Trifluoroiodomethane

Carcinogenicity

Description of key information

Chronic toxicity via inhalation (carcinogenicity study in CByB6F1/Tg rasH2 hemizygous transgenic mice): Discriminating concentration = 40,000 ppm

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Aug 2018 to 15 Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: ICH Guidelines S1C(R2), S1B, S1A, M3 (R2), and S4 and CPMP/SWP/1042/99 Rev 1 Corr

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Trifluoroiodomethane (also known as CF3I, trifluoromethyliodide), CAS No. 2314-97-8
Batch/Lot Nos.: Multiple lot numbers and cylinders from each lot used during animal exposures (BH136-100-4, -6, -8, -9, -11 to -14, -16 to -20; BH140-100-1 to -7; BH141-100-8, -10 to -15; BH142-100-16, -21; BH269-100-2 to -6, -9 to -12, -14; BH271-100-18, -20 to -26, -30 to -39)
Receipt Dates: 12 Jul 2018, 23 Aug 2018, 21 Sep 2018, 06 Nov 2018, 30 Nov 2018, and 16 Jan 2019
Expiration Date: 2 years from date of production (2020 for all cylinders)
Physical Description: Clear, colorless gas
Purity: >99.99%
Storage Conditions: Kept in a room with controls set to maintain 18°C to 24°C
Supplier: Sponsor

Species:

mouse

Strain:

other: CByB6F1-Tg(HRAS)2Jic hemizygous mice

Details on species / strain selection:

The CByB6F1-Tg(HRAS)2Jic hemizygous mouse was chosen as the animal model for this study as they are a preferred rodent species for carcinogenicity testing for non-genotoxic or genotoxic compounds.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Biosciences, Inc., Rensselaer, NY
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 17.9 – 27.9 g
- Housing: solid-bottom cages containing appropriate bedding equipped with an automatic watering valve. On arrival, animals were group housed (2 to 3 animals of the same sex) for 2 days. Thereafter, animals were individually housed. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ health.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet 5002 meal was provided ad libitum throughout the study, except during inhalation exposure periods
- Water: Municipal tap water after treatment by reverse osmosis and ultraviolet irradiation was freely available to each animal via an automatic watering system, including during inhalation exposure periods.
- Acclimation period: prior to initiation of exposure, length not specified

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water was performed. It was considered that there were no known contaminants in the feed nor the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 13 Aug 2018 To: 15 Feb 2019

Route of administration:

inhalation: gas

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Remarks:

filtered

Details on exposure:

INHALATION EXPOSURE METHODS
Filtered air and test substance, atmospheres were administered to CByB6F1/Tg rasH2 hemizygous mice as daily 6-hour and 20-minute (to accommodate the T99period), whole-body inhalation exposures on a 5-day per week basis for 26 weeks (minimum of 130 exposures for each animal). Filtered air and test substance exposures were conducted using four, approximately 1000-L stainless-steel and glass whole-body exposure chambers. One chamber was dedicated to each exposure group. Air supplied to the chambers was provided from the Inhalation Department HEPA-and charcoal-filtered, temperature-and humidity-controlled supply air source. All chambers exhaust was directed to the facility exhaust system, which consists of redundant exhaust blowers preceded by activated-charcoal and HEPA-filtration units. Exposure chamber temperature and relative humidity were monitored using a display supplied with a Vaisala probe. Chamber airflow was monitored using a sharp edge orifice meter and Dwyer Magnehelic® Indicating Transmitter pressure gauge. Each gauge was calibrated for conversion from pressure to airflow in standard litres per minute (SLPM) through the use of a Fox Gas mass flowmeter. Temperature, relative humidity, ventilation rate, and negative pressure within the exposure chambers were continuously monitored and recorded at approximately 60-minute intervals through the use of the Inhalation Exposure Data Collection System (WINH) and a personal computer. Daily means and the grand means for exposure chamber temperature, relative humidity, and ventilation rate during the exposure period were determined. Oxygen content of the exposure atmospheres was measured during the method development phase of the study and was 20.9% for all groups.

EXPOSURE ATMOSPHERE GENERATION METHODS
Test substance exposure atmospheres were generated by releasing the neat test substance gas from the original cylinder. A heat blanket controlled using a J-type thermocouple and an Omega® temperature controller was wrapped around the cylinder and set to 34°C to 39°C to maintain head pressure within the cylinder. A 2-stage regulator equipped with a Dwyer digital pressure gauge was used to monitor the outlet pressure of the cylinder. The test substance gas from the regulator was controlled using a needle valve and monitored using an Omega® mass flowmeter. Test substance gas was directed to the chamber inlet, where it was mixed with supply air to achieve the desired atmosphere concentration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

NOMINAL EXPOSURE CONCENTRATIONS
Nominal exposure concentrations were calculated based on the total amount of test substance consumed during each exposure (as weighed prior to and at the termination of the generation) and the total volume of air passed through the chamber during exposure. Total air volume was calculated by multiplying the daily mean chamber ventilation rate by the duration of generation.

EXPOSURE ATMOSPHERE SAMPLING AND ANALYSIS METHODS
Test substance concentration within the exposure chambers was sampled and analysed at approximately 60-minute intervals using a gas chromatograph (GC). Samples were collected from the approximate animal-breathing zone of the exposure chamber via 1/8-inch Teflon™ tubing. Under the control of the WINH system, sampling and analyses were performed as follows. The program controls an external multi-position valve that permits sequential sampling from the exposure room and each exposure chamber. Gas sampling injection onto the chromatography column occurs via an internal gas-sampling valve with a sample loop, the chromatograph is displayed and the area under the sample peak is calculated and stored. The WINH system then acquires the stored peak area data and uses an ln-quadratic equation based on the GC calibration curve to calculate the measured concentration in ppm.

EXPOSURE ATMOSPHERE HOMOGENEITY ASSESSMENT
Homogeneity of exposure atmospheres was evaluated during the method development phase of the study. Four test locations and a reference location were used for these measurements. Samples were collected and analysed on the GC as rapidly as possible alternating from the reference and then to a test location. For each location, the measured concentration was calculated as a percent difference from the reference location. Homogeneity was performed in triplicate for each test substance exposure chamber and was acceptable for this study. Based on mean differences from the reference location, spatial homogeneity was considered acceptable for all exposure chambers. Temporal stability was evaluated using the concentrations from the reference location. The variability during the homogeneity evaluation was acceptable for all exposure chambers. The generation and exposure trial performed before the start of animal exposure also demonstrated acceptable concentration stability for each exposure chamber.

PURITY ANALYSIS
A sample of the bulk test substance was collected from a test substance cylinder prior to initiation of animal exposures. The purity based on total area percent was determined by the Analytical Chemistry Department of Charles River Ashland using a gas chromatographic method with detection by flame ionization.

STABILITY ANALYSIS
Samples of bulk test substance were also collected from a test substance cylinder at least twice during the animal exposure phase and once following completion of the animal exposure phase. The purity of these samples was also measured. For stability assessment, the in use and end of use percent concentrations were compared to the time zero (pre-use) values.

Duration of treatment / exposure:

6 hours/day on a 5 day/week basis for 26 weeks

Frequency of treatment:

5 days/week (minimum of 130 exposures for each animal)

Dose / conc.:

9 662 ppm (nominal)

Remarks:

Group No. 2; Target concentration: 10,000 ppm

Dose / conc.:

18 024 ppm (nominal)

Remarks:

Group No. 3; Target concentration: 20,000 ppm

Dose / conc.:

34 791 ppm (nominal)

Remarks:

Group No. 4; Target concentration: 40,000 ppm

Dose / conc.:

10 045 ppm (analytical)

Remarks:

Group No. 2; Target concentration: 10,000 ppm

Dose / conc.:

20 031 ppm (analytical)

Remarks:

Group No. 3; Target concentration: 20,000 ppm

Dose / conc.:

40 011 ppm (analytical)

Remarks:

Group No. 4; Target concentration: 40,000 ppm

No. of animals per sex per dose:

25/sex/dose for negative control and test substance treated groups
15/sex for positive control group

Control animals:

yes, concurrent vehicle

Details on study design:

JUSTIFICATION OF ROUTE AND EXPOSURE/DOSE LEVELS
The route of administration for the filtered air control and test substance-treated groups was whole-body inhalation exposure since this is a potential route of unintended human exposure. The route of administration for the positive control group was intraperitoneal injection since it is a known route of exposure to illicit specific tumour formation using the positive control substance. The target exposure concentrations for this study were selected by the Sponsor Representative in consultation with the Study Director based on the results of a 4-week range-finding study. In this study, there were no test substance-related deaths, clinical observations, effects on body weight, food consumption, gross macroscopic findings, or changes in clinical pathology parameters. Higher mean heart weight values in the 20,000 ppm group males and females were possibly test substance-related, but were not considered toxicologically significant. There were no other test substance-related effects on organ weights. Therefore, the Maximum Tolerated Concentration was determined to be higher than the highest concentration tested (20,000 ppm) in the 4-week study. The 4-week range-finding study was extended (Phase II) to include the higher exposure concentration of 40,000 ppm. In Phase 2 of the study, at 40,000 ppm there were no effects noted on body weights or body weight gains. There was a slight decrease in food consumption in females, but not males. Additionally, there were no clinical observations noted at 40,000 ppm. There were no effects noted on serum chemistry parameters and only a slight decrease in reticulocyte counts in males and females at 40,000 ppm. Therefore, the Maximum Tolerated Concentration was determined to be higher than the highest concentration tested (40,000 ppm) in the 4-week study. Based on the results of a Phase II study and the maximum human exposure estimate of approximately 3000-4000 ppm and a10X safety factor, exposure concentrations of 0, 10,000, 20,000 and 40,000 ppm were selected for this study.

SENTINAL ANIMALS
Up to 5 animals/sex from the same shipment of animals as those used for the main study were assigned to sentinel groups. These animals were housed in the same room as the animals assigned to the main study, but were not treated. The sentinel animals were used to provide biological samples for diagnosis of potential disease conditions. The sentinel animals were observed for mortality and moribundity twice daily. Detailed physical observations and body weights were recorded at the same intervals as animals assigned to study groups. Sentinel animals that survived to the end of the study or were euthanized in extremis were anesthetized by isoflurane inhalation followed by exsanguination. Blood was collected from the vena cava into tubes containing K2EDTA as an anticoagulant for the preparation of blood smears for possible future evaluation. Further, a terminal blood sample (at least 0.5 mL) was collected via the vena cava into a tube without anticoagulant from animals anesthetized by isoflurane. Animals were then euthanized by exsanguination. Serum prepared from these whole blood samples was stored in a freezer set to maintain a target of -70°C for possible future evaluation. There was no evidence of disease outbreak in the animal room. Therefore, microscopic and serological examinations were not conducted on the sentinel animals.

Positive control:

MATERIAL INFO
N-nitroso-N-methylurea (NMU)
Lot No.: 2HH0073
Receipt Date: 15 Aug 2018
Expiration Date: 15 Aug 2019
Physical Description: Light yellow solid
Storage Conditions: Kept in a refrigerator set to maintain a target of 5°C
Supplier: Spectrum Chemical Manufacturing Corporation

DOSING
75 mg/kg bw (10 mL/kg dose volume, 7.5 mg/mL dose concentration) was administered once to animals in Group 5 by intraperitoneal injection during the first week of the study.

Observations and examinations performed and frequency:

MORTALITY
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. Animals were not removed from the cage during observations, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS
The animals were removed from the cage, and a detailed clinical observation was performed within 4 days of receipt, on the day of randomization, on Day 1 (prior to exposure), weekly (± 2 days) during the study period, and on the day of the scheduled necropsy.

CAGE SIDE OBSERVATIONS
Cage side observations were performed daily, beginning on the day following randomization and continuing throughout the dosing period. During the exposure period, these observations were performed prior to exposure/dosing and 0-1 hour (+ 0.25 hour) post-exposure/dosing. On non-exposure/dosing days, observations were performed once daily. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

BODY WEIGHTS
Animals were weighed individually within 4 days of receipt, on the day of randomization, on Day 1 (prior to exposure), weekly (± 2 days) during the study period, and on the day of the scheduled necropsy. Terminal body weights were not collected from animals found dead.

FOOD CONSUMPTION
Food consumption was quantitatively measured weekly (± 2 days) starting on Day 1 and continued throughout the exposure/dosing and recovery periods.
On Day 57, a female in the 10,000 ppm group was found without a food jar at the time of loading. Following discussion with the Study Director, this animal received a dosing holiday on this day. The prior to load observation remained in the study records and the food consumption for the week was excluded.

CLINICAL PATHOLOGY
Animals were not fasted overnight prior to blood collection. Blood samples for haematology were collected from the retro orbital sinus from animals anesthetized with isoflurane.
K2EDTA was used for the anticoagulant on samples collected for haematology. Samples were collected in week 27 (week 26 for positive control group).

HAEMATOLOGY
Blood samples were analysed for the parameters specified in Table 1 in 'Any other information on materials and method incl. tables' .

Sacrifice and pathology:

TERMINAL PROCEDURES
Terminal procedures are summarized in Table 2 in 'Any other information on materials and methods incl. tables'.

UNSCHEDULED DEATHS
A necropsy was conducted for animals that died on study, and specified tissues were saved.
If necessary for humane reasons, animals were euthanized. A detailed physical examination was conducted, a terminal body weight was recorded. These animals underwent necropsy, and specified tissues were retained.

SCHEDULED EUTHANASIA
Animals surviving until the scheduled euthanasia were weighed and anesthetized by isoflurane inhalation and euthanized by exsanguination. Animals were not fasted prior to the scheduled necropsy.

NECROPSY
Animals were subjected to a complete necropsy examination, which included evaluation of all external surfaces and orifices; the cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

TISSUE COLLECTION AND PRESERVATION
Representative samples of the tissues identified in Table 3 in 'Any other information on materials and method incl. tables' were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated.

HISTOLOGY
Trimming was performed at the Testing Facility. Tissues identified in Table 3 in 'Any other information on materials and method incl. tables' from animals found dead, euthanized in extremis, and in the control and high-dose groups at the terminal necropsy were embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin. In addition, gross lesions and target tissues (lungs) were prepared from all animals in the low- and mid-dose groups at the terminal necropsy.

HISTOPATHOLOGY
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues identified in Table 3 in 'Any other information on materials and method incl. tables' for microscopic examination were evaluated from all animals found dead, euthanized in extremis, and in the control and high-dose groups at the terminal necropsy. In addition, gross lesions and target tissues (lungs) were examined microscopically from all animals in the low- and mid-dose groups at the terminal necropsy.

Statistics:

Statistics Performed by Charles River Ashland
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and are reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Values may also be expressed as a percentage of predose or control values when deemed appropriate. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. Calculated values on Provantis tables may not be reproducible from the individual values presented because all calculations were conducted using non-rounded values.

Statistics Performed by BioSTAT Consultants, Inc.: see 'Any other information on materials and methods incl. tables'

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related clinical observations of thin fur cover on various parts of the body were noted for a small number of males and females in the 40,000 ppm group and a single male in the 20,000 ppm group during the study. There were no other clear test substance-related clinical signs, as any observations noted were in single animals and/or were not noted in a dose-related manner or were of a physical nature and not toxicological in nature. Masses were noted in a single male in the 10,000 ppm group, a single male in the 20,000 ppm group, and a single female in the 40,000 ppm group.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

None of the unscheduled deaths were considered directly related to exposure to the test substance. There were no statistically significant survival findings in the test substance groups when compared to the filtered air control group. Excluding animals administered the positive control substance N-nitrosomethylurea (NMU), the overall incidence of mortality was low (6.0% in control animals and 4.7% in animals exposed to the test substance). The severe alveolar aggregates of macrophages in the lung in one 40,000 ppm group female were not considered test substance-related. Alveolar macrophage aggregates (also referred to as histiocytic pneumonia or macrophage infiltrates) are a spontaneous finding reported for this strain. The cause of death could not be determined for a small number of animals exposed to the test substance. Two males were found dead on Days 170 and 171, respectively, and had localized hemangiosarcomas (subcutis in one and spleen in the other animal) that were not associated with findings that would explain the cause of death (e.g. massive hemorrhage or widespread metastasis). The only other change in the animals was marked or severe decreased cellularity in the thymus. One female exposed to 10,000 ppm test substance was found dead on Day 136. There was moderate decreased cellularity in the thymus and spleen but also no gross or microscopic findings that would explain the cause of death. One female exposed to 20,000 ppm test substance was euthanized on Day 163. The animal had increased hematopoiesis in the spleen (severe) and liver (moderate) but no gross or microscopic changes that would explain the morbidity. One male exposed to 40,000 ppm test substance was found dead on Day 183. The animal had no gross or microscopic changes that would explain the cause of death.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights were unaffected by test substance administration. There were some weekly intervals during the study where there was a statistically significant difference in mean body weight and/or mean body weight change noted in the 10,000, 20,000, and/or 40,000 ppm groups when compared to the control group. These differences were not considered to be test substance related due to a lack of a dose-responsive pattern, the magnitude of change for mean body weight was often small (less than 10% change, most less than 5%), and some groups gained more weight than the controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption was unaffected by test substance administration. There were some weekly intervals during the study where there was a statistically significant difference noted in the 10,000, 20,000, and/or 40,000 ppm groups when compared to the control group. These differences were not considered to be test substance-related due to a lack of a dose-responsive pattern, and the magnitude of change was often small (less than 20% change).

Food efficiency:

not examined

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 test substance-related changes in haematology parameters for males or females in the test substance-exposed group when compared to the concurrent control group. There were statistically significant differences in some parameters (RBC, MCV, MCH and/or RETIC), however the magnitude of the change was small and the mean values was similar to historical control data for CD-1 mice (Version No. 3.6) and were not considered test substance-related.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Review of the gross necropsy observations revealed no observations that were considered to be associated with exposure to the test substance. All gross findings, including those that correlated microscopically with neoplastic and/or non-neoplastic findings, were considered incidental and/or representative of expected background findings in mice of this strain and age, and without relationship to exposure to the test substance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related non-neoplastic histopathology findings.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related histologic changes. All histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than exposure to the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. There were no test substance-related neoplastic or non-neoplastic histopathology findings. Neoplastic findings noted among the mice administered filtered air (negative control) or the test substance occurred sporadically with a low incidence rate that was non-dose responsive. In addition, the neoplastic findings were generally characteristic of those reported to occur in CByB6F1-Tg( HRAS)2Jic hemizygous mice and, therefore, were considered unrelated to test substance administration. There were no statistically significant tumor findings in the test substance groups when compared to the filtered air control group.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

> 40 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Results positive control substance NMU

Observations

Positive control substance-related clinical observations were noted in males and females and included decreased activity, body cold to touch, hunched posture, body thin, pale skin, labored breathing, shallow breathing, and masses present (noted for 10 males and 13 females).

Mortality

There was a statistically significant decrease in survival rates when comparing the positive control group to the filtered air control group for both males and females. The higher incidence of mortality in males (40%) and females (60%) administered NMU (Group 5 positive control group) was primarily due to malignant lymphoma, an expected finding in positive control animals in this model. The cause of death could not be determined for a small number of animals administered positive control substance (NMU).

Haematology

Positive control substance-related changes in haematology parameters were observed for males and females. In females, these changes included increased absolute neutrophil counts, increased reticulocyte counts, decreased platelet counts, decreased RBC, decreased haematocrit, decreased haemoglobin and increased RDW. All of these changes were statistically significant when compared to the concurrent control group. In males, a similar pattern was repeated, however the only change that was statistically significant was decreased haemoglobin. The magnitude of the changes were less in males than in females. These changes were indicative of a response to a known cytotoxic agent and were a result of chronic inflammation (increased neutrophils) and possible initial bone marrow toxicity (decreased RBC and platelets) followed by a regenerative response (increased reticulocytes with no or minor changes in other leukocytes).

Gross pathology

Many of the gross pathology findings were noted in the mice administered NMU (Group 5 positive control group unscheduled and scheduled euthanasias) and included swelling, mass, or enlargement (colon, kidney, liver, lung, lymph nodes, spleen, stomach, subcutis, thymus, uterus, vagina, and vulva). These gross pathology findings generally correlated microscopically with changes expected to occur in CByB6F1-Tg-(HRAS)2Jic hemizygous mice administered NMU. Corresponding histopathology findings to these gross pathology findings included malignant lymphoma in the thymus, spleen, lymph nodes, liver, and kidney; squamous cell papilloma or squamous cell carcinoma in the subcutis, stomach, vagina, or vulva; hemangiosarcoma in the spleen, uterus, or subcutis; and bronchioloalveolar adenoma or carcinoma in the lung.

Histopathological findings

There was a statistically significant increase in the tumors noted when comparing the positive control group with the filtered air control group. As expected, the positive control substance (NMU) produced an increased incidence of malignant lymphoma (33% of males and 60% of females), as well as squamous cell papilloma and/or squamous cell carcinoma (93% of males and 73% of females). These are well characterized and reported NMU-related histopathology changes which validated the CByB6F1-Tg-(HRAS)2Jic hemizygous mouse model used in this study. Other non-proliferative and proliferative microscopic changes observed (e.g., subcapsular cell hyperplasia in the adrenal gland and bronchioloalveolar hyperplasia, adenoma, and carcinoma in the lung) were considered to be of the nature commonly observed in mice of this age and strain.

Conclusions:

Based on the results of this study, when administered by whole-body inhalation for 6 hours/day on a 5 day/week basis for 26 weeks to CByB6F1/Tg rasH2 hemizygous transgenic mice (minimum of 130 exposures for each animal), the test substance was well tolerated at all exposure concentrations. Test substance-related findings were limited to clinical observations of thin fur cover on various parts of the body noted for a small number of males and females in the 40,000 ppm group and a single male in 20,000 ppm group. No evidence of carcinogenicity or tumorigenesis was noted in test substance-exposed groups.

Executive summary:

The objective of this study was to assess the potential carcinogenicity of the test substance. Control (filtered air) and test substance atmospheres were administered to CByB6F1/Tg rasH2 hemizygous transgenic mice as whole-body inhalation for 6 hours/day on a 5 day/week basis for 26 weeks (minimum of 130 exposures for each animal). The positive control substance (N-nitroso-N-methylurea [NMU]) was administered once to animals in Group 5 by intraperitoneal injection during the first week of the study. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, haematology, gross necropsy findings, histopathologic examinations, and tumour incidence.

No test substance-related effects were noted on survival, body weights, body weight gains, food consumption, haematology, gross necropsy findings, histopathologic examinations, or tumour incidence. A test substance-related effect in clinical observations consisted of thin fur cover on various parts of the body noted for a small number of males and females in the 40,000 ppm group and a single male in 20,000 ppm group. No other clear test substance-related clinical findings were noted. Positive control substance-related clinical observations were noted in males and females and included decreased activity, body cold to touch, hunched posture, body thin, pale skin, laboured breathing, shallow breathing, and masses present (noted for 10 males and 13 females). Positive control substance-related changes in haematology parameters were observed for males and females. In females, these changes included increased absolute neutrophil counts, increased reticulocyte counts, decreased platelet counts, decreased RBC, decreased haematocrit, decreased haemoglobin and increased RDW. In males, a similar pattern was repeated, however the only change that was significant was decreased haemoglobin. The magnitude of the changes were less in males than in females. These changes were indicative of a response to a known cytotoxic agent and were a result of chronic inflammation (increased neutrophils) and possible initial bone marrow toxicity (decreased RBC and platelets) followed by a regenerative response (increased reticulocytes with no or minor changes in other leukocytes). As expected, the positive control substance (NMU) produced an increased incidence of malignant lymphoma (33% of males and 60% of females), as well as squamous cell papilloma and/or squamous cell carcinoma (93% of males and 73% of females). These are well characterized and reported NMU-related histopathology changes which validated the CByB6F1-Tg-(HRAS)2Jic hemizygous mouse model used in this study.

Based on the results of this study, when administered by whole-body inhalation for 6 hours/day on a 5 day/week basis for 26 weeks to CByB6F1/Tg rasH2 hemizygous transgenic mice (minimum of 130 exposures for each animal), the test substance was well tolerated at all exposure concentrations. Test substance-related findings were limited to clinical observations of thin fur cover on various parts of the body noted for a small number of males and females in the 40,000 ppm group and a single male in 20,000 ppm group. No evidence of carcinogenicity or tumorigenesis was noted in test substance-exposed groups. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

320 508.8 mg/m³

Study duration:

chronic

Species:

mouse

Quality of whole database:

The study is a guideline study in compliance with GLP.

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information for carcinogenicity, classification is not warranted in according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and its amendments.

Additional information

The objective of this study was to assess the potential carcinogenicity of the test substance. Control (filtered air) and test substance atmospheres were administered to CByB6F1/Tg rasH2 hemizygous transgenic mice as whole-body inhalation for 6 hours/day on a 5 day/week basis for 26 weeks (minimum of 130 exposures for each animal). The positive control substance (N-nitroso-N-methylurea [NMU]) was administered once to animals in Group 5 by intraperitoneal injection during the first week of the study. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, haematology, gross necropsy findings, histopathologic examinations, and tumour incidence.

No test substance-related effects were noted on survival, body weights, body weight gains, food consumption, haematology, gross necropsy findings, histopathologic examinations, or tumour incidence. A test substance-related effect in clinical observations consisted of thin fur cover on various parts of the body noted for a small number of males and females in the 40,000 ppm group and a single male in 20,000 ppm group. No other clear test substance-related clinical findings were noted. Positive control substance-related clinical observations were noted in males and females and included decreased activity, body cold to touch, hunched posture, body thin, pale skin, laboured breathing, shallow breathing, and masses present (noted for 10 males and 13 females). Positive control substance-related changes in haematology parameters were observed for males and females. In females, these changes included increased absolute neutrophil counts, increased reticulocyte counts, decreased platelet counts, decreased RBC, decreased haematocrit, decreased haemoglobin and increased RDW. In males, a similar pattern was repeated, however the only change that was significant was decreased haemoglobin. The magnitude of the changes were less in males than in females. These changes were indicative of a response to a known cytotoxic agent and were a result of chronic inflammation (increased neutrophils) and possible initial bone marrow toxicity (decreased RBC and platelets) followed by a regenerative response (increased reticulocytes with no or minor changes in other leukocytes). As expected, the positive control substance (NMU) produced an increased incidence of malignant lymphoma (33% of males and 60% of females), as well as squamous cell papilloma and/or squamous cell carcinoma (93% of males and 73% of females). These are well characterized and reported NMU-related histopathology changes which validated the CByB6F1-Tg-(HRAS)2Jic hemizygous mouse model used in this study.

Based on the results of this study, when administered by whole-body inhalation for 6 hours/day on a 5 day/week basis for 26 weeks to CByB6F1/Tg rasH2 hemizygous transgenic mice (minimum of 130 exposures for each animal), the test substance was well tolerated at all exposure concentrations. Test substance-related findings were limited to clinical observations of thin fur cover on various parts of the body noted for a small number of males and females in the 40,000 ppm group and a single male in 20,000 ppm group. No evidence of carcinogenicity or tumorigenesis was noted in test substance-exposed groups.