gestation and lactation. Calculation of the comprehensive intervals excludes all erroneous values such as total food spillage.
Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the estrous cycle of each breeding phase female for 10 days prior to test substance exposure and continuing through the 2-week premating period for all females and for females selected for the breeding phase until evidence of copulation was observed or until termination of the mating period for females with no evidence of mating.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight, seminal vesicle weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a clinical examination on PND 1, 4, 7, 10, and 13. Any abnormalities in nesting and nursing behavior were recorded.
The anogenital distance of all F1 pups was measured on PND 1; the absolute distance and the absolute distance relative to the cube root of body weight were reported for each pup. Pups were individually weighed on PND 1, 4, 7, 10, and 13. Mean pup weights were presented by sex for each litter and by exposure group. Pups were individually sexed on PND 0, 4, and 13. On PND 13, all F1 male offspring were evaluated for the presence of thoracic nipples/areolae. The number of nipples were recorded if nipples were present, and zero was recorded if nipples were absent.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: A complete necropsy was conducted on all F0 parental animals found dead or at the scheduled termination. All surviving F0 adults were anesthetized by isoflurane inhalation followed by euthanasia by exsanguination. Males were euthanized following completion of the mating period or following the 15-day recovery period; blood samples were collected for thyroid hormone analysis immediately prior to euthanasia.

- Maternal animals: A complete necropsy was conducted on all F0 parental animals found dead or at the scheduled termination. All surviving F0 adults were anesthetized by isoflurane inhalation followed by euthanasia by exsanguination. Females that delivered were euthanized on Lactation Day 14; blood samples were collected for thyroid hormone analysis immediately prior to euthanasia (see Section 5.12.). Females that failed to deliver were euthanized on Post-Mating Day 25 (females with evidence of mating); uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.11 Females with total litter loss were euthanized within 24 hours of litter loss.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were examined:
Adrenal glands (2) (weighed)
Aorta
Bone with marrow (sternebrae)
Brain (weighed)
Bulboouretheral gland (weighed)
Coagulating glands (2)
Eyes with optic nerve (2)
Gastrointestinal tract
Epididymides (weighed)
Esophagus
Stomach
Duodenum
Jejunum
Ileum
Cecum
Colon
Rectum
Heart (weighed)
Kidneys (2) (weighed)
Levator ani plus bulbocavernosus (LABC) musle (weighed)
Liver (sections of 2 lobes) (weighed)
Lymph node
Axillary (2)
Mandibular (2)
Mesenteric
Lungs (including bronchi, fixed by inflation under constant pressure with fixative) (weighed)
Ovaries and oviducts (2) (weighed)
Pancreas
Peripheral nerve (sciatic)
Pituitary gland (weighed)
Prostate gland
Salivary gland (mandibular [2])
Nasal cavities with turbinates b
Seminal vesicles (2) (weighed)
Skeletal muscle (rectus femoris)
Skin with mammary gland c
Spinal cord (cervical)
Spleen (weighed)
Testes with epididymides d (1) and vas deferens (weighed)
Thymus gland (weighed)
Thyroids (with parathyroids, if present [2]) (weighed)
Tongue
Trachea
Urinary bladder
Uterus with cervix and vagina
All gross lesions (all groups)
Postmortem examinations (offspring):
SACRIFICE
On PND 13, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital.

GROSS NECROPSY
- Intact offspring that were found dead or euthanized in extremis (by intraperitoneal injection of sodium pentobarbital) from PND 0 to 4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. Tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as deemed necessary by the gross findings. The carcass of each pup was then discarded.

HISTOPATHOLOGY / ORGAN WEIGTHS
Blood samples were collected for thyroid hormone analysis immediately prior to euthanasia from 1 pup/sex/litter; the thyroids (with parathyroids, if present) were weighed (following fixation) and placed in 10% neutral-buffered formalin for possible histopathological examination. Remaining pups (not used for blood collection) were discarded without examination.
Reproductive indices:
Male mating index, female mating index, male fertility index, female fertility index, male copulation index, female copulation index, female conception index, estrous cycle length, pre-coital interval, gestation length.
Offspring viability indices:
Mean live litter size, postnatal survival between birth and PND 0 or PND 4, postnatal survival for all other intervals

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Red material was noted around the nose for F0 males and females in the 1000 and 4000 ppm groups approximately 1 hour following exposure throughout the exposure period. The observation did not persist to the daily examinations on the following day and was not considered adverse.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was no test substance-related mortality noted at any exposure level. One F0 female in the 300 ppm group was found dead on Lactation Day 0; this death was attributed to dystocia and was considered unrelated to test substance exposure. In addition, 1 male in the 4000 ppm group was found dead on the day of the recovery necropsy; the cause of death was undetermined due to the lack of necropsy findings or relevant histopathological changes and therefore was not attributed to test substance exposure. All other F0 and F1 males and females survived to the scheduled necropsies.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A test substance-related lower mean body weight gain was noted for F0 males in the 4000 ppm group compared to the control group for the overall exposure period (Study Days 0–27) due to a lower mean body weight gain in this group during the first week of exposure. However, mean absolute body weights in the 4000 ppm group F0 males were similar to the control group throughout the study. Therefore, the lower mean body weight gains observed in this group were not considered adverse. Mean body weights and body weight gains in the 4000 ppm group F0 males were similar to the control group during the recovery period. Mean body weights and body weight gains in the 4000 ppm group F0 females were unaffected by test substance exposure during the premating, gestation, and lactation periods. Mean body weights in the 4000 ppm group F0 females were similar to the control group throughout the recovery period. Mean body weights and body weight gains in the 300 and 1000 ppm group F0 males and females and mean food consumption in all test substance-exposed groups were unaffected by test substance exposure throughout the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean food consumption, evaluated as g/animal/day, in the 300, 1000, and 4000 ppm groups was unaffected by test substance exposure.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on hematology or coagulation parameters.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on serum chemistry.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no test substance-related effects on the parameters tested in the in the Functional Observation Battery (FOB).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test article-related microscopic findings were noted in the kidney and nasal level II of the 4000 ppm group males at the primary necropsy. The increased incidence and severity of chronic progressive nephropathy persisted in the kidneys of the 4000 ppm group males at the recovery necropsy. In the kidneys at the primary necropsy, lower incidences of tubular basophilia and higher incidences of chronic progressive nephropathy (CPN) were seen in the 4000 ppm group males. As tubular basophilia is the precursor change to the common background finding and spontaneously developing CPN, these incidences demonstrated a nonadverse test article-induced exacerbation in the development of CPN. In addition, CPN and tubular basophilia in the control group males tended to be unilateral while CPN was predominantly bilateral in the 4000 ppm group. A similar effect was not seen in the females. Histopathological examination of the kidneys from the 300 and 1000 ppm group males at the primary necropsy revealed incidences and severities of tubular basophilia and CPN that were considered not appreciably different from those seen in the concurrent control group males; therefore, the effect of test article exposure was limited to the 4000 ppm group males. At the recovery necropsy, CPN was seen in 1 (minimal) control group male and 4 of 5 males (3 minimal and 1 mild; FD male included) from the 4000 ppm group. This persistence of test article-related CPN was expected considering the progressive nature of this spontaneously developing degenerative condition.

In nasal level II at the primary necropsy, higher incidences of mixed cell inflammation were seen in the 4000 ppm group males. The distribution of the inflammation was most commonly associated with the transitional epithelium (maxillary and/or nasal turbinates and the lateral wall of the nasal cavity) and was bilateral in 3 of the 5 cases. The tips of the maxillary and nasal turbinates are often the first and most severely affected by nasal irritants and in this study the
inflammation likely represented non-adverse local irritation directly attributed to test article inhalation. Mild unilateral inflammation involving the lining of the ventral meatus and the lateral wall of the nasal cavity was seen in a 4000 ppm group female (Animal No. 1684); however, this single case was insufficient to be considered a definitive effect of test substance administration. Histopathological examination of nasal level II from the 300 and 1000 ppm group males at the primary necropsy revealed minimal mixed cell inflammation in 2 of 10 rats from each group. These marginally higher incidences compared to the concurrent control group males, absence of a dose-related response, and finding similar inflammation in a control group male at the recovery necropsy suggested that the effect of test article exposure at the primary necropsy was limited to the 4000 ppm group males. Minimal nasal inflammation was observed in 1 rat each from the control and 4000 ppm group males at the recovery necropsy indicative of complete recovery from the prior effects of test article exposure.

There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No test article-related neoplastic histopatholigical findings were observed upon necropsy.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No test article-related effects on estrous cycle were observed.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Testis weight, epididymis weight and seminal vesicle weight were unaffected by test article exposure.
Reproductive performance:
no effects observed
Description (incidence and severity):
No exposure-related effects on reproductive performance were observed at any exposure concentration.

Effect levels (P0)

Key result
Dose descriptor:
NOAEC
Effect level:
ca. 43.89 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
other: Based on the results of the study, the No Observed Adverse Effect Concentration (NOAEC) is 4034 ppm.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by parental test substance exposure. Pups (litters) that were found dead numbered 8(1), 3(3), 0(0), and 3(3) in the control, 300, 1000, and 4000 ppm groups, respectively. Seven, 2, and 1 pups in the control, 1000, and 4000 ppm groups, respectively, were missing and presumed to have been cannibalized.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
The mean number of pups born, live litter size and the percentage of males at birth in the 300, 1000, and 4000 ppm groups were similar to the control group values. Postnatal survival in the 300, 1000, and 4000 ppm groups were unaffected by maternal test substance exposure.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean male and female F1 pup body weights and body weight changes during PND 1–13 in the 300, 1000, and 4000 ppm groups were unaffected by parental test substance exposure. No statistically significant differences from the control group were noted.
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 examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on thyroid weights in the F1 males and females at any dosage level on PND 13. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No internal findings were noted at the necropsy of pups in the 300, 1000, and 4000 ppm group euthanized on PND 13.
Histopathological findings:
not examined

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. 43.89 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
other: Based on the results of the study, the No Observed Adverse Effect Concentration was 4034 ppm.

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
43.89 mg/L air (analytical)
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, the parental systemic and reproductive and developmental No Observed Adverse Effect Concentration for the test article is 43.89 mg/L (4034 ppm).
Executive summary:

The repeat dose and reproductive and developmental toxicity potential of the test article was evaluated when administered by whole body inhalation to male and female Sprague Dawley rats for 28 days. The potential for the test article to affect male and female reproductive performance such as gonadal function, mating behavior and conception through day 13 of postnatal life was evaluated followed by a 14 -day recovery period. The study was conducted in compliance with OECD GLP (1997). The test was conducted according to OECD Guideline 422 (2016). The test substance was administered as a 6-hour, whole-body inhalation exposure daily to 3 groups of male and female Crl:CD(SD) rats. The low- and mid-exposure groups (Groups 2–3) each consisted of 10 rats/sex and the high-exposure group (Group 4) consisted of 15 rats/sex. Target exposure levels were 300, 1000, and 4000 parts per million (ppm). A concurrent control group of 15 rats/sex was exposed to humidified, filtered air on a comparable regimen. Overall mean exposure concentrations were 300, 1004, and 4034 ppm for the 300, 1000, and 4000 ppm groups, respectively. Males and females were approximately 10 weeks of age at the beginning of test substance exposure. Males were exposed to the test substance for 14 days prior to mating. Males continued to be exposed throughout the mating period through 1 day prior to euthanasia for a total of 28 days. Females were exposed to the test substance for 14 days prior to pairing and were exposed through Gestation Day 20. Exposure was suspended from Gestation Day 21 through Lactation Day 4 to avoid potential confounding effects on nesting and nursing behavior caused by separation of dams from their litters. Exposure of the F0 females with evidence of mating was re-initiated on Lactation Day 5 for females that delivered and continued through the day prior to euthanasia (Lactation Day 13) for a total of 50–53 days; females that failed to deliver were exposed through the day prior to euthanasia (Post-Mating day 25) for a total of 35–38 days. The extra 5 males and 5 females in the control and high-exposure groups that were not used for mating were exposed beginning on Study Day 0; following 28 days of exposure for males and 49 days of exposure for females, these animals were assigned to a 15-day nonexposure recovery period. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and motor activity data were recorded for 5 males/group on Study Day 27 and for 5 females/group on Lactation Day 13. All F0 females selected for pairing were allowed to deliver and rear their pups until Lactation Day 14. F1 clinical observations, body weights, and sexes were recorded at appropriate intervals and anogenital distance was recorded on PND 1. To reduce variability among the litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4; blood samples for possible thyroid hormone analysis were collected from the culled pups (1/sex/litter). All F1 male pups were evaluated for areolae/nipple anlagen on PND 13. Remaining F1 pups were euthanized on PND 13; blood samples for thyroid hormone analysis were collected and selected organs were weighed from 1 pup/sex/litter. Clinical pathology evaluations (hematology, coagulation, and serum chemistry) were performed on 5 F0 animals/sex/group at the primary necropsy and 5 animals/sex in the control and high-exposure groups at the recovery necropsy. Blood samples for thyroid hormone analysis were collected from F0 males and females at the primary necropsy; only male samples were analyzed. F0 males were euthanized following completion of the mating period or 15-day recovery period and F0 females were euthanized on Lactation Day 14 for females that delivered, Post-Mating Day 25 for females that failed to deliver, or following the 15-day recovery period. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and high-exposure groups at the primary necropsy. In addition, the kidneys and nasal level II were identified as potential target tissues in males and were examined from all males in all groups at the primary and recovery necropsies.

There was no test substance-related mortality noted at any exposure level. One F0 female in the 300 ppm group was found dead on Lactation Day 0; this death was attributed to dystocia and was considered unrelated to test substance exposure. In addition, 1 male in the 4000 ppm group was found dead on the day of the recovery necropsy; the cause of death was undetermined due to the lack of necropsy findings or relevant histopathological changes and therefore was not attributed to test substance exposure. All other F0 and F1 males and females survived to the scheduled necropsies. Test substance-related red material around the nose was noted for F0 males and females in the 1000 and 4000 ppm groups approximately 1 hour following exposure generally throughout the exposure period. This clinical observation generally did not persist to the daily examinations on the following day and was; therefore, not considered adverse. A test substance-related lower mean body weight gain was noted for F0 males in the 4000 ppm group compared to the control group for the overall exposure period (Study Days 0–27) due to a lower mean body weight gain in this group during the first week of exposure. However, mean absolute body weights in the 4000 ppm group F0 males were similar to the control group throughout the study. Therefore, the lower mean body weight gains observed in this group were not considered adverse. Mean body weights and body weight gains in the 4000 ppm group F0 males were similar to the control group during the recovery period. Mean body weights and body weight gains in the 4000 ppm group F0 females were unaffected by test substance exposure during the premating, gestation, and lactation periods. Mean body weights in the 4000 ppm group F0 females were similar to the control group throughout the recovery period. Mean body weights and body weight gains in the 300 and 1000 ppm group F0 males and females and mean food consumption in all test substance-exposed groups were unaffected by test substance exposure throughout the study. Mean F0 male and female mating and fertility indices, F0 male copulation indices, and F0 female conception indices in the 300, 1000, and 4000 ppm groups were unaffected by test substance exposure. The mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test substance exposure. There were no test substance-related effects noted on mean estrous cycle lengths or the mean numbers of implantation sites and unaccounted-for sites in F0 females at any exposure level. No test substance-related effects were noted during the FOB or motor activity evaluations at any exposure level in the F0 generation. There were no test substance-related gross necropsy observations or alterations in clinical pathology parameters (hematology, coagulation, and serum chemistry) were noted for F0 males and females at any exposure level during the primary and recovery evaluations. Mean serum T4 levels in the F0 males were unaffected by test substance exposure. Test substance-related higher mean absolute and relative (to final body and/or brain weight) lung weights were noted for F0 males and females in the 4000 ppm group at the primary necropsy. There were no microscopic correlates for the higher lung weights and the values were generally within the Charles River Ashland historical control data ranges; therefore, the higher mean lung weights were not considered adverse. Mean lung weights in the 4000 ppm F0 males and females were similar to the control group at the recovery necropsy, indicative of complete recovery. At the primary necropsy, lower incidences of tubular basophilia and higher incidences of chronic progressive nephropathy (CPN) were observed in the 4000 ppm group F0 males. As tubular basophilia is the precursor change to the common background finding and spontaneously developing CPN, these incidences demonstrated a non-adverse test article-induced exacerbation in the development of CPN. 1 In addition, CPN and tubular basophilia in the control group males tended to be unilateral while CPN was predominantly bilateral in the 4000 ppm group. In nasal level II, higher incidences of mixed cell inflammation were seen in the 4000 ppm group F0 males at the primary necropsy. The distribution of the inflammation was most commonly associated with the transitional epithelium and in this study the inflammation likely represented non-adverse local irritation directly attributed to test article inhalation. At the recovery necropsy, test article-related CPN persisted in the 4000 ppm group F0 males which was expected considering the progressive nature of this spontaneously developing degenerative condition while, nasal inflammation was observed in a single male indicative of a complete recovery. There were no test substance-related histologic changes noted for F0 males in the 300 and 1000 ppm groups or F0 females at any exposure level. Mean numbers of F1 pups born, live litter size, percentage of males at birth, postnatal survival, the general physical condition of the F1 pups, pup body weights and body weight gains, anogenital distance, and areolae/nipple anlagen counts (males only) in the 300, 1000, and 4000 ppm groups were unaffected by parental test substance exposure. Necropsy findings for F1 pups that died were not suggestive of any association with maternal administration of the test substance. There were no test substance-related changes in mean serum T4 levels or mean thyroid/parathyroid weights in F1 males and females on PND 13. Based on the results of the study, the parental systemic and reproductive and developmental No Observed Adverse Effect Concentration for the test article is 43.89 mg/L (4034 ppm).