Trifluoroiodomethane

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Feb 2006 to 16 Mar 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 Feb 2006 to 16 Mar 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

adopted 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD 486 (Genetic Toxicology: Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in Vivo).

Version / remarks:

adopted 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

adopted 12 May 1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

TNO Quality of Life, Utrechtseweg 48, P.O. Box 360, 3700 AJ Zeist, The Netherlands

Type of assay:

mammalian bone marrow chromosome aberration test

Specific details on test material used for the study:

Name as cited in study report: CF3I
Colour / appearance: clear colourless liquefied gas
CAS no.: 2314-97-8
Purity: 99.99%
Batch number: BU-342
Storage conditions: ambient temperature
Expiry date: 12 December 2006

A second batch of test material was also used:
Batch number: BV-046-2
Expiry date: 1 February 2007

Species:

rat

Strain:

Wistar

Remarks:

Crl:[WI]WU BR

Details on species / strain selection:

The rat was used as test system, because this species is routinely used at the testing facility for this type of studies and accepted by the relevant authorities.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Choice of animals: Based on information provided by the sponsor, there was no apparent difference in toxicity in male and female animals in previously performed studies with the test substance. Therefore, the CAT was performed in males only.
- Age at study initiation: At the commencement of the treatment period, the rats were 9 weeks old
- Weight at study initiation: Mean body weights of the animals of the 4-week study at the start of treatment (day 0) were 265 g
- Fasting period before study: no
- Housing: Macrolon cages with a bedding of wood shavings (Espen E-001; ABEDD, Köflach, Austria), five rats per cage
- Diet: Feed was provided ad libitum from the arrival of the rats until the end of the study, except during exposure and during overnight fasting prior to necropsy. The animals received a commercial rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) obtained from SDS Special Diets Services, Witham, England.
- Water: Drinking water was provided ad libitum from the arrival of the rats until the end of the study, except during exposure and during overnight fasting prior to necropsy. Tap-water was supplied in polypropylene bottles which were cleaned about weekly and filled up when necessary. Tap water suitable for human consumption (quality guidelines according to Dutch legislation based on EEC Council Directive 98/83/EEC) was supplied by N.V. Hydron Midden-Nederland.
- Acclimation period: 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40 to 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: gas

Vehicle:

- Vehicle used: clean air
Because of the relatively high concentration of test material in the mid- and high-concentration test atmospheres, an additional mass flow controlled stream of oxygen was added to ensure a sufficiently high oxygen concentration.

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Animals were exposed to the test atmosphere in nose-only exposure units. Each unit consisted of a cylindrical PVC column with a volume of ca. 70 litres, surrounded by a transparent hood. The test atmosphere was introduced at the bottom of the central column, and was exhausted at the top. Each column Included two rodent tube sections and each rodent tube section had 20 ports for animal exposure. Additional or empty ports were used for test atmosphere sampling and measurement of temperature, relative humidity and oxygen concentration.
- Method of holding animals in test chamber: The animals were secured in plastic animal holders (Battelle), positioned radially through the outer hood around the central column. The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. In our experience, the animal's body does not exactly fit in the animal holder which always results in some leakage from the high to the low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer hood, which encloses the entire animal holder, air leaks from nose to thorax rather than from thorax to nose and dilution of test atmosphere at the nose of the animals is prevented.
- Method of conditioning air: The test atmosphere for each exposure level was generated by mixing a mass flow controlled amount of gaseous test material with a mass flow controlled stream of humidified compressed air. Because of the relatively high concentration of test material in the mid- and high-concentration test atmospheres, an additional mass flow controlled stream of oxygen was added to ensure a sufficiently high oxygen concentration. The exposure unit for the control animals was supplied with a mass flow controlled stream of humidified compressed air only. The flow from the humidified compressed air and from the oxygen at the settings chosen for the respective test atmospheres was measured usmg volumetric flow meters (DryCal, Bios International Corporation, Butler, NJ, USA). These flows were used to calculate the flows of test material necessary to reach the target concentrations.
- Temperature, humidity, pressure in air chamber: Mean temperature (± standard deviation) was 22.7 (± 0.2), 22.3 (± 0.2), 22.3 (± 0.3) and 22.1 (± 0.2)° C for the control, 10,000 ppm, 20,000 ppm and 40,000 ppm exposure conditions, respectively. Measured minimum and maximum temperatures were 20.2 and 23.7 °C, respectively. Mean relative humidity (± standard deviation) was 42 (± 1), 43 (± 1), 40( ± 1) and 40 (± 1) %, respectively. Measured minimum and maximum relative humidity was 34 and 57 %, respectively. Oxygen concentrations were measured m the chambers dunng preliminary generation tests and were 20.6 %, 20 5 % and 20.6 %, for the 10,000 ppm, 20,000 ppm and 40,000 ppm exposure conditions, respectively. The oxygen concentration in the laboratory room was 20.7 %.
- Air flow rate: The mean total daily airflows through the exposure units were 15.2, 20.1 and 20.2 L/min for the low, mid and high concentration test atmosphere, respectively. For the control unit, the same exposure setting was used each day which was 20.0 L/min.

TEST ATMOSPHERE
- Brief description of analytical method used: The temperature and the relative humidity of the test atmospheres were measured continuously and recorded every minute during exposure using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany)
- Samples taken from breathing zone: yes
MEASUREMENT OF THE ACTUAL CONCENTRATION
- The concentration of test substance in the test atmospheres was measured by total carbon analysis with a flame ionisation detector. The test atmospheres were sampled from the exposure units at the animals' breathing zone and were passed to the total carbon analysers. One total carbon analyser was used for the low concentration test atmosphere (RS55, Ratfisch, Germany) and a second was alternated every 5 minutes between the mid and high concentration test atmospheres (RS55-T, Ratfisch, Germany), respectively. The response of the analysers was recorded on a PC every minute using a CAN transmitter (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany). The daily mean response for each exposure unit was calculated by averagmg values read every minute for the low concentration test atmosphere and averaging the last three values of every five minute sampling period.
Before the start of exposure, the output of the total carbon analysers was calibrated using mass flow controlled mixtures of humidified air, gaseous test material and, in case of the mid and high concentration test atmospheres, oxygen. The output of each mass flow controller was measured at the settings used with a volumetric flow meter (DryCal, Bios International Corporation, Butler, NJ, USA). The concentrations achieved (m ppm) were calculated from the ratios of the flows measured by the TNO volumetric flow meter. The analysers were presented with 3 concentrations per exposure level (at the target concentration and approximately 20% below and 20% above the target concentration). The response of the total carbon analysers was recorded twice at each calibration level used.
- Linear relations between the response Y of the analysers (in % of full scale) and the concentration X of the test material (in ppm) were found and used to were used to convert the readings of the total carbon analysers to test atmosphere concentrations.
- The mass flow controllers were checked weekly during the study with the volumetric flow meter by recording the flows at the settings used during the study. The maximum deviation of the calculated concentrations from the concentrations calculated initially, before the start of the study, was 3.4%. In addition, the output of the TCA's at each target concentration was compared with the calculated concentration. The maximal difference was 4.0%.

MEASUREMENT OF THE NOMINAL CONCENTRATION
The nominal concentration was calculated as the ratio of the flow of test material and the total flow, consisting of the flows of humidified air, test material and, in case of the mid and high concentrations, oxygen. In addition, the amount of test material used during the study, detennined as the weight difference of the cylinder before the start of the study and at the end of the exposure period, was compared to the amount used as calculated from the measured concentration and the total flow of test atmosphere in the four exposure units during the exposure.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours/day, 5 days/week during a period of 4 weeks, resulting in a total number of 20 exposure days

Post exposure period:

Two hours before sacrifice, the animals were given an intraperitoneal injection with colchicine (4 mg/kg bw) in a volume of 10 mL saline/kg-bw, to accumulate cells in the metaphase stage of their cell cycle. Signs of reactions to treatment in the positive control group were recorded once between 1-4 h post treatment.

Dose / conc.:

10 000 ppm

Dose / conc.:

20 000 ppm

Dose / conc.:

40 000 ppm

No. of animals per sex per dose:

Chromosomal aberration test (CA) test: 5 male animals / group including positive control group

Control animals:

yes, concurrent vehicle

Positive control(s):

Intraperitoneal injection with the mutagen Mitomycin C (Mit.C); 0.3 mg/kg bw in a volume of 10 mL saline/kg bw

Tissues and cell types examined:

bone marrow smears

Details of tissue and slide preparation:

Bone marrow sampling:
After sacrifice, the femurs were dissected free of all adherent muscle and tissue. Bone marrow cells were removed from the femurs by flushing with Hank's balanced salt solution (HBBS), exposed to a hypotonic solution (0.075 M potassium chloride, prewarmed to 37 °C) and fixed in freshly prepared 3:1 (v/v) mixture of methanol and glacial acetic acid and processed for chromosomal preparations. The slides were stained with a 2 % Giemsa (Merck-Darmstadt, Damstadt, Germany) solution, air-dried and embedded. Two slides per animal were prepared for the mitotic index scoring and the chromosomal aberration analysis.

Microscopic examination of bone marrow smears:
The slides were randomly coded by a qualified person not involved in the scoring of the slides to enable "blind" scoring. Two slides per animal were examined. One thousand cells (500 cells per slide) were examined to determine the percentage of cells in mitosis (mitotic index). Of each animal, 100 well-spread metaphases (50 metaphases per slide), each containing 40-42 centromeres, were analysed by microscopic examination for both chromatid-type and chromosome-type aberrations and other anomalies such as endoreduplicated cells, polyploid cells or heavily damaged cells.
Endoreduplicated cells, polyploid cells and heavily damaged cells were recorded but not included in the 100 analysed cells per animal. The Vernier readings of all aberrant metaphases observed were recorded.

Evaluation criteria:

- The study was considered valid if the positive control group gave a statistically significant increase in the percentage of cells with structural chromosomal aberrations (in the whole group), when compared 10 the percentage found in the concurrent control group (in the whole group) and if the percentage of aberrant cells, found in the negative control group (in the whole group), were within the historical range.
- There are several criteria for determining a positive result, such as a dose-related increase in the relative number of cells with structural chromosomal aberrations or a clear increase in the number of cells with structural chromosomal aberrations in a single dose group at a single sampling time. Positive results from the in vivo chromosomal aberration test indicate that a test substance induces structural chromosomal aberrations in the bone marrow of the species tested.
- A test substance was considered to be negative in the in vivo chromosomal aberration test if it produces no increase in the number of cells with structural chromosomal aberrations at any of the concentrations and test points analysed. Negative results from the in vivo chromosomal aberration test indicate that a test substance does not induce structural chromosomal aberrations in the bone marrow of the species tested.
- Statistical significance will not be the only determining factor for a positive response. For the evaluation of the test results, the biological relevance will be considered first.

Statistics:

The statistical procedures used in the evaluation of data were as follows:
- For the mitotic index, data were analysed by Analysis of Variance (ANOVA), if necessary after square root transformation (sqrt(x+1)) to 'normalise' the distribution of the counts (Lovell et al., 1989). If the ANOVA yielded significant results, pairwise comparisons between treated and control groups were made. Data from groups A, B, C, D and F were analysed statistically by a non-parametric Anova (Kruskal-WaIIis).
- For the chromosomal aberrations, data from groups A, B, C, D and F were analysed by Fisher's exact probability test (two-sided) to determine significant differences between the treated and control groups.
- All statistical tests were performed using BMDP statistical software (W.J. Dixon, BMDP Statistical Software Manual, University of California Press, Berkeley, 1992).

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The substance was cytotoxic to the bone marrow in a concentration-related way but did not induce structural chromosomal aberrations in the bone marrow.

Conclusions:

It is concluded that the test substance, up to 40000 ppm, was cytotoxic to the bone marrow, but did not induce structural chromosomal aberrations in the bone marrow cells of male rats, under the conditions used in this study.

Executive summary:

The inhalation toxicity of the substance was studied in a sub-acute (4-week) study in Wistar rats, according to OECD guideline 412 and GLP principles and OECD 475 (Mammalian bone marrow chromosome aberration test). Groups of 5 male and 5 female rats were exposed nose-only to target concentrations of 0 (clean air control) 10,000 ppm, 20,000 ppm, or 40,000 ppm for 6 hours a day, 5 days a week during a 4-week period, with a total number of 20 exposure days. Bone marrow of all male animals was used for analysis of chromosomal aberrations. The rats of the positive control group were treated intraperitoneally with the mutagen Mitomycin C (10 mL/kg bw, 0.3 mg/mL) ca. 24 h before necropsy. At ca. 2-3 hours prior to sacrifice, all animals were injected intraperitoneally (10 mL/kg-bw) with colchicine at a concentration of 4 mg/kg-bw to accumulate metaphase cells in the bone marrow. The test substance, up to 40000 ppm, was cytotoxic to the bone marrow, but did not induce structural chromosomal aberrations in the bone marrow cells of male rats, under the conditions used in this study.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

14 Feb 2006 to 16 Mar 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

adopted 12 May 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD 475 (Genetic Toxicology: Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

adopted 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD 486 (Genetic Toxicology: Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in Vivo). 

Version / remarks:

adopted 21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

TNO Quality of Life, Utrechtseweg 48, P.o. Box 360, 3700 AJ Zeist, The Netherlands

Limit test:

no

Specific details on test material used for the study:

Name as cited in study report: CF3I
Colour / appearance: clear colourless liquefied gas
CAS no.: 2314-97-8
Purity: 99.99%
Batch number: BU-342
Storage conditions: ambient temperature
Expiry date: 12 December 2006

A second batch of test material was also used:
Batch number: BV-046-2
Expiry date: 1 February 2007

Species:

rat

Strain:

Wistar

Remarks:

Crl:[WI]WU BR

Details on species / strain selection:

The rat was used as test system, because this species is routinely used at the testing facility for this type of studies and accepted by the relevant authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: At the commencement of the treatment period, the rats were 9 weeks old
- Weight at study initiation: Mean body weights of the animals of the 4-week study at the start of treatment (day 0) were 265 and 175 g for male and female animals, respectively.
- Housing: Macrolon cages with a bedding of wood shavings (Espen E-001; ABEDD, Köflach, Austria), five rats per cage, separated by sex
- Diet: Feed was provided ad libitum from the arrival of the rats until the end of the study, except during exposure and during overnight fasting prior to necropsy. The animals received a commercial rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) obtained from SDS Special Diets Services, Witham, England.
- Water: Drinking water was provided ad libitum from the arrival of the rats until the end of the study, except during exposure and during overnight fasting prior to necropsy. Tap-water was supplied in polypropylene bottles which were cleaned about weekly and filled up when necessary. Tap water suitable for human consumption (quality guidelines according to Dutch legislation based on EEC Council Directive 98/83/EEC) was supplied by N.V. Hydron Midden-Nederland.
- Acclimation period: The duration of the acclimatization period until the experimental start date was 6 days.

DETAILS OF FOOD AND WATER QUALITY:
- Each batch of the diet was analysed by the supplier for nutrients and contaminants.
- The supplier of the test water periodically (twice a year) analysed water samples at the test laboratory location.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40 to 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: gas

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks:

Because of the relatively high concentration of test material in the mid- and high-concentration test atmospheres, an additional mass flow controlled stream of oxygen was added to ensure a sufficiently high oxygen concentration.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Animals were exposed to the test atmosphere in nose-only exposure units. Each unit consisted of a cylindrical PVC column with a volume of ca. 70 litres, surrounded by a transparent hood. The test atmosphere was introduced at the bottom of the central column, and was exhausted at the top. Each column Included two rodent tube sections and each rodent tube section had 20 ports for animal exposure. Additional or empty ports were used for test atmosphere sampling and measurement of temperature, relative humidity and oxygen concentration.
- Method of holding animals in test chamber: The animals were secured in plastic animal holders (Battelle), positioned radially through the outer hood around the central column (males and females alternated). The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. In the laboratories experience, the animal's body does not exactly fit in the animal holder which always results In some leakage from the high to the low pressure side. By securing a positive pressure in the central column and a slightly negative pressure m the outer hood, which encloses the entire animal holder, air leaks from nose to thorax rather than from thorax to nose and dilution of test atmosphere at the nose of the animals is prevented.
- Method of conditioning air: The test atmosphere for each exposure level was generated by mixing a mass flow controlled amount of gaseous test material with a mass flow controlled stream of humidified compressed air. Because of the relatively high concentration of test material in the mid- and high-concentration test atmospheres, an additional mass flow controlled stream of oxygen was added to ensure a sufficiently high oxygen concentration. The exposure unit for the control animals was supplied with a mass flow controlled stream of humidified compressed air only. The flow from the humidified compressed air and from the oxygen at the settings chosen for the respective test atmospheres was measured usmg volumetric flow meters (DryCal, Bios International Corporation, Butler, NJ, USA). These flows were used to calculate the flows of test material necessary to reach the target concentrations.
- Temperature, humidity, pressure in air chamber: Mean temperature (± standard deviation) was 22.7 (± 0.2), 22.3 (± 0.2), 22.3 (± 0.3) and 22.1 (± 0.2)° C for the control, 10,000 ppm, 20,000 ppm and 40,000 ppm exposure conditions, respectively. Measured minimum and maximum temperatures were 20.2 and 23.7° C, respectively. Mean relative humidity (± standard deviation) was 42 (± 1), 43 (± 1), 40( ±1) and 40 (±1) %, respectively. Measured minimum and maximum relative humidity was 34 and 57%, respectively. Oxygen concentrations were measured m the chambers dunng preliminary generation tests and were 20.6%, 20 5% and 20.6%, for the 10,000 ppm, 20,000 ppm and 40,000 ppm exposure conditions, respectively. The oxygen concentration in the laboratory room was 20.7%.
- Air flow rate: The mean total daily airflows through the exposure units were 15.2, 20.1 and 20.2 L/min for the low, mid and high concentration test atmosphere, respectively. For the control unit, the same exposure setting was used each day which was 20.0 L/min.

TEST ATMOSPHERE
- Brief description of analytical method used: The temperature and the relative humidity of the test atmospheres were measured continuously and recorded every minute during exposure using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany)
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

MEASUREMENT OF THE ACTUAL CONCENTRATION
- The concentration of test substance in the test atmospheres was measured by total carbon analysis with a flame ionisation detector. The test atmospheres were sampled from the exposure units at the animals' breathing zone and were passed to the total carbon analysers. One total carbon analyser was used for the low concentration test atmosphere (RS55, Ratfisch, Germany) and a second was alternated every 5 minutes between the mid and high concentration test atmospheres (RS55-T, Ratfisch, Germany), respectively. The response of the analysers was recorded on a PC every minute using a CAN transmitter (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany). The daily mean response for each exposure unit was calculated by averagmg values read every minute for the low concentration test atmosphere and averaging the last three values of every five minute sampling period.
Before the start of exposure, the output of the total carbon analysers was calibrated using mass flow controlled mixtures of humidified air, gaseous test material and, in case of the mid and high concentration test atmospheres, oxygen. The output of each mass flow controller was measured at the settings used with a volumetric flow meter (DryCal, Bios International Corporation, Butler, NJ, USA). The concentrations achieved (m ppm) were calculated from the ratios of the flows measured by the TNO volumetric flow meter. The analysers were presented with 3 concentrations per exposure level (at the target concentration and approximately 20% below and 20% above the target concentration). The response of the total carbon analysers was recorded twice at each calibration level used.
- Linear relations between the response Y of the analysers (in % of full scale) and the concentration X of the test material (in ppm) were found and used to were used to convert the readings of the total carbon analysers to test atmosphere concentrations.
- The mass flow controllers were checked weekly during the study with the volumetric flow meter by recording the flows at the settings used during the study. The maximum deviation of the calculated concentrations from the concentrations calculated initially, before the start of the study, was 3.4%. In addition, the output of the TCA's at each target concentration was compared with the calculated concentration. The maximal difference was 4.0%.

MEASUREMENT OF THE NOMINAL CONCENTRATION
The nominal concentration was calculated as the ratio of the flow of test material and the total flow, consisting of the flows of humidified air, test material and, in case of the mid and high concentrations, oxygen. In addition, the amount of test material used during the study, detennined as the weight difference of the cylinder before the start of the study and at the end of the exposure period, was compared to the amount used as calculated from the measured concentration and the total flow of test atmosphere in the four exposure units during the exposure.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours/day, 5 days/week during a period of 4 weeks, resulting in a total number of 20 exposure days

Dose / conc.:

10 000 ppm

Dose / conc.:

20 000 ppm

Dose / conc.:

40 000 ppm

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The concentration levels were supplied by the sponsor

Positive control:

Only for the UDS and CA test (please refer to section 7.6.2)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. A group-wise observation was made halfway during each exposure day. On working days, all cages were checked again in the afternoon. At weekend days only one check per day was carried out.

BODY WEIGHT:
The body weight of each animal was recorded one day before the start of exposure (day -1), at initiation of treatment (day 0) and generally weekly thereafter (days 7, 14, 21, 27). In addition, animals were weighed before sacrifice for calculation of relative organ weights.

FOOD CONSUMPTION AND FOOD EFFICIENY:
Food consumption was measured per cage, over successive periods of 7, 7, 7 and 6 days. The results were expressed in g per animal per day. The efficiency of food utilization was calculated and expressed in g weight gain per g food consumed

HAEMATOLOGY:
- Time schedule for collection of blood: These examinations were carried out in blood samples taken at necropsy (nominal day 28) from the abdominal aorta.
- Anaesthetic used for blood collection: Nembutal anaesthesia
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked: K2-EDTA was used as anticoagulant. The following determinations were carried out: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time and thrombocyte count
- The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Clinical chemistry determinations were conducted at the end of the treatment period at the same time blood samples for haematology were collected.
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: Blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. The following measurements were made in the plasma: alkaline phosphatase activity (ALP), bilirubin total, aspartate aminotransferase activity (ASAT), cholesterol, alanine aminotransferase activity (ALAT), triglycerides, gamma glutamyl transferase activity (GGT), phospholipids, total protein, calcium (Ca), albumin, sodium (Na), ratio albumin to globulin, potassium (K), urea, chloride (Cl), creatinine, fasting glucose and inorganic phosphate.

Sacrifice and pathology:

SACRIFICE:
At the end of the exposure period, all animals were killed in such a sequence that the average time of killing was approximately the same for each group. Animals were killed on nominal day 28. Animals were killed by exsanguination from the abdommal aorta under Nembutal anaesthesia

GROSS PATHOLOGY:
All animals were examined grossly for pathological changes.

ORGAN WEIGHTS
The following organs were weighed (paired organs together) as soon as possible after dissection (to avoid drying): adrenals, heart, kidneys, liver, brain, spleen, testes, uterus, lungs with trachea and larynx

HISTOPATHOLOGY:
- Samples of the following tissues and organs of all animals were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde (10% solution of formalin). The lungs (after weighing) were infused with the fixative under ca. 15 cm water pressure to insure fixation: adrenals, brain, heart, kidneys, liver, pancreas, spleen, testes, thyroid (with parathyroids), uterus, complete respiratory tract including nasal passages and all relevant gross lesions (i.e. thymus).
- All tissues to be examined microscopically were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin.
- The organs listed above were examined in animals of the high concentration group and control group. In animals of the intermediate groups also the adrenals, spleen and testes were examined. The nasal tissues were examined at 6 levels, the larynx at 3 levels, the trachea at 3 levels (including the bifurcation), and each lung lobe at 1 level.

Other examinations:

Unscheduled DNA Synthesis (UDS) test and Chromosomal Aberration (CA) Test were performed, the details and results on these assays are summarized in section 7.6.2.

Statistics:

The statistical procedures used in the evaluation of data were as follows:
- body weights: one-way analysis of covariance (covariate: body weight on day 0) followed by Dunnett's multiple comparison tests
- food consumption/efficiency: no statistics because of the low number of experimental units per group
- red blood cell and coagulation variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values and organ weights: one-way analysis of variance (Anova) followed by Dunnett's multiple comparison tests
- reticulocytes and relative differential white blood cell counts: KruskaI-Wallis non-parametric Anova followed by Mann-Whitney U-tests
- histopathological changes: Fisher's exact probability test.
All tests were two-sided. Probability values of 0.05 were considered significant.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The group-wise observations during each day's exposure and the individual observations in the mornings, before the start of each day's exposure, revealed no clinical abnormalities. However, in the period from nominal days 3-17 on several exposure days, immediately after exposure, group-wise observations revealed piloerection, coprophagy, reduced food intake, and animals felt cold upon touch. These signs were seen in all groups, controls included, but were not seen on all days. These signs diminished with time and on 2 days, the only sign observed consisted of piloerection in high concentration group animals only.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A concentration-related decrease in body weight was seen in the male animals exposed to 20,000 and 40,000 ppm 7 days after the start of exposure. Thereafter weight gain was seen but this was not as large as in animals of the control and low concentration group, resulting in statistically significantly decreased body weights 14 days after exposure start in male animals exposed to 20,000 ppm and 14, 21 and 27 days after exposure start m male animals exposed to 40,000 ppm. Some body weight decrease was seen In the first week in the female animals of all groups, however, because the control group was included, this was not related to the exposure.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the first week, food consumption was decreased in male and female animals exposed to 20,000 or 40,000 ppm. After the first week, food consumption recovered in the female animals and to a large extent also in the male animals exposed to 20,000 ppm but somewhat less in the male animals exposed to 40,000 ppm.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Food conversion efficiency was negative in the first week in all female groups, including the control group and in in 20,000 and 40,000 ppm male groups.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In female animals, the concentration of thrombocytes was decreased in a concentration related fashion and was significant in the 20,000 and 40,000 ppm group. In males of the high concentration group a decrease was also seen, but this did not reach statistical significance. Haemoglobin was significantly decreased in all exposed female groups and, probably related, also mean corpuscular haemoglobin concentration m the 40,000 ppm female animals. A clear concentration-response in haemoglobin concentration was, however, not observed. White blood cell absolute numbers or the percentage distributions were not significantly different among groups. However, the number of total white blood cells was low in males and females exposed to 40,000 ppm, reaching almost the level of statistical significance males (p=0.055).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Cholesterol was decreased in male and female animals of the 40,000 ppm group. The albumin-globulin ratio was slightly increased in male animals of the 40,000 ppm group. Such a trend, though not significant was also seen in the female animals of the 40,000 ppm group, where it was accompanied by significant decreases in albumin and total protein, the underlying variables. Glucose, alkaline phosphatase and total bilirubin were significantly increased in the female animals of the 20,000 and 40,000 ppm groups. The increase in alkaline phosphatase was concentration related, whereas the increase in glucose and total bilirubin reached a plateau at 20,000 ppm. In the female animals of the 40,000 ppm group, alanine aminotransferase, urea and triglycerides were also increased. Increases in the parameters mentioned, except triglycerides, could also be surmised, though far from statistical significance, in the male animals exposed to 40,000 ppm. The isolated increased level of phospholipids in female animals of the 10,000 ppm group is considered a fortuitous finding.

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

Description (incidence and severity):

- The relative weight of the spleen was significantly decreased in male and female animals of the 40,000 ppm group and in male animals of the 20,000 ppm group. In male animals the decrease included also the absolute weight of the spleen.
- The relative weight of the liver was increased in male and female animals of the 40,000 ppm group. Absolute liver weight was also significantly increased in female animals of the 20,000 and 40,000 ppm groups.
- The slight decrease in relative weight of the brain female animals of the 40,000 ppm group is considered to be a fortuitous finding partly related to differences in body weight.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Males exposed to 40,000 ppm showed a smaller thymus than the other animals. This was also seen in two females of this group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Microscopic examination revealed treatment-related histopathological changes in the adrenals and spleen of both male and female, and in the testes of male animals exposed to 40,000 ppm. The adrenals showed increased vacuolation in the zona fasciculata, which was slightly more prominent in males than in females. The spleen demonstrated very slight to moderate lymphoid depletion of the periarterial lymphocytic sheath (PALS). In the testes of all high concentration group males few multinucleated giant cells were observed, indicative of germ cell degeneration. Microscopic evaluation of the adrenals, spleen and testes of animals exposed to 10,000 or 20,000 ppm did not reveal histopathological changes, except for an increased vacuolation in the zona fasciculata observed in the adrenals of one male exposed to 20,000 ppm.
- The thymus of all males and two females exposed to 40,000 ppm were sampled at necropsy because these appeared small (gross lesion). Histopathological examination of the thymus of these animals demonstrated a moderately to severely decreased size of the medulla, in some cases accompanied by an Increased number of necrotic lymphocytes.
- The other histopathological changes observed were considered to be incidental findings.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The UDS and CA tests were both negative, details are provided in section 7.6.2. In the CA test however, cytotoxicity to bonemarrow (based on a decreased mitotic index) was obeserved.

Dose descriptor:

LOAEL

Effect level:

10 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

food efficiency

haematology

histopathology: non-neoplastic

other: bone marrow cytotoxtcity in all dose groups

Critical effects observed:

yes

Lowest effective dose / conc.:

10 000 ppm

System:

haematopoietic

Organ:

bone marrow

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

40 000 ppm

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

40 000 ppm

System:

endocrine system

Organ:

adrenal glands

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

40 000 ppm

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Analytical verification of test atmosphere

The mean concentrations (± standard deviation) as measured by total carbon analysis, were 9996 (± 27), 20117 ( ± 152) and 40051 ( ±1 26) ppm, for the low, mid- and high-concentration exposures, respectively. These concentrations were close to the target concentrations. The calculated (nominal) concentrations as determined from the settings of the mass flow controllers were 10033, 19779 and 40053 ppm for the low, mid and high concentration exposures, respectively. The corresponding generation efficiencies were 100%, 102% and 100%.

Conclusions:

The sub-acute study (20 exposures) resulted in a LOAEL of 10,000 ppm, based on the cytotoxicity obsreved in the bone marrow of all dose groups.

Executive summary:

The inhalation toxicity of the substance was studied in a sub-acute (4-week) study in Wistar rats, according to OECD guideline 412 and GLP principles. Groups of 5 male and 5 female rats were exposed nose-only to target concentrations of 0 (clean air control) 10,000 ppm, 20,000 ppm, or 40,000 ppm for 6 hours a day, 5 days a week during a 4-week period, with a total number of 20 exposure days. To examine the toxicity of the test material, data on clinical observations, body weight gain, food consumption, food conversion efficiency, haematology and clinical chemistry were used. In addition, animals were examined grossly at necropsy, organs were weighed, and a selection of organs and tissues (Including the complete respiratory tract and nasal passages) was examined microscopically, and the bone marrow of all male animals was used for analysis of chromosomal aberrations. Finally, additional groups of male animals exposed to the control condition, 20,000 or 40,000 ppm were subjected to liver perfusion at necropsy to harvest cells for an Unscheduled DNA Synthesis (UDS). These genotoxicity endpoints are summarized separately in section 7.6.2.

Daily observations of the animals before each exposure or halfway the exposure period did not reveal any abnormalities. However, on several days during the first two weeks of exposure, after each day's exposure, clinical signs such as piloerection, coprophagy, and reduced food intake were observed and animals felt cold upon touch. These signs were seen m all groups, controls included.

Seven days after exposure start, reduced body weight was seen in male animals exposed to 20,000 or 40,000 ppm. Thereafter weight gain was observed, however, body weights were still significantly lower than in controls 14 days after exposure start (20,000 ppm group), and 14, 21 and 27 days after exposure start (40,000 ppm group). Some body weight decrease was also seen during the first week in all female groups, but this was also seen in controls.

In the first exposure week, food consumption was decreased in males and females exposed to 20,000 or 40,000 ppm; it recovered thereafter in female animals whereas food intake was still lower in these male groups when compared to controls. Food conversion efficiency was negative in males exposed to 20,000 or 40,000 ppm and in all female groups (including controls) during the first week.

Statistically significant changes in red blood cell parameters consisted of a decrease m thrombocytes in females exposed to 20,000 or 40,000 ppm, a decrease in mean corpuscular haemoglobin concentration (MCHC) in females exposed to 40,000 ppm, and a decrease in haemoglobin concentration in all exposed female groups. A clear concentration-related decrease in haemoglobin concentration in these animals was, however, not observed. No such changes were observed in males. Decreased white blood cell numbers were observed in animals exposed to 40,000 ppm; a statistically significant degree was almost reached in male rats.

Statistically significant changes in clinical chemistry parameters included a decrease in cholesterol in male and female animals exposed to 40,000 ppm, an increase in glucose, alkaline phosphatase (ALP), and total bilirubin in female animals exposed to 20,000 or 40,000 ppm, and an Increase in alanine aminotransferase (ALAT), urea and triglycerides in females exposed to 40,000 ppm. Such increases could also be observed m males exposed to 40,000 ppm, but a statistically significant level was not reached. An increase in albumin/globulin ratio was seen in males and females exposed to 40,000 ppm; in males it reached statistical significance, in females it was accompanied by significant decreases in albumin and total protein. Absolute and relative spleen weight was decreased In males exposed to 20,000 or 40,000 ppm, and relative spleen weight in females exposed to 40,000 ppm. Absolute liver weight was increased in females exposed to 20,000 or 40,000 ppm; relative liver weight was increased in males and females exposed to 40,000 ppm. Macroscopic examination at necropsy revealed a smaller thymus in all males and two females exposed to 40,000 ppm. Histopathological treatment-related changes were found in the adrenals and the spleen of males and females exposed to 40,000 ppm, and in the testes of males exposed to 40,000 ppm. These changes consisted of increased vacuolation in the zona fasciculate of the adrenals, which was slightly more prominent in males than in females, very slight to moderate lymphoid depletion of the periarterial lymphocytic sheath (PALS) of the spleen, and a few multinucleated giant cells in the testes, indicative of germ cell degeneration. The changes in the thymus of all males and two females exposed to 40,000 ppm (collected at necropsy because of gross lesions) revealed a moderately to severely decreased size of the medulla, in some cases accompanied by an increased number of necrotic lymphocytes. No other exposure-related findings were observed. In the CA assay the substance was found to be cytotoxic to the bone marrow in a concentration-related way.

In conclusion, because of the changes induced in lymphoid organs, the should be considered an immunotoxic compound. A LOAEL of 10,000 ppm is derived based on the cytotoxicity in bone marrow in all dose groups.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

adopted 12 May 1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

adopted 21 July 1997

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Version / remarks:

adopted 21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

TNO Quality of Life, Utrechtseweg 48, P.o. Box 360, 3700 AJ Zeist, The Netherlands

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

Trifluoroiodomethane

EC Number:

219-014-5

EC Name:

Trifluoroiodomethane

Cas Number:

2314-97-8

Molecular formula:

CF3I

IUPAC Name:

trifluoroiodomethane

Specific details on test material used for the study:

Name as cited in study report: CF3I
Colour / appearance: clear colourless liquefied gas
CAS no.: 2314-97-8
Purity: 99.99%
Batch number: BU-342
Storage conditions: ambient temperature
Expiry date: 12 December 2006

A second batch of test material was also used:
Batch number: BV-046-2
Expiry date: 1 February 2007

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:[WI]WU BR

Details on species / strain selection:

The rat was used as test system, because this species is routinely used at the testing facility for this type of studies and accepted by the relevant authorities.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Choice of animals: Based on information provided by the sponsor, there was no apparent difference in toxicity in male and female animals in previously performed studies with the test substance. Therefore, the UDS test was performed in males only.
- Age at study initiation: At the commencement of the treatment period, the rats were 9 weeks old
- Weight at study initiation: Mean body weights of the animals of the 4-week study at the start of treatment (day 0) were 265 g
- Fasting period before study: no
- Housing: Macrolon cages with a bedding of wood shavings (Espen E-001; ABEDD, Köflach, Austria), five rats per cage
- Diet: Feed was provided ad libitum from the arrival of the rats until the end of the study, except during exposure and during overnight fasting prior to necropsy. The animals received a commercial rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) obtained from SDS Special Diets Services, Witham, England.
- Water: Drinking water was provided ad libitum from the arrival of the rats until the end of the study, except during exposure and during overnight fasting prior to necropsy. Tap-water was supplied in polypropylene bottles which were cleaned about weekly and filled up when necessary. Tap water suitable for human consumption (quality guidelines according to Dutch legislation based on EEC Council Directive 98/83/EEC) was supplied by N.V. Hydron Midden-Nederland.
- Acclimation period: 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40 to 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: gas

Vehicle:

- Vehicle used: clean air
Because of the relatively high concentration of test material in the mid- and high-concentration test atmospheres, an additional mass flow controlled stream of oxygen was added to ensure a sufficiently high oxygen concentration.

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Animals were exposed to the test atmosphere in nose-only exposure units. Each unit consisted of a cylindrical PVC column with a volume of ca. 70 litres, surrounded by a transparent hood. The test atmosphere was introduced at the bottom of the central column, and was exhausted at the top. Each column Included two rodent tube sections and each rodent tube section had 20 ports for animal exposure. Additional or empty ports were used for test atmosphere sampling and measurement of temperature, relative humidity and oxygen concentration.
- Method of holding animals in test chamber: The animals were secured in plastic animal holders (Battelle), positioned radially through the outer hood around the central column. The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. In our experience, the animal's body does not exactly fit in the animal holder which always results in some leakage from the high to the low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer hood, which encloses the entire animal holder, air leaks from nose to thorax rather than from thorax to nose and dilution of test atmosphere at the nose of the animals is prevented.
- Method of conditioning air: The test atmosphere for each exposure level was generated by mixing a mass flow controlled amount of gaseous test material with a mass flow controlled stream of humidified compressed air. Because of the relatively high concentration of test material in the mid- and high-concentration test atmospheres, an additional mass flow controlled stream of oxygen was added to ensure a sufficiently high oxygen concentration. The exposure unit for the control animals was supplied with a mass flow controlled stream of humidified compressed air only. The flow from the humidified compressed air and from the oxygen at the settings chosen for the respective test atmospheres was measured usmg volumetric flow meters (DryCal, Bios International Corporation, Butler, NJ, USA). These flows were used to calculate the flows of test material necessary to reach the target concentrations.
- Temperature, humidity, pressure in air chamber: Mean temperature (± standard deviation) was 22.7 (± 0.2), 22.3 (± 0.2), 22.3 (± 0.3) and 22.1 (± 0.2)° C for the control, 10,000 ppm, 20,000 ppm and 40,000 ppm exposure conditions, respectively. Measured minimum and maximum temperatures were 20.2 and 23.7 °C, respectively. Mean relative humidity (± standard deviation) was 42 (± 1), 43 (± 1), 40( ± 1) and 40 (± 1) %, respectively. Measured minimum and maximum relative humidity was 34 and 57 %, respectively. Oxygen concentrations were measured m the chambers dunng preliminary generation tests and were 20.6 %, 20 5 % and 20.6 %, for the 10,000 ppm, 20,000 ppm and 40,000 ppm exposure conditions, respectively. The oxygen concentration in the laboratory room was 20.7 %.
- Air flow rate: The mean total daily airflows through the exposure units were 15.2, 20.1 and 20.2 L/min for the low, mid and high concentration test atmosphere, respectively. For the control unit, the same exposure setting was used each day which was 20.0 L/min.

TEST ATMOSPHERE
- Brief description of analytical method used: The temperature and the relative humidity of the test atmospheres were measured continuously and recorded every minute during exposure using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany)
- Samples taken from breathing zone: yes

MEASUREMENT OF THE ACTUAL CONCENTRATION
- The concentration of test substance in the test atmospheres was measured by total carbon analysis with a flame ionisation detector. The test atmospheres were sampled from the exposure units at the animals' breathing zone and were passed to the total carbon analysers. One total carbon analyser was used for the low concentration test atmosphere (RS55, Ratfisch, Germany) and a second was alternated every 5 minutes between the mid and high concentration test atmospheres (RS55-T, Ratfisch, Germany), respectively. The response of the analysers was recorded on a PC every minute using a CAN transmitter (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany). The daily mean response for each exposure unit was calculated by averagmg values read every minute for the low concentration test atmosphere and averaging the last three values of every five minute sampling period.
Before the start of exposure, the output of the total carbon analysers was calibrated using mass flow controlled mixtures of humidified air, gaseous test material and, in case of the mid and high concentration test atmospheres, oxygen. The output of each mass flow controller was measured at the settings used with a volumetric flow meter (DryCal, Bios International Corporation, Butler, NJ, USA). The concentrations achieved (m ppm) were calculated from the ratios of the flows measured by the TNO volumetric flow meter. The analysers were presented with 3 concentrations per exposure level (at the target concentration and approximately 20% below and 20% above the target concentration). The response of the total carbon analysers was recorded twice at each calibration level used.
- Linear relations between the response Y of the analysers (in % of full scale) and the concentration X of the test material (in ppm) were found and used to were used to convert the readings of the total carbon analysers to test atmosphere concentrations.
- The mass flow controllers were checked weekly during the study with the volumetric flow meter by recording the flows at the settings used during the study. The maximum deviation of the calculated concentrations from the concentrations calculated initially, before the start of the study, was 3.4%. In addition, the output of the TCA's at each target concentration was compared with the calculated concentration. The maximal difference was 4.0%.

MEASUREMENT OF THE NOMINAL CONCENTRATION
The nominal concentration was calculated as the ratio of the flow of test material and the total flow, consisting of the flows of humidified air, test material and, in case of the mid and high concentrations, oxygen. In addition, the amount of test material used during the study, detennined as the weight difference of the cylinder before the start of the study and at the end of the exposure period, was compared to the amount used as calculated from the measured concentration and the total flow of test atmosphere in the four exposure units during the exposure.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours/day, 5 days/week during a period of 4 weeks, resulting in a total number of 20 exposure days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 000 ppm

Doses / concentrations 2

Dose / conc.:

40 000 ppm

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control group was treated by gavage with the mutagen 2-acetylaminofluorene (2-AAF; 20 mL/kg bw; 2.5 mg/mL in corn oil)

Examinations

Tissues and cell types examined:

hepatocyte cultures

Details of tissue and slide preparation:

Since in one day only a limited number of animals can be handled in the UDS test, the animals were necropsied on several consecutive days, i.e. 14, 15, and 16 March 2006. Dosing of the animals with 2-AAF was done on 15 March 2006 and sacrifices on 16 March 2006. Signs of reactions to treatment (positive control group) were recorded once between 1-4 h post-treatment.

PREPARATION OF HEPATOCYTE CULTURES
Within 24 hours after the last exposure period, negative control animals and exposed animals were sacrificed for isolation of hepatocytes. Because of necessity, on 15 March 2006, two reserve animals of group A (negative control) were taken out of their restraining tubes approx. 3 hours after the start of their clean air exposure and were sacrificed approx. 3 hours later. The positive control animals (2-AAF) were sacrificed 12-16 h after treatment. Hepatocytes were isolated from the liver using the perfusion technique described by Williams et al. (1977) with minor modifications. Briefly, the liver of each rat was perfused in situ with a Ca- and Mg-free HEPES buffer (0.01 M) whilst under Nembutal anaesthesia, followed by an in vitro perfusion with a HEPES-buffered (0.1 M) collagenase solution.

After isolation, the dissociated cells were incubated for 5-10 minutes in a shaking water bath at 37°C. Thereafter, they were filtered over a 200 mesh nylon filter, centrifuged and resuspended in WEC medium [= Williams medium E complete, which consisted of Williams medium E containing Glutamax supplemented with 10 % fetal calf serum and gentamycin (50 µg/mL)]. Cell counts were made with a haemocytometer. The viability of the hepatocytes was determined by trypan blue exclusion to confirm viability greater than 50 % in case of negative control cells.

Suspensions containing 5 x 10^5 cells/mL were prepared in WEC medium. Aliquots (one mL) were seeded onto Themanox 25 mm round plastic cover slips in 35 mm 6-well dishes, which already contained 1 mL of WEC medium. The cultures were then incubated at 37°C in a humidified incubator containing ca 5 % CO2 and 95 % air to allow cells to attach.

LABELLING OF CULTURES
Within 2-4 h after seeding of the cells, the medium was removed and cells were washed twice with 2 mL Williams E medium leaving only attached viable cells. Immediately after washing, 2 mL WEI [= Williams E medium incomplete, which consisted of Williams medium E containing Glutamax, gentamycin (50 µg/mL), hydrocortison (36 µg/mL) and insuline (8 µg/mL)] and 10 µCi methyl]-3H]thymidine (specific activity 5 Ci.mmol^-1 [185 MBq.mmol^-1]) per mL were added to the cultures.

The hepatocyte cultures were incubated for ca 18 hours at 37°C. Thereafter, the cover slips were rinsed in three successive washes of Williams E medium. The cover slips were then immersed in 2 mL of a 1% sodium citrate solution for 10 min to allow the cells to swell. Subsequently, cells were fixed in three 30-min changes of absolute ethanol-acetic acid (3:1), air-dried, and mounted on glass slides.

AUTORADIOGRAPHY
Slides were processed for autoradiography using Ilford K5D emulsion. After 7 and 14 days of exposure to the emulsion at < -18°C, three slides per animal were developed with Kodak D19, fixed in Kodak Fixer and washed with water. They were then stained with haematoxylin and eosin and embedded in Pertex. Two slides of each animal at the better exposure duration (7 days) were selected and coded by a qualified person not involved in analysing the slides to enable 'blind' scoring. The remaining slide of each animal was kept as reserve.

UDS ANAYLIS: GRAIN COUNTING AND CALCULATIONS
An Artek electronic counter with microscopic attachment (Zeiss nucroscope connected to a high resolution TV camera) was used for grain counting in nuclei and cytoplasm. Fifty cells (randomly selected from top to bottom) per slide and 2 slides per animal were counted. For the positive control group three slides of each animal were counted. Cells with abnormal morphology (pyknotic or lysed nuclei) or heavily-labeled S-phase cells were not counted. Cytoplasmic labeling was determined by a duplicate count of a nucleussized area of cytoplasm adjacent to the nucleus. The mean cytoplasmic count was subtracted from the nuclear count to give the net nuclear grams (NNG). To set the nonspecific (background) labeling on the slides, a triplicate count of a nuclei-sized area outside the cells was determined; the mean background labeling was between 5 and 12 grains. These values were not used in further calculations.

A cut-off of NNG = 5 was chosen as a conservative estimate of whether any particular cell is in repair

The following calculations were made for each slide:
a) the population average NNG ± S.D. (cell to cell)
b) the percent of cells in repair
c) the population average NNG ± S.D. for the subpopulation of cells that are in repair

The following calculations were made for each animal:
a) the population average NNG ± S.D. (slide to slide)
b) the percent of cells in repair ± S.D. (slide to slide)

The following calculations were made for each test group:
a) the population average NNG ± S.D. (animal to animal)
b) the percent of cells in repair ± S.D. (animal to animal)

Evaluation criteria:

- The study is considered valid if the positive control gives a positive response and if the negative controls give a clear negative response.
- A response at a data point is considered positive if the population average for NNG ≥ 5 and if at least 20 % of the cells are "in repair".
- A response is considered weakly positive if the population average for NNG is between 0 and 5.
- A test substance is considered to cause DNA damage and Induce DNA repatr in liver cells if the concentration levels result in a positive or weakly positive response.
- A test substance is considered non-genotoxic under the conditions of the test if all concentration levels produce NNG ≤ 0.
- Both numencal significance and biological relevance are considered together in the evaluation

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Clinicals signs, and data on body weight and food intake were sinular to those of the animals of the main study (see sectiion 7.5.2). One animal of the positive control group was slightly lethargic shortly after dosing.

Both the test substance and the negative control (clean air) yielded net nuclear grains (NNG) ≤ 0. Since the test substance did not induce NNG ≥ 5, it is demonstrated that the test substance did not induce unscheduled DNA synthesis in rat hepatocytes.

In this study, the positive control substance 2-AAF did not induce NNG ≥ 5. However, the percentage 'cells in repair' was (slightly) higher than 20%. The mean NNG determined for 2-AAF (-2.05) was clearly higher than the mean NNG of the negative control (-9.94) and the exposure groups (-8.81 and -8.78). In addition, the percentage 'cells in repair' was also clearly higher for the positive control group (21.83%) versus the negative control (0.20%) and the exposure groups (1.00% and 2.50%).
The reason for the less pronounced effect of the positive control substance is unclear. The lower specific activity of the methyl-[3H]thymidine used (5 Ci.mmol^-1 instead of ca. 40-60 Ci.mmol^-1 ) might have resulted in a reduced sensitivity, since the radioactive intensity of the built-in thymidine might be lower. However, in another study the positive control substance 2-AAF did induce a clear positive response (mean NNG = 6.68 and 62 0/0 'cells in repair') with at this specific activity of 5 Ci.mmol^-1. Furthermore, no technical problems with respect to slide analysis were observed, i.e. negative controls and background counts were within historical ranges (historical data (mean ± S.E.M.) negative control: NNG = -7.6 ± 0.6 and 'cells in repair' 1.9 ± 0.6% (n=15); positive control 2-AAF: NNG = 6.2 ± 1.4 and 'cells in repair' 56 ± 7% (n=14-15)). Therefore, there is no reason to assume that the responses observed in the groups exposed to test substance were not correct. Hence, the unscheduled DNA synthesis test was considered to be valid.

Applicant's summary and conclusion

Conclusions:

The test substance did not induce unscheduled DNA synthesis in rat hepatocytes.

Executive summary:

The inhalation toxicity of the substance was studied in a sub-acute (4-week) study in Wistar rats, according to OECD guideline 412 and GLP principles and OECD guideline 486 (Genetic Toxicology: Unscheduled DNA Synthesis (UDS) with Mammalian Liver Cells in Vivo). Groups of 5 male rats were exposed nose-only to target concentrations of 0 (clean air control), 20,000 ppm, or 40,000 ppm for 6 hours a day, 5 days a week during a 4-week period, with a total number of 20 exposure days. These animals were subjected to liver perfusion at necropsy to harvest cells for an Unscheduled DNA Synthesis CUDS). Unscheduled DNA Synthesis (UDS) in liver cells of male rats was not observed.