Multi-Walled Carbon Nanotubes (MWCNT), synthetic graphite in tubular shape

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-OCT-04 to 2022-SEP-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: United States Environmental Protection Agency (EPA) Health Effects Test Guideline OPPTS 870.3550 Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

carbon

EC Number:

936-414-1

Molecular formula:

C

IUPAC Name:

carbon

Test material form:

solid: nanoform

Details on test material:

Name of substance: MWCNT NC-7000™ (Nanocyl™ NC7000)
EC No. 936-414-1
Batch No. A2270
Purity: 90%
Storage: 18°C to 24°C

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:
Nanocyl, SA (Belgium); Batch # A2270
- Purity, including information on contaminants, isomers, etc.:
90%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions of test material:
18°C to 24°C
- Stability and homogeneity of the test material under test conditions (e.g. in the exposure medium) and during storage:
Stability analyses determining an expiration date for the lot of test substance used on this study were not performed; however, when stored under the proper conditions, the test
substance is not expected to degrade over time.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding):
For exposure of the test substance-treated groups, a dry powder aerosol exposure was generated using a jet mill.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
: dry powder aerosol

INFORMATION ON NANOMATERIALS
- Chemical Composition:
multi-walled carbon nanotubes (90% purity); EC# 936-414-1
- Density:
Not specified
- Particle size & distribution:
D10: 53 µm; D50: 197 µm; D90: 622 µm; Range: 5-1971 µm (Provided as an attachment)
- Specific surface area:
Not specified
- Isoelectric point: Not specified
- Dissolution (rate): Not specified

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The Crl:WI(Han) rat is recognized as appropriate for reproduction studies. The CRO has reproductive historical control data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Raleigh, NC)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) x 10-11 wks
- Weight at study initiation: (P) Males: 231 and 287 g; Females: 163 and 197 g
- Fasting period before study: Not specified
- Housing: In Solid-bottom cages containing appropriate bedding material as follows:
Pre-mating period: In groups of 2-3 animals per cage, by sex
Post-mating period: Single/Individually following positive signs of mating or the end of the mating period.
- Use of restrainers for preventing ingestion (if dermal): Not applicable since inhalation exposure but nose-only exposure restraint tubes were used in the study.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum except during the exposure period and acclimation to nose-only restraint tubes
- Water (e.g. ad libitum): Municipal tap water, treated by reverse osmosis and ultraviolet irradiation ad libitum via an automatic watering system, except during the exposure period and acclimation to nose-only restraint tubes.
- Acclimation period: male and female rats were acclimated for 7 and 21 days, respectively, prior to initiation of exposure

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68°F to 78°F (20°C to 26°C)
- Humidity (%): 30% to 70%
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2021-OCT-05 (female) and 2021-OCT-19 (male) To: 2021-DEC-17

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

nose only

Mass median aerodynamic diameter (MMAD):

> 1.2 - <= 1.9 µm

Remarks on MMAD:

The particle size was expressed as the mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD). Please see Table 10.

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

Description of Inhalation Exposure System:

Exposures were conducted using 3-tier stainless-steel, conventional nose-only exposure systems (11.0-L CNOS). One exposure system was dedicated to each group for the duration of the study.

Exposure Environmental Conditions:

The exposure systems were operated under dynamic conditions and at a slight negative pressure. Pressure was verified using an open T-fitting on the CNOS exhaust line.

Exposure system airflow (ventilation rate) was monitored using a venturi tube and Dwyer Magnehelic® Indicating Transmitter pressure gauge (Model No. 605-2 or 605-2C, Dwyer Instruments, Inc.; Michigan City, IN). Each gauge was calibrated for conversion from pressure to airflow in liters per minute through the use of a Dry Test Meter (Model DTM-200A, Elster American Meter Co.; Nebraska City, NE). Temperature and relative humidity of the exposure atmospheres was monitored using a display (Model No. HMT120, Vaisala; Helsinki, Finland) supplied with a Vaisala probe (Model No. HMP110).

Exposure system airflow, temperature and relative humidity was monitored and manually recorded ranging from 28 minutes to 93 minutes. The mean temperature and relative humidity of the exposure atmospheres were targeted at 19°C to 25°C and 30% to 70%, respectively. Environmental conditions for the male and female exposures are summarized in Table 8.

Oxygen content of the exposure atmospheres measured during the method development phase of the study using a Dräger PAC III equipped with a calibrated oxygen sensor was determined to be 20.9% for all groups.

Control Exposure System:

For exposure of the filtered air control group, dry compressed air and humidified air was mixed to achieve a similar humidity and airflow to that in the test substance-exposed groups.

Dry, compressed air was supplied to the exposure system using a Coilhose regulator (Model No. 8802K, Coilhose Pneumatics Inc.; East Brunswick, NJ) and a Dwyer rotameter-type flowmeter (Model No. VFB-69-BV).

Humidified air was generated by passing dry compressed air through a muffler-type bubbler submerged in a 2-L Erlenmeyer flask filled with filtered water. The flowrate of air delivered to the exposure system was controlled using a Coilhose regulator (Model No. 8802K) and a Dwyer rotameter-type flowmeter (Model No. DR4141M). The flask was heated to approximately 30°C using Omega® heat tapes (Model No. STH101-040L, FGS051-040L, STH051-080 or STH051-080L, Omega Engineering, Inc.; Stamford, CT). The temperature of the heat tape(s) was regulated using an Omega® temperature controller (Model No. CN370) and a J-type thermocouple. Water within the flask was emptied and refilled daily. Airflows used for the control exposure system are presented in Table 5.

Test Substance-Treated Exposure Systems:

For exposure of the test substance-treated groups, a dry powder aerosol exposure atmosphere was generated using a jet mill.

To aerosolize the test substance dry, compressed air was supplied to the inlet and micronizing ports of the jet mill (Model No. 00, Jet-O-Mizer, Fluid Energy Equipment Division; Telford, PA) using Coilhose regulators (Model No. 8802K) at an inlet airflow of 27.1-28.3 LPM and a micronizing airflow of 15 LPM.

The test substance was delivered to the jet mill using an AccuRate screw-type powder feeder (Schenck AccuRate Inc.; Whitewater, WI). The Accurate feeder was equipped with a stainless-steel auger and stainless-steel delivery tube. Syntron vibrators (Model No. V-2-B, FMC Technologies; Houston, TX) were connected to the lid of the AccuRate and auger sheath to help facilitate delivery of test substance.

The output from the jet mill was directed through a stainless steel cyclone to a 4.8 L glass chromatography jar. The chromatography jar pressure was controlled using a ½” gate valve and monitored using a Dwyer Magnehelic Gauge (Model No. 2302). From the chromatography jar, compressed air powered, adjustable Vaccon transvector air pumps (Model No. VDF-100, Vaccon Company, Inc.; Medway, MA) were used to control a portion of test substance aerosol delivered to each exposure system. For Exposure Systems 2 and 3, a siphon was placed in line after the transvectors to remove excess test substance aerosol. The siphon was controlled using a needle valve and a Gilmont type flowmeter (Model No. 12, Barnant Co./Gilmont Instruments; Barrington, IL) for CNOS 2 and a Dwyer type flowmeter (Model No. VFB-67-SSV) for CNOS 3. The excess test substance removed using the siphon was directed to a Solberg filter prior to entering the facility exhaust system.

The resulting aerosol atmosphere was directed to a Y-fitting where it mixed with humidified air to achieve the desired atmosphere concentration prior to being delivered to the nose-only exposure system. Humidified air was generated by passing dry compressed air through a muffler-type bubbler submerged in a 2-L Erlenmeyer flask filled with filtered water. Humidified airflow delivered to the exposure systems was controlled using a Coilhose regulator (Model No. 8802K) and a Dwyer rotameter-type flowmeter (Model No. DR4141M). The flask was heated to approximately 30°C using Omega® heat tapes (Model No. STH101-040L, FGS051-040L, STH051-080 or STH051-080L). The temperature of the heat tape(s) were regulated using an Omega® temperature controller (Model No. CN370) and a J-type thermocouple. Water within the flasks was emptied and refilled daily. Airflows used for the test substance generation for the test substance exposure systems are presented in Table 6.

TEST ATMOSPHERE
Aerosol Particle Size:

Aerosol particle size was determined using a 7-stage IN-TOX Brass Cascade Impactor (Model No. 02-140, IN-TOX Products; Moriarty, NM). Pre-weighed, 25-mm glass-fiber filters (Type A/E) was used as substrates for stages 1 through 7.

Samples were collected at the approximate breathing zone of the nose-only exposure systems. One sample was collected each week for the test substance exposure systems during the study period. Samples were collected over 1080, 360, and 240 minutes for Exposure Systems 2 (0.1 mg/m3), 3 (0.4 mg/m3), and 4 (1.5 mg/m3) respectively. For Exposure System 2, due to the long sampling period, the impactor was capped between exposure days. Sample flow was controlled using a needle valve connected to the facility vacuum source and was measured using a mini-Buck calibrator. Following sample collection, the substrates were re-weighed and the particle size was calculated based on the impactor stage cut-offs. The particle size was expressed as the mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD).

Aerosol Monitoring:

Real-time aerosol monitoring was performed for the test substance exposure systems using a light scattering aerosol monitor (Casella CEL-712 or equivalent) but only as a guide and not for data collection.

Exposure Atmosphere Homogeneity Assessment:

Homogeneity of exposure concentration for Exposure System 4 was evaluated during method development. Two test locations per tier and a reference location were used for these determinations. Samples were collected using standard gravimetric techniques. The measured concentration was calculated as a percent difference for each position from the reference location. Homogeneity was performed in triplicate for the exposure system and was acceptable for this study. As an identical setup was used for Exposure Systems 2 and 3, similar homogeneity was to be expected. Due to the low target concentration for Exposure Systems 2 and 3, homogeneity was only performed for Exposure System 4. The homogeneity assessment for Exposure System 4 is summarized in Table 7.

Nominal Exposure Concentrations:

Nominal exposure concentrations were not calculated due to the configuration of the exposure atmosphere generation systems. Documentation of test substance usage was performed by calculating the total amount of test substance used. The amount used is defined as the difference between the weight of the accurate feeder prior to and following use over a weekly period.

Actual Exposure Concentrations:

Standard gravimetric methods were used for determination of actual test substance exposure concentrations. Samples were collected from the approximate animal breathing zone on pre-weighed 25-mm glass-fiber filters (Type A/E, PALL Corporation; Ann Arbor, MI) held in a filter holder.

Sample flow was controlled using a needle valve attached to the facility vacuum source and was measured using a mini-Buck Calibrator (A.P. Buck, Inc.; Orlando, FL). Following each sample collection, the filter was re-weighed and the exposure concentrations in mg/m3 was calculated by dividing the gravimetrically determined mass by the sample volume. Sample volume was calculated by multiplying the sample flowrate (LPM) by the length of the sampling period (minutes). The approximate sampling conditions during the exposures are listed in Table 4.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum 14 days allowed for mating
- Proof of pregnancy: evidence of a copulatory plug in the vagina or by evidence of sperm in a vaginal lavage referred to as day 0 of pregnancy.
- After 14 days of unsuccessful pairing, if no evidence of mating was obtained, the animals were separated without further opportunity for mating, and the female was placed in a solid-bottom cage containing bedding material
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): female was returned to an individual solid-bottom cage
- Any other deviations from standard protocol: Not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Actual Exposure Concentrations

Standard gravimetric methods were used for determination of actual test substance exposure concentrations. Samples were collected from the approximate animal breathing zone on pre-weighed 25-mm glass-fiber filters (Type A/E, PALL Corporation; Ann Arbor, MI) held in a filter holder.

Sample flow was controlled using a needle valve attached to the facility vacuum source and was measured using a mini-Buck Calibrator (A.P. Buck, Inc.; Orlando, FL). Following each sample collection, the filter was re-weighed and the exposure concentrations in mg/m3 was calculated by dividing the gravimetrically determined mass by the sample volume. Sample volume was calculated by multiplying the sample flowrate (LPM) by the length of the sampling period (minutes). The approximate sampling conditions during the exposures are listed in Table 4.

Duration of treatment / exposure:

Males: minimum 28 days

Females: for at least 14 consecutive days prior to mating and continuing through Lactation Day 12
Exposure was suspended from Gestation Day 21 through Lactation Day 4, inclusive.

Frequency of treatment:

6-hours once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

Group 1 (Control - Filtered Air)

Doses / concentrations 2

Dose / conc.:

0.1 mg/m³ air (nominal)

Remarks:

Group 2 (Low Concentration)

Doses / concentrations 3

Dose / conc.:

0.4 mg/m³ air (nominal)

Remarks:

Group 3 (Intermediate Concentration)

Doses / concentrations 4

Dose / conc.:

1.5 mg/m³ air (nominal)

Remarks:

Group 4 (High Concentration)

No. of animals per sex per dose:

10/sex/concentration

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:

Dose levels were selected based on information provided by the Sponsor and published literature using similar multi-walled carbon nanotubes in an attempt to produce graded responses to the test substance. It was anticipated that the high-exposure level of 1.5 mg/m˄3 would show test substance-related effects but not produce an incidence of fatalities that would prevent a meaningful evaluation. The lower exposure levels were selected at intervals that were predicted to be narrow enough to reveal any dose-related trends.

- Rationale for animal assignment (if not random):

Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. Animals not exhibiting normal, 4- to 5-day estrous cycles were not assigned to groups.

- Fasting period before blood sampling for clinical biochemistry:
Not specified

- Other:

- Justification for Route of Exposure: The inhalation route of exposure was selected because this a possible route of human exposure.

- Justification for number of animals: The number of animals was based on the OECD Guideline for the Testing of Chemicals: Guideline 421, Reproduction/Development Toxicity Screening Test, 29 Jul 2016 (as modified), which recommends that evaluation of each group be initiated with at least 10 males and 12–13 females per group. Females were evaluated for estrous cyclicity during the pretest period and any females that failed to exhibit normal 4–5 day estrous cycles during the pretest period, were excluded from the study, therefore, the extra females were included to yield at least 10 females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 8 at termination.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily; prior to exposure on days of scheduled dosing and approximately 1–2 hours post-exposure.
- Mortality: At least twice daily (morning and afternoon) beginning upon arrival through termination (Except on days of receipt and necropsy where frequency was at least once daily).

DETAILED CLINICAL OBSERVATIONS: Yes; observations included, but were not limited to, evaluations for changes in appearance of skin and fur, eyes, mucous membranes,
respiratory and circulatory system, autonomic and central nervous systems, somatomotor activity, and behavior.
- Time schedule: Weekly, beginning on the first day of exposure for males, and beginning at the start of estrous lavages for females, through euthanasia. On days of scheduled exposure, the
observations were collected prior to dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: F0 males: Twice weekly, beginning with the initiation of exposure through euthanasia. Body weights were also recorded on the day of
euthanasia; F0 females: Twice weekly beginning with the initiation of exposure until evidence of copulation was observed, or until euthanasia for females with no evidence of mating. Body weights were also recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20, and Lactation Days 1, 4, 7, 10, and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
F0 males: Twice weekly, beginning with the initiation of exposure through the initiation of breeding. Measured on a per cage basis, except during periods of individual housing.
Normalized to the number of animals/cage and reported in g/animal/day. Not recorded following the initiation of the mating period.
F0 females: Twice weekly, beginning with the initiation of exposure until the initiation of breeding; Food consumption was also recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20; and Lactation Days 1, 4, 7, 10, and 13. Measured on a per cage basis, except during periods of individual housing. Normalized to the number of animals/cage and reported in g/animal/day. Not recorded during the mating period or following the mating period for females with no evidence of mating.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): N/A

OTHER:

- THYROID HORMONE ANALYSIS::
Blood samples for thyroid hormone assessments were collected (from all F0 males (Day 28) and F0 females (Lactation Day 13)) prior to noon on each day of collection, and around the same time each day to reduce variability due to normal diurnal variations in physiological levels of thyroid hormones. Blood samples were collected (at least 1.0 mL), without anticoagulants, from the jugular vein of adult rats using the hand-held restraint method. Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in a refrigerated centrifuge (approximately 3000 rpm for 10 minutes at 4°C) and stored in a freezer set to maintain a target of -70°C. Samples were analyzed for Triiodothyronine (T3) and Thyroxine (T4) using a validated UHPLC/MS/MS assay and for Thyroid Stimulating Hormone (TSH) using a validated Luminex Bead-Based (TSH) assay.

Oestrous cyclicity (parental animals):

All F0 females: Daily vaginal lavages performed beginning during the pretest period and continuing through the 2-week premating and mating treatment periods until evidence of mating was observed.

Sperm parameters (parental animals):

Not specified

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: 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:
mortality checks daily, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD) on Day 1, pup weights on Days 1, 4, 7, 10 and 13, presence of nipples/areolae in male pups on Day 13. Detailed clinical observations on Days 1, 4, 7, 10 and 13 (pups were evaluated for changes in appearance of skin and fur, eyes, mucous membranes, respiratory and circulatory system, autonomic and central nervous systems, somatomotor activity, and behavior).

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

OTHER:

THYROID HORMONE ANALYSIS:

Blood samples (at least 1.0 mL) for thyroid hormone analyses were collected without anticoagulants via cardiac puncture under isoflurane anesthesia from F1 pups at the time of culling on PND 4 from 2 culled pups/litter and samples were pooled regardless of sex. Blood samples were also collected at termination on PND 13 from 1 pup/sex/litter (samples analyzed separately, by sex). Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in a refrigerated centrifuge (approximately 3000 rpm for 10 minutes at 4°C) and stored in a freezer set to maintain a target of -70°C. Samples were analyzed for Triiodothyronine (T3) and Thyroxine (T4) using a validated UHPLC/MS/MS assay and for Thyroid Stimulating Hormone (TSH) using a validated Luminex Bead-Based (TSH) assay.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on Day 28 were euthanized by inhalation of isoflurane followed by exsanguination
- Maternal animals: All surviving animals on Lacation Day 13 were euthanized by inhalation of isoflurane followed by exsanguination

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

F0 animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. The numbers of former implantation sites were recorded for females that delivered. Uteri of females without macroscopic evidence of implantation were opened and placed in 10% ammonium sulfide solution for detection of early implantation loss.

HISTOPATHOLOGY / ORGAN WEIGHTS

ORGAN WEIGHTS: Organs listed in Table 2 were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together, unless otherwise indicated. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.

HISTOPATHOLOGY: The tissues/samples indicated in Table 3 were collected from all animals and preserved in 10% neutral buffered formalin.

Tissues identified in Table 3 with the exception of the adrenal gland, heart, spleen, and thymus, from all F0 animals/sex in the control and high-dose groups, as well as gross lesions and lungs from all groups, were embedded in paraffin, sectioned, mounted on glass slides, stained with hematoxylin and eosin (PAS staining was used for the testes and epididymides) and examined.

Postmortem examinations (offspring):

SACRIFICE
- F1 offspring culled on Day 4 were discarded without necropsy.

Scheduled Euthanasia
On PND 13, surviving F1 pups were euthanized via an intraperitoneal injection of sodium pentobarbital.

Unscheduled Deaths
A necropsy was conducted for animals that died on study, and gross lesions were saved. Intact offspring that were found dead, during PND 0–4, were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. Pups with external abnormalities that would warrant further skeletal examination were eviscerated and stained for subsequent skeletal evaluation and findings were recorded as developmental variations or malformations, as appropriate.

GROSS NECROPSY
- On PND 13, 1 pup/sex/litter was subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system. All other animals were discarded without examination.

HISTOPATHOLOGY / ORGAN WEIGTHS

ORGAN WEIGHTS: Not evaluated

HISTOPATHOLOGY:

Representative samples of the Thyroid (with parathyroids, if present) were collected from 1 pup/sex/litter at the scheduled euthanasia and preserved in 10% neutral buffered formalin.

Statistics:

Please see 'Any other information on materials and methods incl. tables' for information on statistics.

Reproductive indices:

1) Female Mating Index = (Number of Females with Evidence of Mating (or no confirmed mating date and pregnant) / Number of Females Paired)

2) Female Fertility Index = (Number of Pregnant Females / Number of Females with Evidence of Mating (or no confirmed mating date and pregnant))

3) Female Pregnancy Index = (Number of Pregnant Females / Number of Females Paired)

4) Male Mating Index = (Number of Males with Evidence of Mating (or female partner confirmed pregnant) / Number of Males Paired)

5) Male Fertility Index = (Number of Males Impregnating a Female / Number of Males with Evidence of Mating (or female partner confirmed pregnant))

6) Male Pregnancy Index = (Number of Males Impregnating a Female / Number of Males Paired)

Offspring viability indices:

1) Live Birth Index (Percentage of pups born alive) = (Number of Live Newborn Pups / Number of Newborn Pups) x 100

2) Viability Index (Percentage of pups born that survive 4 days postpartum) = (Number of Live Pups on Day 4 Postpartum / Number of Liveborn Pups) x 100

3) Survival Index (Percentage of pups that survive 13 days postpartum) = (Number of Live Pups on Day 13 Postpartum / Number of Live Pups on Day 4 Postpartum) x 100

4) Sex Ratio (% males) = (Number of Live Male Pups / Total Number of Live Pups) x 100

5) Post-Implantation Loss/Litter = Number of Implants – Total Newborn Pups

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical effects were observed in any group during the daily examinations or 1–2 hours following exposure.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All F0 males and females survived to schedule necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males: Mean body weights and body weight gains in the 0.1, 0.4, and 1.5 mg/m3 group males were unaffected by treatment and none of the differences from the control group were statistically significant.

Females: Mean body weights and body weight gains in the 0.1, 0.4, and 1.5 mg/m3 group females during pre-mating, gestation, and lactation were unaffected by treatment and none of the differences from the control group were statistically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Males: Mean food consumption, evaluated as g/animal/day in the 0.1, 0.4, and 1.5 mg/m3 group males were unaffected by treatment and none of the differences from the control group were statistically significant.

Females: Mean food consumption, evaluated as g/animal/day in the 0.1, 0.4, and 1.5 mg/m3 group females during pre-mating, gestation, and lactation were unaffected by treatment and none of the differences from the control group were statistically significant.

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

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects on thyroid hormone values were observed in the F0 males at any exposure level. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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):

Treatment-related findings were observed in the lungs in the 0.1, 0.4, and 1.5 mg/m3 group male and female rats. This included a dose-dependent increase in severity and/or incidence of lipoproteinosis, mixed cell inflammation, increased alveolar macrophages, and/or pigment in the 0.1, 0.4, and 1.5 mg/m3 group male and female rats.

Mixed cell inflammation was observed in the 0.4 and 1.5 mg/m3 group males and females and was characterized as primarily mononuclear in nature with fewer neutrophils with occasional multifocal aggregates of mononuclear cells. Inflammation was most prominent in areas containing pigment either free or within alveolar macrophages. Additional changes associated with the inflammation included prominent dilated and/or congested capillaries, uncommon intra-alveolar erythrocytes/hemorrhage, and slight type II pneumocyte hyperplasia. At the dosages of 0.1 and 0.4 mg/m3, a mixed cell infiltrate of similar composition of predominantly mononuclear cells was observed but without evidence of tissue damage.

Lipoproteinosis was observed in the 0.4 and 1.5 mg/m3 group males and females and characterized by the accumulation of basophilic to amphophilic lightly granular substance present within alveolar spaces/ducts or intracytoplasmically in alveolar macrophages. Increased amounts of lipoproteinosis were typically seen in areas containing increased alveolar macrophages and/or pigment either free or within alveolar macrophages.

A dose-dependent increase in alveolar macrophage numbers and/or incidence was observed in the 0.1, 0.4, and 1.5 mg/m3 group male and female rats. Alveolar macrophages were typically enlarged with cytoplasm expanded by vacuoles containing a granular amphophilic substance consistent with what is commonly seen in association with lipoproteinosis. In some macrophages, accumulation of black pigment was observed and considered phagocytosed test material. The increase in alveolar macrophages was considered to be in response to inhaled test material, adaptive, and non-adverse.

Pigment was observed in the 0.1, 0.4 and 1.5 mg/m3 group male and female rats. Pigment was black, globular to angular ranging from individual fragments to pigment aggregates and was present both free within the alveolar spaces and intra-cytoplasmically within alveolar macrophages. The black pigment was consistent with the nature of the test substance (i.e., carbon nanotubules). In areas of increased pigment, a concomitant increase in mixed cell inflammation or infiltration, lipoproteinosis, and/or increased alveolar macrophages was typically observed.

Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to exposure to the test material.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The mean lengths of estrous cycles in the treated groups were similar to the control group value and differences from control were slight and not statistically significant.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

No treatment-related effects on reproductive performance were observed at any exposure level. No statistically significant differences were noted between the control and treated groups. A single mating pair in the 0.4 mg/m3 group showed evidence of mating but the female was not gravid.

The mean numbers of days between pairing and coitus in the test substance-exposed groups were generally similar to the control group value. The pre-coital interval in the 1.5 mg/m3 group was statistically significantly shorter than the control group, but the difference occurred in a direction that is generally not considered toxicologically relevant.

Mean gestation lengths in the 0.1, 0.4, and 1.5 mg/m3 groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were observed in these groups.

The mean numbers of implantation sites in the 0.1, 0.4, and 1.5 m/m3 groups were similar to the control group value.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEC

Effect level:

ca. 0.1 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic Toxicity

2

Key result

Dose descriptor:

NOAEC

Effect level:

ca. 1.5 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive Toxicity

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.4 other: mg/m3

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The general physical condition (defined as the occurrence and severity of clinical observations) of all F1 pups in this study was unaffected by treatment. Noted clinical observations occurred in single pups or litters and/or in a manner that was not dose-responsive.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The mean number of pups born and the total number of newborn pups at birth in the 0.1, 0.4, and 1.5 mg/m3 groups were similar to the control group values. Postnatal survival in these same groups was unaffected by test material administration. One pup each in the control, 0.1, 0.4, and 1.5 mg/m3 groups were found dead. One pup in the 1.5 mg/m3 group was missing and presumed to have been cannibalized.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean pup body weights and body weight changes during PND 1–13 in the 0.1, 0.4, and 1.5 mg/m3 groups were unaffected by parental exposure to the test substance. Any statistically significant differences from the control group did not occur in a dose responsive manner.

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:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on thyroid hormone values in the F1 males and females at any dose 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.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distances (absolute and relative to the cube root of pup body weight) in the 0.1, 0.4, and 1.5 mg/m3 groups were similar to the control group values. Differences from the control group were slight and not statistically significant.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Areolae/nipple anlagen in the F1 male pups was unaffected by parental exposure to the test material when evaluated on PND 13. None of the male pups were noted with retained areolas/nipples on PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross necropsy did not reveal any remarkable macroscopic findings in pups at the scheduled necropsy on PND 13.

One (1) pup (litters) each in the control, 0.1, 0.4, and 1.5 mg/m3 groups were found dead. No internal findings that could be attributed to parental test material exposure were observed at the necropsies of pups that were found dead. Aside from the presence or absence of milk in the stomach, findings were limited to malformations of small mandible and small mouth in 1 pup in the 0.4 mg/m3 group. No other internal findings were observed.

Histopathological findings:

not examined

Other effects:

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. 1.5 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental Toxicity

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

 

Table 9. Overall Mean Exposure Concentrations
Group	1 (Control – 0 mg/m^3)		2 (0.1 mg/m^3)		3 (0.4 mg/m^3)		4 (1.5 mg/m^3)
	Male	Female	Male	Female	Male	Female	Male	Female
Target Concentration (mg/m^3)	0	0	0.1	0.1	0.4	0.4	1.5	1.5
Mean Concentration (mg/m^3)	0.0	0.0	0.11	0.12	0.44	0.44	1.5	1.5
Standard Deviation	0.00	0.00	0.04	0.04	0.06	0.14	0.11	0.15
Number of Sampling Days	7	15	28	49	28	50	28	48

 

Table 10. Mean Aerosol Particle Size
Group	2 (0.1 mg/m^3)		3 (0.4 mg/m^3)		4 (1.5 mg/m^3)
	Male	Female	Male	Female	Male	Female
Mean MMAD (microns)	1.2	1.4	1.8	1.9	1.3	1.4
Mean GSD	3.56	3.42	2.80	2.67	2.94	3.16
N	4	8	4	8	4	8

 

Table 11. Summary of Organ Weight Data – Terminal Euthanasia^a
	Male				Female
Group	1	2	3	4	1	2	3	4
Dose (mg/m^3)	0	0.1	0.4	1.5	0	0.1	0.4	1.5
No. Animals per Group	10	10	10	10	10	10	10	10
Lung (No. Weighed)^b	(10)	(10)	(10)	(10)	(10)	(10)	(9)	(10)
Absolute value	1.18	11.25	32.45**	54.38**	1.04	14.97**	27.53**	65.28**
% of body weight	0.38	8.03	26.91**	51.01**	0.38	13.57	24.90**	68.13**
% of brain weight	63.35	11.43	32.13**	53.07**	59.27	13.65**	24.95**	63.78**

^a Organ weight values rounded to the appropriate decimal place to account for the size of the organ (values presented to all decimal places in Provantis tables).

^b Control group expressed as mean values; all other values expressed as percent difference from the control group means.

** = Statistically significantly different from the control group at ≤ 0.01 using Dunnett’s or Dunn’s test.

Bold = Values considered to be treatment-related.

 

Table 12. Summary of Microscopic Findings – Terminal Euthanasia
	Male				Female
Group	1	2	3	4	1	2	3	4
Dose (mg/m^3)	0	0.1	0.4	1.5	0	0.1	0.4	1.5
No. Animals per Group	10	10	10	10	10	10	10	10
Lung (No. Examined)	(10)	(10)	(10)	(10)	(10)	(10)	(10)	(10)
Inflammation, mixed cell	0	0	3	10	0	0	8	10
Minimal	-	-	1	1	-	-	3	0
Mild	-	-	2	3	-	-	4	4
Moderate	-	-	0	6	-	-	1	6
Infiltration, mixed cell; alveolus/duct	0	3	3	0	0	8	2	0
Minimal	-	3	3	-	-	8	2	-
Lipoproteinosis	0	0	7	10	0	0	5	10
Minimal	-	-	6	3	-	-	5	0
Mild	-	-	1	7	-	-	0	8
Moderate	-	-	0	0	-	-	0	2
Alveolar macrophages, increased	0	8	10	10	1	8	10	10
Minimal	-	8	7	0	1	8	4	0
Mild	-	0	3	7	0	0	4	0
Moderate	-	0	0	3	0	0	2	9
Marked	-	0	0	0	0	0	0	1
Pigment	0	10	10	10	0	10	10	10
Minimal	-	10	4	0	-	10	2	0
Mild	-	0	6	3	-	0	8	3
Moderate	-	0	0	7	-	0	0	7

- = No noteworthy findings

 

Table 13. Results of Reproductive Performance
Parameter	Dose Level (mg/m^3)				CRL HC^a (Mean Range)
	0	0.1	0.4	1.5
Male Mating Index (%)	100.0	100.0	100.0	100.0	99.4 (94.7 – 100.0)
Female Mating Index (%)	100.0	100.0	100.0	100.0	99.8 (96.0 – 100.0)
Male Fertility Index (%)^b	100.0	100.0	90.0	100.0	94.8 (84.0 – 100.0)
Female Fertility Index (%)^c	100.0	100.0	90.0	100.0	94.5 (84.0 – 100.0)
Male Pregnancy Index (%)^d	100.0	100.0	90.0	100.0	94.2 (84.0 – 100.0)
Female Pregnancy Index (%)^e	100.0	100.0	90.0	100.0	94.4 (84.0 – 100.0)
Estrous Cycle Length (days)	4.22	4.57	4.23	4.70	4.2 (3.6 – 4.8)
Pre-Coital Interval (days)	2.5	2.5	2.3	1.4*	2.7 (1.9 – 3.6)

^a Charles River Ashland historical control data (version 2020.02).

^b Equivalent to Male Copulation Index in the Charles River Ashland historical control data.

^c Equivalent to Female Conception Index in the Charles River Ashland historical control data.

^d Equivalent to Male Fertility Index in the Charles River Ashland historical control data.

^e Equivalent to Female Fertility Index in the Charles River Ashland historical control data.

^* = Statistically significant at 0.05 compared to the control group.

 

Table 14. Summary of Litter Observations: F1 Generation
Sex: Female   Day(s) Relative to Littering (Litter: A)		Dose Level
		Group1 0 (mg/m^3)	Group 2 0.1 (mg/m^3)	Group 3 0.4 (mg/m^3)	Group 4 1.5 (mg/m^3)
Group Size - Females		10	10	10	10
Females with Liveborn	N+ve	10	10	9	10
Viability Index Birth-PND4 (%) [k]	Mean	99.0	99.0	98.14	99.17
	SD	3.16	3.16	3.72	2.64
	N	10	10	9	10
	% Diff	-	0.00	-0.87	0.17
Survival Index PND4Postcull-PND13 (%) [k]	Mean	100.0	100.0	100.0	100.0
	SD	0.00	0.00	0.00	0.00
	N	10	10	9	10
	% Diff	-	0.00	0.00	0.00
Dead Pups PND 0	Sum	0	0	0	0
Dead Pups PND 1	Sum	0	0	1	0
Dead Pups PND 2	Sum	0	0	0	0
Dead Pups PND 3	Sum	1	1	0	1
Dead Pups PND 4	Sum	0	0	0	0
Dead Pups PND 5	Sum	0	0	0	0
Dead Pups PND 6	Sum	0	0	0	0
Dead Pups PND 7	Sum	0	0	0	0
Dead Pups PND 8	Sum	0	0	0	0
Dead Pups PND 9	Sum	0	0	0	0
Dead Pups PND 10	Sum	0	0	0	0
Dead Pups PND 11	Sum	0	0	0	0
Dead Pups PND 12	Sum	0	0	0	0
Dead Pups PND 13	Sum	0	0	0	0

[k] - Kruskal-Wallis & Dunn

Table 15. Summary of Litter Mean Pup Body Weights: F1 Generation
Sex: Female   Day(s) Relative to Littering (Litter: A)			Dose Level
			Group1 0 (mg/m^3)	Group 2 0.1 (mg/m^3)	Group 3 0.4 (mg/m^3)	Group 4 1.5 (mg/m^3)
Males
Mean Pup BW	1 [G]	Mean	6.05	6.30	6.47	6.34
		SD	0.38	0.37	0.60	0.55
		N	10	10	9	10
		% Diff	-	4.06	6.85	4.74
	4 [G]	Mean	8.56	9.27	9.41	8.87
		SD	0.74	0.57	1.00	1.05
		N	10	10	9	10
		% Diff	-	8.34	9.92	3.67
	7 [G]	Mean	12.85	13.29	13.82	12.59
		SD	1.02	0.93	1.22	1.55
		N	10	10	9	10
		% Diff	-	3.45	7.56	-1.97
	10 [G]	Mean	17.21	18.00	18.63	16.95
		SD	1.29	1.62	1.40	2.02
		N	10	10	9	10
		% Diff	-	4.64	8.26	-1.50
	13 [G]	Mean	22.01	22.87	23.60	21.78
		SD	1.74	2.38	1.59	1.97
		N	10	10	9	10
		% Diff	-	3.92	7.24	-1.03
Females
Mean Pup BW	1 [G]	Mean	5.77	5.92	6.22	5.97
		SD	0.36	0.30	0.56	0.46
		N	10	10	9	10
		% Diff	-	2.62	7.82	3.50
	4 [G]	Mean	8.20	8.81	9.12	8.50
		SD	0.67	0.39	0.94	0.91
		N	10	10	9	10
		% Diff	-	7.43	11.27	3.63
	7 [G]	Mean	12.25	12.74	13.68*	12.22
		SD	0.90	0.57	1.22	1.39
		N	10	10	9	10
		% Diff	-	4.02	11.73	-0.20
	10 [G]	Mean	16.53	17.25	18.47*	16.61
		SD	1.21	1.24	1.55	1.79
		N	10	10	9	10
		% Diff	-	4.36	11.74	0.50
	13 [G]	Mean	21.33	22.04	23.56*	21.43
		SD	1.63	1.73	1.83	1.81
		N	10	10	9	10
		% Diff	-	3.34	10.47	0.47

[G] - Anova & Dunnett: * = p ≤ 0.05

[G1] - Kruskal-Wallis & Dunn

 

Table 16. Summary of Thyroid Hormone Values: F1 Generation - PND 13 Pups
Day: 13 Relative to Start Date   Dose Group		Thyroid Hormones
		TSH (pg/mL) [G]	T3 (pg/mL) [G1]	T4 (pg/mL) [G]
Male
Group 1 0 mg/m^3	Mean	1071.600	406.3	93360.0
	SD	272.226	41.2	8687.3
	N	10	10	10
Group 2 0.1 mg/m^3	Mean	1272.000	367.7	86650.0
	SD	416.801	58.2	9464.6
	N	10	10	10
	tCtrl	1.19	0.90	0.93
Group 3 0.4 mg/m^3	Mean	1784.222	400.3	108588.9
	SD	724.768	67.7	21612.5
	N	9	9	9
	tCtrl	1.67	0.99	1.16
Group 4 1.5 mg/m^3	Mean	1219.778	411.5	107710.0
	SD	278.897	31.7	24091.1
	N	9	10	10
	tCtrl	1.14	1.01	1.15
Female
		TSH (pg/mL) [G]	T3 (pg/mL) [G]	T4 (pg/mL) [G]
Group 1 0 mg/m^3	Mean	1142.100	415.3	96010.0
	SD	264.306	61.6	12611.8
	N	10	10	10
Group 2 0.1 mg/m^3	Mean	1272.100	388.8	88300.0
	SD	410.568	80.5	12826.3
	N	10	10	10
	tCtrl	1.11	0.94	0.92
Group 3 0.4 mg/m^3	Mean	1887.000**	403.2	100388.9
	SD	688.296	51.3	17220.0
	N	9	9	9
	tCtrl	1.65	0.97	1.05
Group 4 1.5 mg/m^3	Mean	1272.400	381.0	104240.0
	SD	423.117	41.1	12729.5
	N	10	10	10
	tCtrl	1.11	0.92	1.09

Males: [G] - Kruskal-Wallis & Dunn; [G1] - Anova & Dunnett

Females: [G] - Anova & Dunnett: ** = p ≤ 0.01

 

Table 17. Summary of Macroscopic Pathology: F1 Generation - PND 13 Pups
Removal Reason(s): Terminal Euthanasia	Male				Female
Summary: Incidence	Group1 0 (mg/m^3)	Group 2 0.1 (mg/m^3)	Group 3 0.4 (mg/m^3)	Group 4 1.5 (mg/m^3)	Group1 0 (mg/m^3)	Group 2 0.1 (mg/m^3)	Group 3 0.4 (mg/m^3)	Group 4 1.5 (mg/m^3)
Number of Animals	10	10	9	10	10	10	9	10
GLAND, PARATHYROID
Submitted	10	10	9	10	10	10	9	10
No. of Visible Lesions	10	10	9	10	10	10	9	10
GLAND, THYROID
Submitted	10	10	9	10	10	10	9	10
No. of Visible Lesions	10	10	9	10	10	10	9	10

 

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the lack of effects on F0 male and female reproductive performance and F1 litters, an exposure level of 1.5 mg/m3 was considered to be the No-Observed-Adverse-Effect-Concentration (NOAEC) for F0 reproductive toxicity and F1 neonatal toxicity of MWCNT NC-7000™ when administered by nose-only inhalation exposure to Crl:WI(Han) rats. The NOAEC for F0 systemic toxicity was considered to be 0.1 mg/m3 based on adverse lung findings noted at pathology in the 0.4 and 1.5 mg/m3 groups.

Executive summary:

A key OECD Guideline 421 reproductive / developmental toxicity screening study was conducted to evaluate the potential adverse effects of the test material (MWCNT NC-7000™), on male and female reproduction, when administered to Crl:WI(Han) rats via nose-only inhalation. This encompassed gonadal function, mating behavior, conception, parturition, and lactation of the parental generation and the development of offspring from conception through Day 13 of postnatal life. 

Crl:WI(Han) rats (10/sex/concentration) were exposed to the test material at concentrations of 0, 0.1, 0.4, or 1.5 mg/m³ in air via nose-only inhalation for 6 hours daily. Male rats were exposed for 14 days prior to mating and continuing through 1 day prior to euthanasia, for a minimum of 28 days. Female rats were exposed for 14 days prior to mating and continuing through Lactation Day 12. Exposure was suspended from Gestation Day 21 through Lactation Day 4, inclusively. The control group received filtered air only.

 

Mortality, clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen retention, thyroid hormones, macroscopic findings, organ weights, and microscopic examinations were evaluated in the study.

 

All F0 males and females survived to the scheduled necropsies and no treatment-related clinical observations were observed at any concentration at the daily examination or 1–2 hours following exposure. No treatment-related effects on body weight, body weight gain, food consumption, estrous cycles, reproductive performance, gestation length, parturition findings, or thyroid hormone levelswere observed in F0 males and/or females at any concentration.

 

Treatment-related dose-dependent findings observed in the lungs included mixed cell inflammation or infiltration, lipoproteinosis, increased alveolar macrophages containing proteinaceous fluid and/or pigment, and black pigment consistent with the test material. Findings were considered adverse at the 0.4 and 1.5 mg/m³ concentration levels due to the mild to moderate severity of inflammation and lipoproteinosis observed at these dosages. Higher mean lung weights observed were due to the increased cellularity (mixed cell inflammation, infiltration, and/or increased alveolar macrophages) and/or lipoproteinosis within the lungs. Gross observation of dark red discoloration correlated with the mixed cell inflammation. The increase in alveolar macrophages was considered to be in response to inhaled test material, was adaptive, and therefore, non-adverse. The pigment free or in alveolar spaces was observed across all dosage groups, considered to represent inhaled test material, and was non-adverse. A single male rat in the 1.5 mg/m³ group exhibited an enlarged tracheobronchial lymph node at gross examination. However, in the absence of a microscopic correlate this finding was considered to be secondary to changes within the lung.

 

In the F1 litters, no treatment-related effects on the mean number of pups born, mortality, clinical observations, body weights, body weight changes, anogenital distance (absolute and relative to the cube root of pup body weight), areolae/nipple retention, thyroid hormone levels, or macroscopic findings were observed at any concentration.

 

In conclusion, based on the lack of effects on F0 male and female reproductive performance and F1 litters, an exposure level of 1.5 mg/m3 was considered to be the No-Observed-Adverse-Effect-Concentration (NOAEC) for F0 reproductive toxicity and F1 neonatal toxicity of MWCNT NC-7000™ when administered by nose-only inhalation exposure to Crl:WI(Han) rats. The NOAEC for F0 systemic toxicity was considered to be 0.1 mg/m3 based on adverse lung findings noted at pathology in the 0.4 and 1.5 mg/m3 groups.