Trifluoroiodomethane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity in bacterial cells (Ames test): Positive

Mutagenicity in mammalian cells: (+/- mouse lymphoma cell mutagenesis assay, similar to OECD 476): negative in the absence and presence of metabolic activation.

Chromosome aberration test in Chinese Hamster Lung (CHL)/IU cells: Positive

Micronucleus test in human peripheral blood lymphocytes: Negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Oct 2018 to 19 Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Qualifier:

according to guideline

Guideline:

other: ISO/IEC 17025:2005

Version / remarks:

2005

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his (Salmonella), trp (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The S9 (Lot No. 3999, Exp. Date: 29 Aug 2020) was purchased commercially from MolTox (Boone, NC).
- method of preparation of S9 mix: Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice.
- concentration S9 in the final culture medium: 10% (v/v)
- quality controls of S9: Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

Test concentrations with justification for top dose:

1.4, 2.8, 6.9, 14, 21, 28 and 37 mmol/L

Vehicle / solvent:

Air

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

air

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

FREQUENCY AND ROUTE OF ADMINISTRATION
The test system was exposed to the test substance via the desiccator methodology, a modification of the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983). The desiccator methodology has been shown to be an effective method for detecting the genotoxic activity of volatile and gaseous test substances (Wagner et al., 1992).

INITIAL TOXICITY-MUTATION ASSAY TO SELECT DOSE LEVELS
The initial toxicity-mutation assay was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the untreated control, positive controls and seven dose levels of the test substance, in duplicate, in the presence and absence of Aroclor-induced rat liver S9. Dose levels for the confirmatory mutagenicity assay were based upon post-treatment toxicity.

CONFIRMATORY MUTAGENICITY ASSAY
The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test substance. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the untreated control, positive controls and seven dose levels of the test substance, in triplicate, in the presence and absence of Aroclor-induced rat liver S9.

UNTREATED AND POSITIVE CONTROL TREATMENT
One half milliliter (0.5 mL) of S9 mix or Sham mix, 100 μL of tester strain and 50.0 μL of positive control were added to 2.0 mL of molten selective top agar at 45±2°C. When plating the untreated controls (2 plates), the addition of positive control was omitted. The mixture was vortex mixed and overlaid onto the surface of a minimal bottom agar plate. After the overlay was solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. One set of air control plates was plated as indicated for the untreated control plates. The overlaid plates was inverted and placed uncovered in the appropriate number of 9-liter desiccators.

TEST SUBSTANCE TREATMENT
One half milliliter (0.5 mL) of S9 mix or Sham mix and 100 μL of tester strain were added to 2.0 mL of molten selective top agar at 45±2°C. The mixture was vortex mixed and overlaid onto the surface of a minimal bottom agar plate. The overlaid plates was inverted and placed uncovered in the appropriate number of 9-liter desiccators. An appropriate quantity of the test substance was introduced into each desiccator by withdrawing an appropriate amount of air and replacing it with test substance. The desiccators were incubated for 24±1 hours at 37±2°C. Following the 24-hour incubation, the plates were removed from the desiccators and incubated with the lids replaced at 37±2°C for a total of 48 to 72 hours.

SCORING
The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the untreated control plate using the codes shown in the following table. As appropriate, colonies were enumerated either by hand or by machine.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:

- Strains TA1535 and TA1537: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean untreated control value and above the corresponding acceptable untreated control range.

- Strains TA98, TA100 and WP2 uvrA: Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean untreated control value and above the corresponding acceptable untreated control range.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

STERILITY RESULTS
No contaminant colonies were observed on the sterility plates for the S9 and Sham mixes.

INITIAL TOXICITY-MUTATION ASSAY
No precipitate was observed. Toxicity was observed as a reduction in revertant counts, in Salmonella strains TA100 and TA1535 in the absence and presence of S9-mix at 37 mmol/L and in TA100 in the absence of S9-mix also at 28 mmol/L and higher. Positive mutagenic responses (2.6- through 20.3- fold, maximum increases) were observed with tester strains TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation and TA1535 in the absence of S9 activation.

CONFIRMATORY MUTAGENICITY ASSAY
Based upon the results of the initial toxicity-mutation assay, the dose levels selected for the confirmatory mutagenicity assay were 1.4, 2.8, 6.9, 14, 21, 28 and 37 mmol/L. No precipitate was observed. Toxicity was observed as a reduction in revertant counts, in Salmonella strains TA100 and TA1535 in the absence and presence of S9-mix at 37 mmol/L and in TA100 in the absence of S9-mix also at 28 mmol/L and higher. Positive mutagenic responses (3.3- through 21.1- fold, maximum increases) were observed with tester strains TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation.

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test substance did cause a positive mutagenic response with TA98, TA1537 and WP2 uvrA in the presence or absence of Aroclor-induced rat liver S9.

Executive summary:

The gaseous test substance was tested to evaluate its mutagenic potential according to OECD TG 471 and in compliance with GLP by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

In the initial toxicity-mutation assay, the dose levels tested were 1.4, 2.8, 6.9, 14, 21, 28 and 37 mmol/L. No precipitate was observed. Toxicity, as a reduction in revertant count, was observed at 28 and/or at 37 mmol/L with tester strains TA100 and TA1535 in the presence and absence of S9 activation. Positive mutagenic responses (2.6- through 20.3- fold, maximum increases) were observed with tester strains TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation and TA1535 in the absence of S9 activation. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 37 mmol/L. In the confirmatory mutagenicity assay, the dose levels tested were 1.4, 2.8, 6.9, 14, 21, 28 and 37 mmol/L. No precipitate was observed. Toxicity, as a reduction in revertant count, was observed at 28 and/or at 37mmol/L with tester strains TA100 and TA1535 in the presence and absence of S9 activation. Positive mutagenic responses (3.3- through 21.1- fold, maximum increases) were observed with tester strains TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation.

These results indicate that the test substance was positive for the ability to induce reverse mutations at selected loci of some strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Mar 1994 to 20 May 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

The test was conducted according to the procedures described by Mitchell et al. (1988).

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro gene mutation study in mammalian cells

Specific details on test material used for the study:

- Name of test substance: iodotrifluoromethane
- Physical state, colorless gas
- Storage condition of test material: in their original steel containers at room temperature

Target gene:

TK +/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The TK+/- heterozygote of L5178Y mouse lymphoma cells was used.

- Source of cells: Dr. Donald Clive, Burroughs Wellcome Co., Research Triangle Park, NC
- Clone 3.7.2C
- Storage: in liquid nitrogen
- Method followed in cell culture: The cells were grown as a suspension culture in FIOHP medium, cleansed of homozygous (TK -/-) cells with medium containing 0.1 µg/ml methotrexate, as described by Mitchell et al., 1988, and used as target cells for chemical exposure.
- Media used: L5178Y cells were cultivated in Fischer's medium for leukemic cells of mice supplemented with 31 Ug/ml penicillin (1650 units/mg), 50 ug/ml streptomycin sulfate, 0.1% Pluronic F68, 0.22 mg/ml sodium pyruvate, 25 mM HEPES buffer, and 10% heat-inactivated horse serum to make FIOHP- FSHP/ containing 5%, rather than 10%, heat-inactivated horse serum, was the medium used for exposures in the presence of exogenous metabolic activation. Fiop was the medium used for exposures in the absence of exogenous metabolic activation and Fiop was used during the expression period. The horse serum concentration was 20% in incomplete cloning medium (ICM), which did not contain agar. BBL agar (0.22%, final concentration) was added to ICM to make complete cloning medium (CCM). The selective cloning medium contained TFT at a final concentration of 1 µg/ml.

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 from Aroclor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

Main test: 79610, 176884, 305887, 425606, 454184, 517777, 496814 ppm (analytical concentrations;based on preliminary cytotoxicity tests):
- In the first preliminary concentration range-finding assay of CF3I, relative suspension growth of the cultures appeared to be slightly depressed at the highest concentration tested, a nominal concentration of 800,000 ppm, but no concentration-related depression in RSG was observed for lower tested concentrations. Because this assay yielded insufficient information for selecting concentrations for mutagenesis testing, the concentration range-finding assay was repeated.
- In the second concentration range-finding assay of CF3I, concentration related depressions in RSG were obtained in the absence and presence of metabolic activation, from 96.2% RSG at 55,000 ppm CF3I to 28.4% RSG at 900,000 ppm CF3I in the absence of activation, and from 70% RSG at 225,000 ppm CF3I to 16.5% RSG at 900,000 ppm CF3I in the presence of activation. Therefore, the mutagenesis assay was conducted over a similar range of concentrations.

Vehicle / solvent:

No vehicle: the closed tubes to be exposed to the CF3I were treated as followes: using a syringe a predetermined volume of air was withdrawn from each tube and an equal volume of the pure test material was added.

Untreated negative controls:

yes

Remarks:

air exposed

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: hycanthone

Details on test system and experimental conditions:

TEST PROCEDURE
Media:
L5178Y cells were cultivated in Fischer's medium for leukemic cells of mice supplemented with 31 µg/mL penicillin (1650 units/mg), 50 µg/mL streptomycin sulfate, 0.1 %. Pluronic F68, 0.22 mg/ml sodium pyruvate, 25 mM HEPES buffer, and 10 % heat-inactivated horse serum to make F10HP. F5HP, containing 5 %, rather than 10 %, heat-inactivated horse serum, was the medium used for exposures in the presence of exogenous metabolic activation. F10P was the medium used for exposures in the absence of exogenous metabolic activation and F10P was used during the expression period. The horse serum concentration was 20 % in incomplete cloning medium (ICM), which did not contain agar. BBL agar (0.22 %, final concentration) was added to ICM to make complete cloning medium (CCM). The selective cloning medium contained TFT at a final concentration of 1 µg/mL.
Exposure of Cell Cultures:
- For testing this volatile material in the preliminary concentration range-finding and mutagenesis assays, three sterile 15 mL round-bottom glass blood tubes, sealed with red rubber serum stoppers, were prepared for each concentration level: a tube for the culture tested without activation, a tube for the culture tested with activation, and a sham tube that contained medium only (no cells or S9) which was used to estimate postexposure infrared (IR) analysis of the concentration of test material in the other two tubes.
- To provide a maximum available volume for the test material, each culture contained approximately 2.5 x 10^6 cells in 5 mL of F10HP for cultures tested without exogenous metabolic activation, or in 1.5 mL S9 mix plus 3.5 mL F5HP for cultures tested with metabolic activation. Therefore, at least 10 mL/tube was available for the volatile test material. After the cultures had been placed in the tubes at Genesys and the stoppers replaced, the tubes to be exposed to the CF3I were transported to ManTech/RTP where, using a syringe, a predetermined volume of air was withdrawn from each tube and an equal volume of the pure test material was added. (This was not necessary for the negative control [air] or the positive controls; the latter were added directly to the cell culture before the tubes were sealed.) The three tubes per test material concentration were then returned to Genesys, and exposure was initiated by placing the tubes in a roller drum, and rotating them (~40 rpm) for 4 hours at 37 °C.
- After the exposure period, the cultures containing cells tested without and with S9 were transferred from the blood tubes to 15 ml plastic centrifuge tubes for subsequent steps in the assays. The sham tubes were allowed to cool to room temperature prior to analysis of the concentrations of the test materials by IR.

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 14 days

SELECTION AGENT
1 µg/mL 5-trifluorothymidine

NUMBER OF REPLICATIONS:
3

Evaluation criteria:

The results were evaluated according to the categories of responses utilized by the U.S. EPA Gene-Tox Workgroup (Mitchell et all, in preparation), as follows:

++ : Strong positive response with evidence of a dose-response and an induced mutation frequency of at least 100 x 10^-6 (∆100) at a relative total growth (RTG) ≥ 20 %.

+ : Positive response with evidence of a dose-response and an induced mutation frequency of at least 70 x 10^-6 (∆70) at a RTG ≥ 10 %.

- : Negative response for which toxicity is evidenced by a RTG of 10 - 20 %, and the positive control mutation frequency demonstrates that there are no inherent problems with the assay.

= : Negative response with no toxicity, and the positive control mutation frequency demonstrates that there are no inherent problems with the assay.

E : Equivocal response in which positive and negative results are obtained in repeated experiments, and no reason is found to give greater weight to the positive or the negative result.

# : Not-testable. The test material can not be tested to sufficiently high concentration to obtain a conclusive result in the MLA because of limited solubility, acidic pH shifts, the test material's dissolving plastic, etc.

Hence, biological significance was considered in the evaluation of the results.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- It was concluded that test substance induced mutation frequencies of only 14 to 21 x 10^-6 for the four highest tested concentrations in the absence of activation, and only 1 to 14 x 10^-6 for the five highest tested concentrations in the presence of activation, and these increases were not concentration-related. Therefore, there was no evidence to suggest that induced mutation frequencies of at least 70 x 10^-6 (required for evaluating the result as positive, +) would be obtained with greater toxicity, e.g., in the range of 10 to 20 % Relative suspension Growth (RSG). For this reason, and considering biological significance, the results obtained for CF3I in the L5178Y / tk +/- mouse lymphoma cell mutagenesis assay in the absence and presence of activation are evaluated as negative (-).

Conclusions:

The test substance in the L5178Y/ tk+/- mouse lymphoma cell mutagenesis assay was found to be negative in inducing mutations in the absence and presence of metabolic activation.

Executive summary:

In a GLP compliant study similar to OECD 476 the potential of test substance to produce a increase in mutant frequency in the mouse lymphoma cell assay (L5178Y, TK+/-) was investigated. The test substance was initially tested in two preliminary concentration range-finding assays with concentrations ranging from 55000 to 900000 ppm. The test substance appeared to be soluble in cell culture media and concentration-related increases in toxicity were obtained in each assay. In the mutagenesis assay the concentrations of the test substance ranged from 125000 to 1000000 ppm conducted in the absence and presence of metabolic activation. For the mutation test the following analytical concentrations were assessed: 79610, 176884, 305887, 425606, 454184, 517777, 496814 ppm. In this assay, the negative control cloning efficiency and spontaneous mutation frequency met the criteria for acceptability, and positive control mutant frequencies were within the historical ranges for the laboratory, and when tested to the maximum concentration that could be obtained, test substance was negative in the presence of toxicity. Therefore, the test substance did not induce gene or chromosomal mutations in mammalian cells in vitro.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Principles of method if other than guideline:

The test material (bp. -22 °C) is gas, so proliferating cells are treated with test chemical in the presence and absence of a metabolic activation system in the gas phase.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Purity: 99.9%
- Physical state, colorless gas
- Lot No. 010126

Species / strain / cell type:

mammalian cell line, other: Chinese Hamster Lung(CHL)/IU

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

20 %, 40 %, 60 % and 80 %
Justification for top dose: To breed mammalian cell, at least 5 % oxygen concentration in the system is necessary. In the case of treatment with S9, about 5 % of oxygen concentration is necessary to activate metabolic system. Accordingly test gas concentration 80 % for cytostatic test is set the maximum concentration to keep 4 % oxygen concentration in the system by diluted with 20 % air

Vehicle / solvent:

vehicle: air
Justification for choice of solvent/vehicle:Test sample is gas, chemically stable and easily mixed with any gas. Thus high purity air is selected as vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: air

Positive control substance:

benzo(a)pyrene

mitomycin C

other: Methyl chloride, vinyl chloride

Details on test system and experimental conditions:

Cultivating conditions:
- Broth Eagle MEM which contains 10% inactivated fetal bovine serum; conditions cultivated with 5 % CO2 at 37 °C and contiuously cultivated each two to four days.

Proliferation:
- Monolayer cultivation on the plate
- Doubling time: about 15 hours

Chromosome
- Chromosome number mode: 25

Evaluation criteria:

The criteria is set up as belows based on Sofuni 's criteria and background data in our laboratory which contains chromosomal structural aberration (not involve gaps) and numerical aberration (ploidy). As for structural aberration, cells which have gap only was not involved.

Chromosomal aberration incidence Judge
Les than 5% -
From equal and more than 5% to less than 10% ±
Equal or more than 10% +

Statistics:

Not used

Key result

Species / strain:

mammalian cell line, other: Chinese Hamster Lung (CHL)/IU

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Short term treatment test
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Cell proliferation test: Cell proliferation rates without S9 were 108% and 80% at test material concentration 40% and 80% respectively. Cell proliferation rates with metabolic activator S9 were 100% and 86% at test material concentration 40% and 80% concentration respectively.
- Main test: In case of treatment without S9, cell proliferation rates were 96, 84, 69 and 53% at test material concentration 20, 40, 60 and 80% respectively. In case of treatment with S9, cell proliferation rates were 102, 103, 94 and 75% at test material concentration 20, 40, 60 and 80% respectively.
CHROMOSOMAL ABERRATIONS:
- Preliminary chromosomal aberration test : In case of treatment without S9, the frequencies of polyploid cells were 2.9 and 7.4 % at 40 and 80% of test material concentration respectively. The frequencies of structural aberration cells were less than 5%.
In case of treatment with S9, both frequencies of structural aberration cells and polyploid cells were less than 5% at every concentration. Based on these results, the test concentrations for main test were set as 20%, 40%, 60% and 80% with and without S9.
- Chromosomal aberration test : In case of short term treatment without S9, 3, 9% and 10.7 % frequencies of polyploid cells were observed at 60 and 80% concentration of test material. The frequencies of structural aberration cells were less than 5%. In case of treatment with S9, both frequencies of polyploid cells and structural aberration cells were less than 5%. D20 (without S9) of induced polyploid cells was beyond 100% (180%).

Continuous treatment test
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Cell proliferation test : In case of 24 -hour treatment, cell proliferation rates were 101% and 83% at test material concentration 40% and 80% respectively. In case of 48-hour treatment, cell proliferation rates were 56%, 34% and 5% at test material concentration 20%, 40% and 80% concentration respectively.
- In consideration of cell proliferation test results, concentrations of the test material for the main test were set as 4 levels, as for 24-hour treatment, 20, 40, 60 and 80%, and as for 48-hour treatment test, 20, 30, 40 and 50%
- Chromosomal aberration test: Cell proliferation rates were 98, 68, 64, and 42% at the concentration of 20, 40, 60 and 80% respectively for 24-hour treatment. As for 48-hour treatment, the rates were 83, 71, 41 and 30% at the concentration of 20, 30, 40 and 50%.
CHROMOSOMAL ABERRATIONS:
-  Preliminary chromosomal aberration test: In case of 24-hour treatment, both frequencies of structural aberration cells and polyploid cells were less than 5% at every concentration. In case of 48-hour treatment, 7.4% and 11.5% frequencies of polyploid cells were observed at test material concentration 40 and 50% respectively. Frequencies of structural aberration cells were less than 5% at every concentration. At the test material concentration 80% no metaphase cells were observed.
- Chromosomal aberration test: In case of 24-hour treatment, 6.5% frequencies of polyploid cell were observed at 60% test material concentration. Frequencies of structural chromosomal aberration were less than 5%. In case of 48-hour treatment,  7.4 and 11.5% frequencies of polyploid cells were observed at 40% and 50% concentration respectively. The frequencies of structural chromosomal aberration were less than 5%. D20 to induce polyploid cells for 48-hour treatment was determined as 99%.

CONTROL DATA
All data obtained for solvent control and positive control in the preliminary chromosomal test and main test was within background data obtained in our laboratory. This shows the test was conducted adequately.

Conclusions:

Short term treatment without S9 and the 48-hour continuous treatment tests gave more than 10% of frequencies of polyploid cells, since these results are exceeding the criteria of a positive result (more than 10%) this test substance was judged as positive under the conditions of the test.

Executive summary:

An in vitro chromosome aberration assay in Chinese Hamster Lung (CHL)/IU was performed. The cells were exposed to 20 %, 40 %, 60 % and 80 % of test gas concentration with and without metabolic activation (S-9 mix). Two tests were performed; a short term treatment test and a continuous treatment test (24- and 48-hours). The test concentrations for the main tests were based on preliminary chromosomal aberration tests. As positive controls benzo(a)pyrene, mitomycin C, Methyl chloride and vinyl chloride were used.

In case of short term treatment without S9, 3.9 % and 10.7 % frequencies of polyploid cells were observed at 60 and 80 % concentration of test material. The frequencies of structural aberration cells were less than 5 %. In case of treatment with S9, both frequencies of polyploid cells and structural aberration cells were less than 5 %. D20 (without S9) of induced polyploid cells was beyond 100 % (180 %). In case of treatment without S9, cell proliferation rates were 96, 84, 69 and 53 % at test material concentration 20, 40, 60 and 80% respectively. In case of treatment with S9, cell proliferation rates were 102, 103, 94 and 75 % at test material concentration 20, 40, 60 and 80 % respectively.

In case of 24-hour treatment, 6.5 % frequencies of polyploid cell were observed at 60 % test material concentration. Frequencies of structural chromosomal aberration were less than 5 %. In case of 48-hour treatment, 7.4 and 11.5 % frequencies of polyploid cells were observed at 40 % and 50 % concentration respectively. The frequencies of structural chromosomal aberration were less than 5 %. D20 to induce polyploid cells for 48-hour treatment was determined as 99 %. Cell proliferation rates were 98, 68, 64, and 42 % at the concentration of 20, 40, 60 and 80 % respectively for 24-hour treatment. As for 48-hour treatment, the rates were 83, 71, 41 and 30 % at the concentration of 20, 30, 40 and 50 %.

It was concluded that short term treatment without S9 and 48-hour continuous treatment test gave more than 10% of frequencies of polyploid cells. The criteria of positive result are more than 10%. Under this test conditions, chromosomal aberration induced property to CHL/IU cells of this test substance was judged as positive.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Dec 2017 to 19 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

26 September 2014

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Sex, age and number of blood donors: male, 25 years, single donor
- Whole blood was used
- Mitogen used for lymphocytes: phytohemagglutinin

MEDIA USED
- Peripheral blood lymphocytes were cultured in complete medium (RPMI-1640 containing 15% heat inactivated fetal bovine serum, 2mM L-glutamine, 100 units penicillin and 100 µg/mL streptomycin) and 25 mM HEPES by adding 0.5 mL heparinized blood to a centrifuge tube containing 15 mL of complete medium with 2% phytohemagglutinin. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 44-48 hours. The RPMI 1640 used was supplemented with 25 mM HEPES.

Cytokinesis block (if used):

Cytochalasin B (cytoB) was dissolved in DMSO to a stock concentration of 2 mg/mL. It was used at 6 µg/mL concentration to block cytokinesis.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced liver S9 from male Sprague-Dawley rats

Test concentrations with justification for top dose:

Based upon the results of the preliminary toxicity assay, the doses selected for the micronucleus assay were as follows:
Non-activated:
- 4h treatment: 25, 250, 500, 1000, 1800, 2000 µg/mL
- 24h treatment: 5.0, 25, 250, 400, 450, 500, 550, 600, 700, 800 µg/mL
S9-activated:
- 4h treatment: 25, 250, 500, 1000, 1200, 1400, 1600, 1800, 2000 µg/mL

Vehicle / solvent:

Solvent used: acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

vinblastine

Details on test system and experimental conditions:

PRELIMINARY TOXICITY TEST FOR SELECTION OF DOSE LEVELS
HPBL were exposed to vehicle alone and to nine concentrations of test substance with half- log dose spacing using single cultures. Precipitation of test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the vehicle, the highest dose, and the highest soluble dose was measured. Dose levels for the micronucleus assay were based upon post- treatment toxicity (CBPI relative to the vehicle control) or lack thereof.

MICRONUCLEUS ASSAY
Based upon these results, the doses chosen for the micronucleus assay ranged from 25 to 2000 µg/mL for the non-activated and S9-activated 4-hour exposure groups; and from 5.0 to 800 µg/mL for the non-activated 24-hour exposure group. Precipitation of the test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose evaluated for the micronuclei was selected based the limit dose for this assay (4 hour – S9) or 55 ± 5% cytotoxicity (CBPI relative to the vehicle control; 4 hour + S9 and 24 h – S9). Two additional doses were included in the evaluation of micronuclei.

TREATMENT OF TARGET CELLS (PRELIMINARY TOXICITY TEST AND MICRONUCLEUS ASSAY)
The pH of the highest dose of dosing solution in the treatment medium was measured using test tape. Treatment was carried out by refeeding the cultures as shown in Table 1 in ‘Any other information on materials and methods, incl. tables’. After the 4-hour treatment in the non-activated and the S9-activated studies, the cells were centrifuged, the treatment medium was aspirated, the cells were washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium containing cytoB at 6.0 µg/mL and returned to the incubator under standard conditions. For the 24-hour treatment in the non-activated study, cytoB (6.0 µg/mL) was added at the beginning of the treatment.

COLLECTION OF CELLS (PRELIMINARY TOXICITY TEST AND MICRONUCLEUS ASSAY)
Cells were collected after being exposed to cyto B for 24 hours (± 30 minutes), 1.5 to 2 normal cell cycles, to ensure identification and selective analysis of micronucleus frequency in cells that have completed one mitosis evidenced by binucleated cells (Fenech and Morley, 1986). The cyto B exposure time for the 4 hour treatment in the non-activated and the S9-activated studies was 20 hours (± 30 minutes). Cells were collected by centrifugation, swollen with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 25:1 v/v), capped and the slides were prepared immediately after harvest. To prepare slides, the cells were collected by centrifugation and the suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with acridine orange and identified by the study number, treatment condition, dose level, test phase, harvest date, activation system, and replicate tube design.

CELL CYCLE KINETICS SCORING (PRELIMINARY TOXICITY TEST AND MICRONUCLEUS ASSAY)
For the preliminary toxicity test, at least 500 cells were evaluated to determine the CBPI at each dose level and the control. For the micronucleus assay, at least 1,000 cells (500 cells per culture) were evaluated to determine the CBPI at each dose level and the control. The CBPI was determined using the following formula:

CBPI = (1 x Mononucleated cells + 2 x Binucleated cells + 3 x Multinucleated cells) / Total number of cells scored

% Cytostasis (cytotoxicity) = 100 -100 {(CBPIt-1) /(CBPIc-1)}

t = test substance treatment culture
c = vehicle control culture

MICRONUCLEUS SCORING (MICRONUCLEUS ASSAY)
The slides from at least three test substance treatment groups were coded using random numbers by an individual not involved with the scoring process and scored for the presence of micronuclei based on cytotoxicity. A minimum of 2000 binucleated cells from each concentration (if possible, 1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei. Micronuclei in a binucleated cell (MN-BN) were recorded if they met the following criteria:
- the micronucleus should have the same staining characteristics as the main nucleus
- the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges)
- the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus

Evaluation criteria:

The test substance was considered to have induced a positive response if
- at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
- the increase was concentration-related (p ≤ 0.05), and
- results were outside the 95% control limit of the historical negative control data.

The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the percentage of micronucleated cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
Visible precipitate was observed in treatment medium at the beginning of the treatment period for all treatment conditions at a concentration of 2000 µg/mL. At the conclusion of the treatment period, no precipitation was observed.
The osmolality of the test substance doses in treatment medium was considered acceptable. The pH of the highest dose of test substance in treatment medium was 7.5.
Cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at 2000 µg/mL in the non-activated and S9-activated 4-hour exposure groups and at doses ≥ 600 µg/mL in the non-activated 24-hour exposure group. Based upon the results of the preliminary toxicity assay, the doses selected for the micronucleus assay were as follows:
Non-activated:
- 4h treatment: 25, 250, 500, 1000, 1800, 2000 µg/mL
- 24h treatment: 5.0, 25, 250, 400, 450, 500, 550, 600, 700, 800 µg/mL
S9-activated:
- 4h treatment: 25, 250, 500, 1000, 1200, 1400, 1600, 1800, 2000 µg/mL

MICRONUCLEUS ASSAY
Visible precipitate was observed in treatment medium at the beginning of the treatment period for the 4 h treatment conditions both in the absence and presence of metabolic activation at a concentration of 1000 µg/mL and higher. For all other treatment conditions, no precipitation was observed.
The pH of the highest dose of test substance in treatment medium was 7.5. Cytotoxicity (CBPI relative to the vehicle control) was observed as follows:
Non-activated:
- 4h treatment: 29 % cytotoxicity at 2000 µg/mL
- 24h treatment: 54% cytotoxicity at 500 µg/mL
S9-activated:
- 4h treatment: 51% cytotoxicity at 1600 µg/mL

No significant or dose-dependent increases in micronuclei induction were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).
The results for the positive and vehicle controls indicate that all criteria for a valid assay were met.

In addition to the micronucleus treatments, a concurrent non-GLP experiment was conducted by the Sponsor in order to determine actual test substance concentrations present in the culture media during treatment of the micronucleus assay in the sham cultures. Stock formulations of the test substance were added to sham cultures (i.e. cultures identical to those for micronucleus assessment but that did not contain any human lymphocytes) and incubated under the same conditions as the micronucleus cultures.Sham culture treatments were performed concurrently with the micronucleus treatments at the test facility. The test substance was detected in the sham cultures (stored for 1 week at room temperature following exposure) at approximately 12% of the targeted concentrations. During micronucleus and sham treatments at 2000 µg/mL, a release of bubbles was noticed; this is suggestive of a loss of test article at the point of initial contact into the aqueous culture media and is the likely explanation for the low recovery. In the S9-activated 4 hour and non-activation 24 hr micronucleus treatments, >50% cytotoxicity was observed at several high concentrations, thus indicating that even if there was a substantial loss of the test substance at the point of test item addition to the treatment cultures, the remaining aqueous concentration of the test substance was sufficient to cause dose-limiting cytotoxicity. In the non-activation 4 hr treatment, the highest dose tested (limit dose of 2000 µg/mL nominal) induced 29% cytotoxicity, however, in the preliminary toxicity assay 58% cytotoxicity was observed under the same treatment condition; a similar shift in toxicity between experiments was also noted for the other treatment conditions. Overall it is considered that the data show that the highest achievable concentration of the test substance in aqueous media caused dose limiting cytotoxicity under all experimental conditions, including the non-activation 4 hour treatment, but that the same limiting cytotoxic concentrations did not induce micronuclei.

Conclusions:

Under the conditions of the assay described in this report, the test substance was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.

Executive summary:

The test substance was tested according to OECD TG 487 and in compliance with GLP to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. HPBL cells were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Acetone was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 µg/mL, which was the limit dose for this assay. Cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at 2000 µg/mL in the non-activated and S9-activated 4-hour exposure groups and at doses ≥ 600 µg/mL in the non-activated 24-hour exposure group. Based upon these results, the doses chosen for the micronucleus assay ranged from 25 to 2000 µg/mL for the non-activated and S9-activated 4-hour exposure groups; and from 5.0 to 800 µg/mL for the non-activated 24-hour exposure group. In the micronucleus assay, cytotoxicity (≥ 50% CBPI relative to the vehicle control) was observed at doses ≥ 1600 µg/mL in the S9-activated 4-hour exposure group and at doses ≥ 500 µg/mL in the non-activated 24-hour exposure group. In the non-activated 4-hour exposure group, there was a shift in cytotoxicity profile (compared to the preliminary assay, which showed >50% CBPI at the same concentration), with 29% cytotoxicity observed at 2000 µg/mL. The doses selected for evaluations of micronuclei were 250, 1000, and 2000 µg/mL for the non-activated 4-hour exposure group; 250, 1000, and 1600 µg/mL for the S9-activated 4-hour exposure group; and 25, 250, and 500 µg/mL for the non-activated 24-hour exposure group. No significant or dose-dependent increases in micronuclei induction were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

These results indicate, under the study design, the test substance was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Transgenic rodent mutation assay for Mutagenicity: Negative

The weight-of-evidence approach, applied on the data obtained from multiple in vivo chromosomal aberration and micronucleus studies, results in the following conclusion: Positive

Unscheduled DNA Synthesis test: Negative

At high exposures (>20000 ppm) CF₃I induces micronuclei in immature erythrocytes of exposed rats and mice. However, there is convincing evidence that this is a consequence of chemically-induced hypothermia rather than any direct DNA-damaging mechanism. Based on this proposed Mode of Action, the overall conclusion for in vivo genotoxicity is considerd to be negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Jul 2016 to 18 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)

GLP compliance:

yes (incl. QA statement)

Type of assay:

transgenic rodent mutagenicity assay

Specific details on test material used for the study:

Name as cited in study report: CF3I
Lot no.: 1080616A-1120L
Purity: 99.51 %
ID no.: 16023F
Storage conditions: room temperature
Expiry date: 30 June 2017

Species:

rat

Strain:

Fischer 344

Remarks:

Big Blue homozygous transgenic rats [Taconic nomenclature: F344-TgN(lamda/lacI)]

Details on species / strain selection:

Rats have been used historically in safety evaluation and genotoxicity studies and are recommended by regulatory agencies. Because this study was conducted in accordance with regulatory guidelines and regulatory agency request, alternatives could not be considered.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Biosciences, Inc., Hudson, NY
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: ranged from 169 g to 286 g
- Fasting period before study: no
- Housing: 2 animals per cage in clean, solid bottom cages containing ground corncob bedding material (Bed O’Cobs; The Andersons, Cob Products Division, Maumee, OH).
- Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet 5002, provided ad libitum throughout the study, except during acclimation to the nose-only restraint and the exposure periods
- Water: Reverse osmosis-treated (on-site) drinking water, provided ad libitum throughout the study, except during acclimation to the nose-only restraint and the exposure periods
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3
- Humidity (%): 50.4 to 63.8
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Vehicle:

filtered air

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 7.9 L stainless steel nose-only exposure systems with grommets in the exposure ports to engage animal holding tubes.
- Method of holding animals in test chamber: held in restraint tubes for 15 to 41 minutes before the initiation of exposure
- Acclimation: All animals were acclimated to nose-only exposure tubes five times (one acclimation/day) prior to their first day of animal exposure. Acclimation was performed for approximately 1 hour on the first day, approximately 2 hours on the second day, approximately 3 hours on the third day, approximately 4 hours on the fourth day, and approximately 6 hours on the fifth day. Following acclimation to the restraint tubes, each animal was
observed for clinical signs of injury or stress.
- Source and rate of air: Air supplied to each nose-only system was provided from the Inhalation Department breathing quality, in-house compressed air source and a HEPA- and charcoal-filtered, temperature- and humidity-controlled supply air source.
- Method of monitoring aerosol content: light scattering type real-time aerosol monitor
- Control exposure system: The control exposure system (Group 1) was operated as follows. Humidified supply air was delivered to the CNOS from the facility humidified supply air source and metered using a rotameter-type flowmeter. Dry dilution air from the Inhalation breathing-quality compressed air system was delivered to the control group in addition to humidified supply air. Dry air was controlled using a regulator and metered using a rotameter-type flowmeter.
- Test substance exposure system: The test substance exposure systems (Groups 2 to 4) were operated as follows. Exposure atmospheres were generated by releasing pure test substance gas (1,000,000 ppm) from the Sponsor-supplied cylinder. A 2-stage regulator and a pressure gauge were used to monitor cylinder pressure and control the flow of test substance gas from the cylinder. A heat blanket was placed on the cylinder to facilitate maintaining adequate head pressure within the cylinder. The heat blanket was controlled using a temperature controller. From the regulator, test substance gas was delivered to a manifold system equipped with a pressure gauge to monitor manifold pressure. The manifold was used to distribute CF3I gas to each exposure system. Test substance flow from the manifold was controlled using a needle valve and monitored using a mass flowmeter. The test substance gas was directed to each CNOS inlet through a “T” fitting where the test substance was mixed with dilution supply air to achieve the desired exposure concentrations. The humidified dilution air was delivered to each CNOS using a rotameter-type flowmeter.
- Temperature, humidity: 20 °C to 26 °C, 30 % to 70 %.
- Treatment of exhaust air: All nose-only system exhaust passed through the facility exhaust system, which consists of redundant exhaust blowers preceded by activated-charcoal and HEPA-filtration units.

TEST ATMOSPHERE
Analytical exposure concentrations / analytical method used: Exposure atmospheres were sampled and concentrations were analyzed approximately every 35 to 66 minutes using a gas chromatography (GC) method. Additional analyses were conducted as required to monitor and control exposure concentrations. Samples were manually collected from the approximate animal-breathing zone of each exposure chamber via one-eighth inch Teflon® tubing. An external multi-position valve permits sequential sampling from the exposure room and each exposure chamber. Gas sample injection onto the chromatography column occurred via an internal gas-sampling valve with a sample loop. The chromatograph was displayed, the area under the sample peak was calculated and stored, and the concentration in parts per million (ppm) was calculated.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

6 hours/day, 7 days/week (total of 28 exposures)

Dose / conc.:

1 250 ppm (nominal)

Dose / conc.:

2 500 ppm (nominal)

Dose / conc.:

5 000 ppm (nominal)

No. of animals per sex per dose:

- control, mid dose (2500 ppm) & positive control: 5 males
- low dose (1250 ppm): 4 males
- high dose (5000 ppm): 6 males

Control animals:

yes, concurrent vehicle

Positive control(s):

20 mg/kg/day ENU, days: 1, 2, 3, 12, 19 and 26

Tissues and cell types examined:

Lung was evaluated as it is a site of first contact for inhalation exposure. In addition, liver and bone marrow were being evaluated. Testes were collected, frozen and held in reserve in case of germ cell mutagenesis was needed.

Details of tissue and slide preparation:

JUSTIFICATION FOR SELECTION OF EXPOSURE ROUTE EN LEVELS
In a 28-day nose-only inhalation study with CF3I, test substance-related effects including decreased body weights, body weight gains and food consumption, along with changes in red blood cell parameters and clinical
chemistry parameters were noted at exposure concentrations of 2% and 4% (20,000 and 40,000 ppm, respectively). In addition, increased liver weight and decreased spleen weight were noted. Reduced white blood cell count was noted at 40,000 ppm. Microscopic changes were noted in the adrenals, spleen, thymus and testes of animals exposed to 40,000 ppm. Cytotoxicity to the bone marrow was observed in males at all levels tested (TNO, 2007). Based on these results, 40,000 ppm was initially selected to be the maximum concentration for this study along with additional exposure levels 20,000 and 10,000 ppm (4%, 2% and 1%). The same exposure route (nose-only) was selected, in order to maximize exposure. Seven days per week exposure and 6 hours per day were selected for dose administration to maximize test substance exposure to the test system. The inhalation route was selected as this was the route used in previous animal studies and is a relevant route of human occupational exposure. Lung was evaluated as it is a site of first contact for inhalation exposure.
Liver and bone marrow were selected following OECD TG 488 guidelines. Liver was selected as a tissue with relatively slow cell turnover that is a major site of mammalian metabolism. Bone marrow was selected as a
representative fast dividing tissue, and to provide a mutational mode of action endpoint in the bone marrow compartment. Testes were collected, frozen and held in reserve, should analysis of germ cell mutagenesis be
needed.

SAMPLE COLLECTION FOR FLOW CYTOMETRY
6 males/group (Groups 1-4) on Study Day 28 via the jugular vein into 2.0 mL K2 EDTA tubes. The blood samples were diluted in MicroFlow anticoagulant (supplied by Litron Laboratories), divided into 2 aliquots of
approximately 180 μL each (primary and backup samples) into separate vials containing cold fixative, and then fixed in cold methanol for approximately 72 hours. After fixation, samples were removed from frozen storage,
washed out of fixative, placed into Long Term Storage Solution and stored frozen (-65°C to -85°C). The primary and backup blood samples were shipped separately, using frozen cool packs (packaged in a way to avoid
direct contact with the sample containers) to the Study Director at BioReliance Corporation, Rockville, MD, for possible future analysis by flow cytometry.

SACRIFICE, NECROPSY, ORGAN WEIGHTS & TISSUE COLLECTION
In order to minimize the in situ degradation of the DNA, all animals were euthanized by CO2 inhalation. Liver, lungs and testes were collected from all animals. In addition, bone marrow was collected from the right femur of all animals, suspended using PBS and split into three aliquots. Each aliquot was centrifuged, supernatant decanted and the pellet was frozen. Collected tissues were weighed (except for bone marrow), flash frozen in liquid nitrogen, stored at approximately -70 °C, and shipped on dry ice to BioReliance Corporation, Rockville, MD, for cII mutant analysis.

EXTRACTION OF GENOMIC DNA
Post-life analysis of frozen tissues was performed by BioReliance Corporation. Liver, lung and bone marrow tissue samples were processed for DNA isolation from frozen tissues of the first 5 surviving animals in the filtered air control group (Group 1) and the test substance-treated groups (Groups 2, 3, and 4). At the discretion of the Study Director, and in consultation with the Sponsor, analysis of tissue from the sixth animal in a group for one or more tissues may be performed for added statistical resolution or should replacement of one of the initial five animals be needed for any reason.
In addition, DNA from liver and lung tissue samples from 5 positive control-treated animals dosed, necropsied and frozen by BioReliance Corporation. This positive control group was identified in post-life data as Group 5. This use of “packaging controls” is permitted by OECD TG 488. The goal of the positive control group in this case is to demonstrate the ability to recover induced mutants from the study target tissues. Positive control tissues from target organs were collected on Study Day 31 after start of dosing from Big Blue F344 male rats exposed by oral gavage to 40 mg/kg of ethyl nitrosourea (ENU) on Study Days 1, 2, 3, 12, 19 and 26 as part of BioReliance Corporation Study is a potent direct acting mutagen demonstrated to be mutagenic in the target tissues. Replacement of positive control tissue from another rat study dosed with ENU is permitted upon documentation in the raw data and report.
Tissues were extracted following BioReliance SOP that is based on methods described for Agilent product RecoverEase (Agilent, 2009a) for somatic tissues. Isolated DNA were stored at 2° to 8°C.

IN VITRO PACKAGING OF DNA AND PLATING OF PHAGE
Isolated DNA was processed using Agilent Transpack packaging extract to isolate the recoverable lambda shuttle DNA vectors from the genomic DNA and to package the lambda shuttle vector DNA into empty phage capsids creating infectious phage particles. Methods followed SOP OPGT9031 that is based on Agilent instruction manual titled “λ Select-cII Mutation Detection System for Big Blue Rodents” (Agilent, 2009b) and Agilent instruction manual titled “Transpack Packaging Extract for Lambda Transgenic Shuttle Vector Recovery” (Agilent 2009c).
Frozen stocks of E. coli strain G1250 provided and characterized by Agilent as part of λ Select-cII Mutation Detection System for Big Blue Rodents were used to prepare master bacterial plates. One or more colonies were picked from master plates and used to prepare overnight suspension cultures for plating of phage for plaque formation.
Packaged phage were evaluated for overall phage titer and mutant phage titer to permit calculation of a mutant frequency for each tissue following a SOP. Packaged phage were incubated overnight at 37° ± 1.0°C, then scored for plaque formation and titer determination; cII mutant selection plates were incubated at 24° ± 0.5°C, then scored for mutant plaques starting after 40 to 48 hours of incubation. At least 125,000 phage will be evaluated from at least 2 packagings. Due to the stringency of mutant selection, confirmation of mutant phenotype is not routinely performed.

Evaluation criteria:

Mutant Frequency Assay Evaluation Criteria
- Criteria for a Positive Response: The test substance will be considered to have produced a positive response if it induces a statistically significant increase in the frequency of cII mutants in any dose level outside the 99% confidence interval of the
historical background mutant frequency range.
- Criteria for a Negative Response: A test substance will be considered to have produced a negative response if no significant increase in cII mutant frequency is observed.
- Criteria for an Equivocal Response: Equivocal responses will be evaluated by the Study Director on a case-by-case considering both statistical significance and biological relevance.
- Other Considerations: Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical or literature control values, biological significance, etc.). In such cases, the Study Director will use sound scientific judgment and clearly report and describe any such considerations.

Statistics:

Data Analysis:
The individual animal is considered the experimental unit. The mutant frequency (MF) was calculated (number of mutant phage / number of total phage screened) for each tissue analyzed from each animal. Since this ratio is extremely small and may not be normally distributed, a log10 transformation of the MF data was performed. The statistical analysis of MF was conducted in two parts. Initially, the positive control group was compared to the filtered air control for each tissue. In the second part of the analysis, all the groups (except the positive control) were compared to the filtered air control. In both cases, log10 transformed MF data from the filtered air control and treated groups were evaluated using a 1-Way Analysis of Variance (ANOVA). The suitability of using the parametric ANOVA was confirmed by testing parameters of the log10 transformed MF data for normality and equal variance. If the data are normally distributed and exhibit equal variance, the parametric ANOVA analysis would be used. Otherwise, if either test fails, a nonparametric analysis of variance would be used. Statistical analysis was performed using Minitab 16.1.0.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Test substance-related lower body temperatures were noted in the 2500 and 5000 ppm groups.

Additional information on results:

Sufficient quantity and quality of DNA was obtained to permit 2 to 5 packagings of each DNA sample, yielding more than the OECD-specified minimum of 125,000 phage/tissue/animal. Since the experimental unit is the animal, the total number of plaque-forming units, the number of mutants and the mutant frequency for each tissue per animal are reported. The number of packaging cycles used to achieve the reported data is also reported for each tissue from each animal.

BONE MARROW
For bone marrow, the Big Blue® F344 rat mutation assay gave background mutant frequencies in the filtered air control (Group 1) of 27.4 ± 21.9 x 10^-6 (mean ± standard deviation), comparable to historical experience of 29.5 ± 11.7 x 10^-6 for bone marrow. Mean cII mutant frequencies in bone marrow from iodotrifluoromethane treated animals were 18.8 ± 6.3 x 10^-6, 16.4 ± 3.7 x 10^-6, and 29.5 ± 32.5 x 10^-6 (Groups 2, 3 and 4, respectively). A few individual animal bone marrow mutant frequencies in animals exposed to iodotrifluoromethane were outside (at both ends) of the historical range for bone marrow vehicle control animals, exhibiting some variation common to biological systems. Statistical analysis of the iodotrifluoromethane treated groups relative to the filtered air control by 1-Way ANOVA revealed that the mean mutant frequency of the test substance-treated groups was not significantly different. than the control group (p = 0.830). The normality test performed on the residuals (p = 0.050) and the equal variance test (p = 0.427) confirmed that the ANOVA criteria were met, therefore the analysis was considered adequate.
Results from bone marrow DNA from ENU treated animals (Group 5) demonstrated reproducible mutant frequencies of 267.9 ± 74.1 x 10^-6; the results were comparable to historical experience of 277.5 ± 88.9 x 10^-6 for
bone marrow. 1-Way ANOVA revealed that the mean mutant frequency of the ENU-treated group was significantly elevated over the control group (p < 0.001). However, the normality test performed on the residuals failed (p = 0.021), therefore the data were not normally distributed, and the ANOVA test was not considered appropriate. Data was further analyzed using the Kruskal-Wallis test (non-parametric alternative to ANOVA); this test confirmed that the median MF of the ENU-treated group was significantly elevated over the control group (p = 0.009).

LIVER
For liver, the Big Blue® F344 rat mutation assay gave background mutant frequencies in the filtered air control (Group 1) of 76.2 ± 34.3 x 10^-6 (mean ± standard deviation), comparable to historical experience of 42.2 ± 16.1 x 10-6 for liver. Mean cII mutant frequencies in liver from iodotrifluoromethane treated animals were 60.5 ± 19.3 x 10^-6, 55.1 ± 21.4 x 10^-6, and 63.0 ± 14.5 x 10^-6 (Groups 2, 3 and 4, respectively). A few individual animal liver mutant frequencies in animals exposed to iodotrifluoromethane were outside (slightly higher) of the historical range for liver vehicle control animals, exhibiting some variation common to biological systems. Statistical analysis of the iodotrifluoromethane treated groups relative to the filtered air control by 1-Way ANOVA revealed that the mean mutant frequency of the test substance-treated groups was not significantly different than the control group (p = 0.611). The normality test performed on the residuals (p > 0.100) and the equal variance test (p = 0.689) confirmed that the ANOVA criteria were met, therefore the analysis was considered adequate.
Results from liver DNA from ENU treated animals (Group 5) demonstrated reproducible mutant frequencies of 206.5 ± 28.6 x 10^-6; the results were comparable to historical experience of 154.5 ± 51.7 x 10^-6 for liver. 1-Way ANOVA revealed that the mean mutant frequency of the ENU-treated group was significantly elevated over the control group (p = 0.001). The normality test performed on the residuals (p > 0.100) and the equal variance test (p = 0.124) confirmed that the ANOVA criteria were met, therefore the analysis was considered adequate.

LUNG
For lung, the Big Blue® F344 rat mutation assay gave background mutant frequencies in the filtered air control (Group 1) of 55.1 ± 25.5 x 10^-6 (mean ± standard deviation), comparable to historical experience of 34.4 ± 18.2 x 10^-6 for lung. Mean cII mutant frequencies in lung from iodotrifluoromethane treated animals were 30.9 ± 24.8 x 10^-6, 48.9 ± 33.2 x 10^-6, and 39.4 ± 16.2 x 10^-6 (Groups 2, 3 and 4, respectively). Individual animal lung mutant
frequencies in animals exposed to iodotrifluoromethane were generally within the historical range for lung vehicle control animals, but did exhibit some variation common to biological systems. Statistical analysis of the iodotrifluoromethane treated groups relative to the filtered air control by 1-Way ANOVA revealed that the mean mutant frequency of the test substance-treated groups was not significantly different than the control group (p = 0.362). The normality test performed on the residuals (p > 0.100) and the equal variance test (p = 0.723) confirmed that the ANOVA criteria were met, therefore the analysis was considered adequate.
Results from lung DNA from ENU treated animals (Group 5) demonstrated reproducible mutant frequencies of 188.5 ± 83.9 x 10^-6; the results were comparable to historical experience of 75.7 ± 10.0 x 10^-6 for lung. 1-Way ANOVA revealed that the mean mutant frequency of the ENU-treated group was significantly elevated over the control group (p = 0.002). The normality test performed on the residuals (p > 0.100) and the equal variance test (p = 0.779) confirmed that the ANOVA criteria were met, therefore the analysis was considered adequate.

MORTALITY AND IN-LIFE OBSERVATIONS

Following 28 consecutive exposures/animal at target exposure concentrations of 0, 1250, 2500 and 5000 ppm in the Phase 3 study, no test substance-related mortality was noted, although two 5000 ppm males were found dead following blood collection. These deaths were not considered test substance-related but were likely procedural, as a result of jugular blood collection. Test substance-related clinical observations noted in the 5000 ppm group included partial or complete closure of the eyes, cool extremities, unkempt appearance, dermal atonia, thin body condition, red or clear discharge from the eyes, red material around the eyes, brown material around the anogenital area and yellow material around the urogenital area throughout the exposure period. Cool extremities were noted in the 2500 ppm group during the exposure period. During the 3-day non-exposure period prior to necropsy, thin body condition, red material around the nose, and/or yellow material around the urogenital area were noted prior to the scheduled necropsy in the 2500 and/or 5000 ppm groups.

IN-LIFE MEASUREMENTS

Test substance-related lower body weights were noted in the 2500 and 5000 ppm groups compared to the control group. These correlated with test substance-related lower food consumption throughout the exposure period. Test substance related lower body temperatures were noted in the 2500 and 5000 ppm groups throughout the exposure period compared to the control group. There were no test substance-related effects noted for the 1250 ppm group.

POST-LIFE DATA

No gross lesions were noted for any animals at the scheduled necropsy. Test substance-related lower liver and lung weights were noted in the 5000 ppm group compared to the control group. Lower testes weights were noted in the 2500 and 5000 ppm groups compared to the control group.

Conclusions:

Test substance is negative for the induction of cII mutants in bone marrow, liver and lungs of Big Blue male rats under the conditions of testing. Therefore, test substance is considered not mutagenic in this TGR mutation assay, under the conditions of testing.

Executive summary:

A Transgenic Rodent (TGR) mutation assay (BioReliance 2017), was carried out according to OECD Test Guideline TG 488 and GLP regulations. The test substance was administered via nose-only inhalation exposure for 6 hours per day to male F344 rats. Initially, the study was conducted at exposure concentrations of 0, 10,000, 20,000 and 40,000 ppm (Groups 1-4, respectively; 6 males/group). Due to overt signs of toxicity and a severe decrease in body temperatures after Day 0 exposure (prior to exposure on Day 1), Groups 3 and 4 were euthanized in extremis. Groups 1 and 2 did not receive any further exposures. This was deemed Phase 1 of the study. The study was restarted with new group assignments, at exposure concentrations of 2500, 5000 and 10,000 ppm (Groups 2-4, respectively). Due to overt signs of toxicity and a severe decrease in body temperatures during the first week of exposure to 10,000 ppm, Group 4 was euthanized in extremis on Day 5.

During the final Phase, test substance was administered for 28 consecutive days at target exposure concentrations of 1250, 2500 and 5000 ppm (Groups 2, 3 and 4, respectively). The control group (Group 1) was exposed to humidified, filtered air on a comparable regimen. All animals survived the in-life exposure phase and all animals were euthanized on Test Site Day 30 (Test Facility Day 31), 3 days following the final exposure with the exception of animals 5825 and 5828 in Group 4. These two animals died shortly after blood collection on Test Site Day 28, the day after the last exposure. In order to minimize the in situ degradation of the DNA, all surviving animals were euthanized by CO2 inhalation and necropsied; the liver, lungs and testes were removed and weighed (for prediction of the possible number of DNA extractions from a tissue). Bone marrow was collected from one femur, pelleted by centrifugation and the supernatant decanted. All tissues were immediately flash frozen in liquid nitrogen, and stored at approximately -70 °C.

Exposure of male Fisher 344 Big Blue rats to test substance using the above described regimen for Phase 3 resulted in no test substance-related mortality. Based on Phase 1 and 2 findings, the 5000 ppm level was determined to be the highest dose that would be tolerated over a 28-day exposure period, using 7 days per week dosing for 6 hours per day. However, test substance-related clinical findings (most notably, cool extremities and thin body condition), lower body weights, food consumption, body temperatures, and organ weights were noted in the 2500 and 5000 ppm groups. There were no test substance-related effects noted for the 1250 ppm group. In conclusion, treatment with test substance did not cause statistically elevated mutant frequencies at the cII gene in bone marrow, liver and lungs of Big Blue male rats under the conditions of testing. Therefore, test substance is considered not mutagenic in this TGR mutation assay, under the conditions of testing.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

A micronucleus test was performed as part of a sub-chronic toxicity study. Fifteen male and 15 female rats were exposed 2 hr/day, 5 days/week, for up to 13 weeks (65 exposures over 90 days) to 0, 2, 4, or 8% test substance. Prior to initiation of the exposures, rats were acclimated to the nose-only chamber restraint system, breathing air only, 2 hr/day for 1 week. Bone marrow cells were collected from the femur and smears were prepared from 5 rats/sex/group at both the 30-day and 90-day animal sacrifices to investigate the mutagenic potential of CF3I via induction of micronuclei in bone marrow polychromatic erythrocytes. Two or three control rats/sex were administered a single dose of cyclophosphamide (7.5 mg/kg) intraperitoneally 24 hrs prior to sacrifice to serve as positive controls. Slides were stained by the Giemsa/May-Greenwald method and observed microscopically at 1000x. The frequency of micronucleated cells was evaluated by random observation of 1000 polychromatic erythrocytes (PCE) per sample. The ratio between PCE and normochromatic erythrocytes (NCE) was also determined by scoring approximately 1000 erythrocytes as an indicator of toxicity.

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Raleigh, NC)
- Age at study initiation: 8 to 9 weeks at the start of exposure
- Weight at study initiation: not reported for acute study. In the concurrent 13 week study (same animal batch) the mean weight was between 180 to 185 g (males) or 135 to 140 g (females) at the initiation of the treatment.
- Housing: single housed in in clear plastic cages with wood chip bedding (Betta-Chip, Northeastem Products Corp., WalTensburg, NY).
- Diet: Purina Formulab No. 5002 or 5008 was available ad libitum, except during exposure
- Water: ad libitum, except during exposure
- Acclimation period: 2 weeks. Prior to initiation exposures, rats were acclimated to the nose-only chamber restraint system, breathing air only, 2 hr/day for 1 week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 25
- Humidity (%): 40 to 60
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: gas

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The chamber selected for nose-only exposure was a stainless-steel flow-past system designed by Cannon et al. (1983). Each chamber had 52 ports for exposure of animals. Ports for rat exposure were selected randomly using a computer program diat generates random numbers.
- Method of holding animals in test chamber: Rat restraining tubes made of Lucite were plugged into the animal ports, resulting in the outward extension of the tubes radially from the main body of the chamber
- Source and rate of air: The test substance and air for dilution were delivered from pressurized systems and were controlled through flow meters. fine control of chamber concentration was made by minor adjustment of the substance flow in response to chemical analysis of the chamber atmosphere. Total chamber air flow resuited in the delivery of more than 300 mL/min of mixed substance and air at each animal port.
- Method of conditioning air: A portion of the diluent air passed through a gas washing bottle (Ace Glass, Vinland, NJ, Model 7166-26) containing water to provide adequate relative humidity to the chamber input air stream.
- Temperature, humidity, pressure in air chamber: Relative humidity and temperature of the exposure atmosphere were constantly monitored and recorded using HY-CAL dual probes (Models HY-CAL, Atlanta. GA) and a data acquisition system.

Duration of treatment / exposure:

- Five male and 5 female rats per group were necropsied after 4 weeks on study (20 exposures).
- Remaining animals: up to 13 weeks (65 exposures over 90 days)

Frequency of treatment:

2 hr/day, 5 days/week

Dose / conc.:

20 000 ppm

Remarks:

corresponding to 2%

Dose / conc.:

40 000 ppm

Remarks:

corresponding to 4%

Dose / conc.:

80 000 ppm

Remarks:

corresponding to 8%

No. of animals per sex per dose:

15 (micronucleus assay was performed on 5 animals/ sex/dose at both the 30 and 90-day timepoints)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (7.5 mg/kg) intraperitoneally 24 hrs prior to sacrifice

Tissues and cell types examined:

erythrocytes in bone marrow

Details of tissue and slide preparation:

MICRONUCLEUS ASSAY IN BONE MARROW
Bone marrow cells were collected from the femur and smears were prepared from five rats/sex/group at both the 4- and 13-week animal terminations to investigate the mutagenic potential via induction of micronuclei in bone marrow. Slides were stained by the Giemsa/May-Greenwald method and observed microscopically at 1000x. The frequency of micronucleated cells was evaluated by random observation of 1000 polychromatic etythrocytes (PCE) per sample. The ratio between PCE and nonnochromatic erythrocytes (NCE) was also determined by scoring approximately 1000 erythrocytes as an indicator of toxicity.

Statistics:

Chi-square analysis and one way analysis (ANOVA) was used to evaluate data for the micronuclei induction.

Key result

Sex:

male/female

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

part of repeated dose toxicity: inhalation 90 days study

Additional information on results:

Bone marrow micronuclei induction
The results of the micronuclei examination of bone marrow polychromatic erythsocytes of male and female rats euthanized following 4 weeks of exposure indicate that the substance at concentrations of 4 and 8 % increased the micronuclei frequency in male rats by 2.7- and 3.9-fold, respectively, and in female rats, 3.5- and 4.9-fold, respectively. Rats administered the positive control agent cyclophosphamide increased micronuclei frequency 7- to 8-fold above the air-only control values. Female rats examined from the 2% group did not show an increase in the mean percentage of micronucleated cells compared to the control value. Bone marrow toxicity (indicated by a statistically significant reduction in the ratio of number of polychromatic erythrocytes to number of normochromatic erythrocytes, PCE/NCE) was observed in male rats exposed to 4 % and in male and female rats exposed to 8%.
Following 13 weeks of exposure, the substance increased the micronuclei frequency in polychromatic erythrocytes of male rats by 2.4-, 4.2-, and 5.5-fold and of female rats by 1.9-, 2.6-, and 4.3-fold at concentrations of 2, 4. and 8 %, respectively. The PCE/NCE ratios, compared to control values, were reduced by 39, 51, and 57 % in male rats and by 50, 56, and 60 % in female rats of the 2, 4, and 8 % groups, respectively.

Table: Micronuclei Induction by CF3I in Rat Bone Marrow Erythrocytes

Group	Micronucleated Cells (%)^a	Ratio
Male Rats 30 Days
Control (3)^b	0.23 ± 0.05	1.00
4 % (5)	0.64 ± 0.10	2.74
8 % (4)	0.90 ± 0.12	3.86
Male Rats 90 Days
Control (2)	0.25 ± 0.05	1.00
2 % (5)	0.60 ± 0.09	2.40
4 % (5)	1.06 ± 0.17	4.24
8 % (5)	1.38 ± 0.27	5.52
Female Rats 30 Days
Control (2)	0.20 ± 0.08	1.00
2 % (5)	0.20 ± 0.06	1.00
4 % (5)	0.70 ± 0.13	3.50
8 % (4)	0.98 ± 0.18	4.88
Female Rats 90 Days
Control (2)	0.30 ± 0.10	1.00
2 % (5)	0.58 ± 0.16	1.93
4 % (5)	0.78 ± 0.16	2.60
8 % (5)	1.30 ± 0.24	4.33

a: Mean ± SD

b: (N)

Analytical verification of test atmosphere

Daily mean exposure concentrations were maintained within ± 2% of the target concentrations. The daily mean reladve humidity ranged between 47 and 52%, while the daily mean temperature ranged from 67 to 73°F.

Conclusions:

The positive evaluation in the mouse bone marrow erythrocyte micronucleus test indicated that test substance was positive in vivo.

Executive summary:

As part of a sub-chronic (90 day) repeated dose toxicity study incl. a 4 week interim sacrifice a erythrocyte micronucleus assay was performed. In total fifteen male and 15 female rats were exposed 2 hr/day, 5 days/week, for up to 13 weeks (65 exposures over 90 days) to 0 (air control), 2, 4, or 8% test substance. Prior to initiation of the exposures, rats were acclimated to the nose-only chamber restraint system, breathing air only, 2 hr/day for 1 week. Bone marrow cells were collected from the femur and smears were prepared from five rats/sex/group at both the 4- and 13-week animal terminations to investigate the mutagenic potential of the substance via induction of micronuclei in bone marrow polychromatic erythrocytes. Increases in the frequency of micronucleated bone marrow polychromatic erythrocytes were observed in rats of all three groups. Therefore the substance was considered positive under the experimental conditions.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Dec 1996 to Jan 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

An erythrocyte micronucleus assay was performed as part of a combined repeated dose and reproductive toxicity screening study was performed in Sprague-Dawley Crl:CD(SD)BR rats. In total the study had 16 male and 16 female rats/group to yield at least 12 pregnant females at term. The animals were assigned to one of four groups (0, 0.2, 0.7, and 2.0%) and exposed to substance vapor for 4 wk (6 h/day, 5 days/wk) prior to mating. An additional six male and three female rats were assigned to a fifth group only to serve as positive controls in the micronuclei assay. Animals were exposed 6 h/day, 7 days/wk, during the mating, gestation, and lactation phases of the study. However, dams were not exposed from gestation day 21 through lactation day 4 to allow for parturition and early lactation. The remaining 8 male rats/group and 16 female rats/group were terminated over a 5-day period subsequent to when the last female rat on study reached lactation day 21. Animals were killed by CO2 inhalation overdose followed by exsanguination. Bone marrow cells were collected from the femur and smears were prepared from all rats. Positive control rats were administered a single dose of cyclophosphamide (7.5 mg/kg) intraperitoneally 24 hrs prior to termination. Slides were stained by the Giemsa/May-Greenwald method and observed microscopically at 100x.

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, MA.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 11 weeks at the start of exposure
- Weight at study initiation: around 225 g at SD 0. Body weights of males are not reported.
- Housing: The rats were housed in wire-bottom cages during exposure (one per cage except during the mating period) and in plastic shoebox cages with bedding (two males per cage, one female per cage, except during the mating period) during nonexposure periods. Exposure cages were rotated in a clockwise manner within the chamber each exposure day.
- Diet: certified rodent diet (PMI Feeds, Inc., St. Louis, MO) was available ad libitum during nonexposure periods.
- Water: water was available ad libitum during nonexposure periods.
- Acclimatization period: 2 to 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 21 and 26
- Humidity (%): between 35 and 65
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Whole-body inhalation exposures were performed in 690-L chambers made of stainless steel and glass.
- Source and rate of air: The substance and air for dilution were controlled through flow meters
- Temperature, humidity, oxygen and ammonia in air chamber: Relative humidity and temperature of the exposure atmosphere were constantly monitored and recorded. Oxygen concentration of the high concentration chamber was analyzed on occasion using a Hudson O2 sensor model 82T (MDA, Lincolnshire, IL). Ammonia levels during exposure were estimated not to be of biological importance
- Air flow and air change rate: To conserve the use of test material and minimize cost, total chamber air flow was reduced to 60 L/min (approximately 5 air changes/h).

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

- 6 hours/day, 5 days/wk prior to mating
- 6 hours/day, 7 days/ wk during the mating, gestation, and lactation phases of the study.
- Dams were not exposed from gestation day 21 through lactation day 4 to allow for parturition and early lactation.
- Pups were not exposed

Dose / conc.:

2 000 ppm

Remarks:

equivalent to 0.2%

Dose / conc.:

7 000 ppm

Remarks:

equivalent to 0.7%

Dose / conc.:

20 000 ppm

Remarks:

equivalent to 2.0%

No. of animals per sex per dose:

16 (to yield at least 12 pregnant females); positive control: 6 male and 3 female animals

Control animals:

yes, concurrent vehicle

Positive control(s):

Single dose of cyclophosphamide (7.5 mg/kg) intraperitoneally 24 hrs prior to termination.

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

Bone marrow cells were collected from the femur and smears were prepared from all rats. Positive control rats were administered a single dose of cyclophosphamide (7.5 mg/kg) intraperitoneally 24 hrs prior to termination. Slides were stained by the Giemsa/May-Greenwald method and observed microscopically at 100x.

Evaluation criteria:

The frequency of micronucleated cells were evaluated by random observation of 1000 polychromatic erythrocytes (PCE) per sample. The ratio between PCE and normochromatic erythrocytes (NCE) was determined by scoring approximately 1000 erythrocytes as an indicator oftoxicity ofthe test agent.

Statistics:

For micronuclei data the Wilcoxon Rank Sum test was used to increase the statistical sensitivity when comparing control group values to treated group values.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Micronuclei scores for male and female rats exposed to CF3I for 7 or 14 wk were similar to micronuclei scores in the control animals. Positive control animals had mean micronuceli scores that were two- to five-fold higher than negative control values. The weak response in the positive control animals was due to the administration of a small dose of cyclophosphamide to induce micronuclei formation. The ratio ofPCE/NCE (an indicator of bone marrow cell toxicity) was similar in all study groups, control or CF3I exposure.

Conclusions:

Bone marrow micronucleus assay did not no show any dose related mutagenic alterations in male or female Sprague Dawley rats at the high exposure dose of 2.0 %.

Executive summary:

An erythrocyte micronucleus assay was performed as part of a combined repeated dose and reproductive toxicity screening study was performed in Sprague-Dawley Crl:CD(SD)BR rats (detailed description in Section 7.8.1). In total the study had 16 male and 16 female rats/group to yield at least 12 pregnant females at term. The animals were assigned to one of four groups (0, 0.2, 0.7, and 2.0%) and exposed to substance vapor for 4 wk (6 h/day, 5 days/wk) prior to mating. An additional six male and three female rats were assigned to a fifth group only to serve as positive controls in the micronuclei assay (Single dose of cyclophosphamide (7.5 mg/kg) intraperitoneally 24 hrs prior to termination.). Animals were exposed 6 h/day, 7 days/wk, during the mating, gestation, and lactation phases of the study. However, dams were not exposed from gestation day 21 through lactation day 4 to allow for parturition and early lactation. The remaining 8 male rats/group and 16 female rats/group were terminated over a 5-day period subsequent to when the last female rat on study reached lactation day 21. Animals were killed by CO2 inhalation overdose followed by exsanguination. Bone marrow cells were collected from the femur and smears were prepared from all rats. Positive control rats were administered a single dose of cyclophosphamide (7.5 mg/kg) intraperitoneally 24 hrs prior to termination. Slides were stained by the Giemsa/May-Greenwald method and observed microscopically at 100x. The frequency of micronucleated cells was evaluated by random observation of 1000 polychromatic erythrocytes (PCE) per sample. The ratio between PCE and normochromatic erythrocytes (NCE) was determined by scoring approximately 1000 erythrocytes as an indicator of toxicity of the test agent. Micronuclei scores for male and female rats exposed to CF3I for 7 or 14 wk were similar to micronuclei scores in the control animals.

In the current investigation, CF3I was negative in the in vivo micronucleus test since there was no increase in micronuclei frequency in the bone marrow erythrocytes of rats exposed to the test item.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 May 1994 to 18 Aug 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Four groups of 5 Swiss Webster mice/ sex were exposed six hours per day for three consecutive days at concentrations of either 0, 2.5%, 5.0% or 7.5% CF3I via nose-only inhalation. An additional group served as a positive control and was dosed 0.4 mg/kg triethylenemelamine on exposure day 3 via i.p. injection. The CF3I mice were exposed in Cannon 52-port nose only chambers (Lab Products, Maywood, NJ), and the negative control mice were exposed in a nose-only chamber made by IN-TOX Products (Albuquerque, NM) due to a shortage of the Cannon Chambers. Forty of the 52 ports of the Cannon chambers were sealed to allow for reduced air flow in an attempt to conserve the test material. The nominal concentration for each exposure was determined by dividing the total amount of the test material consumed (weight of the gas cylinder determined before and after each exposure) by the total exposure chamber airflow. Slides of peripheral blood smears were made for all animals at 24 ± 3 hours after the last exposure by the following procedure. Micronuclei were scored in slides from male and female mice from CF3I exposure group, and in slides from the positive and negative control animals.

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

- Name of test substance: iodotrifluoromethane
- Physical state, colorless gas
- Storage condition of test material: in their original steel containers at room temperature

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: ManTech/RTP, from Charles River Laboratories in Raleigh, NC.
- Age at study initiation: approximately 42 days
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: during all non-exposure periods, the mice were individually housed in suspended stainless steel wire-mesh cages with dimensions of 7" x 4" x 5" (L x W x H)
- Diet: Purina Certified Rat Chow 5002, St. Louis MO, ad libitum except during the actual inhalation exposure periods.
- Water: ad libitum
- Quarantine period: approximately 1 week; mice were examined carefully to ensure their health and suitability as test subjects

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 46-64
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: gas

Vehicle:

air

Details on exposure:

TYPE OF INHALATION EXPOSURE
Nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The CF3I mice were exposed in Cannon 52-port nose only chambers (Lab Products, Maywood, NJ), and the negative control mice were exposed in a nose-only chamber made by IN-TOX Products (Albuquerque, NM) due to a shortage of the Cannon Chambers. Forty of the 52 ports of the Cannon chambers were sealed to allow for reduced air flow in an attempt to conserve the test material.
- Generation of test atmosphere: For each exposure group, the test atmosphere was generated by metering the CF31 gas from the cylinder into either a 2000 or 4000 ml Erlenmeyer flask that served as a mixing plenum. Air was metered into the flask to provide the desired exposure concentration. Each flowmeter used to deliver the gases to the generation system was calibrated with the gas for which it was to be used. The air/CF31 mixture exited the flask and entered the top of the exposure chambers.
- Flow rate: The test mixture was distributed to each animal port at a rate of approximately 52 mL/min (total chamber flow was 621 ml/min).
- Treatment of exhaust air: To maintain proper chamber airflow, the exhaust line controller was adjusted so the static pressure inside the chamber was approximately zero, indicating that the amount of flow entering the chamber matched the amount of flow exiting the chamber. This approach was required because it was not possible to accurately measure the exhaust flow using a rotameter since the flow properties change as the percentage of CF3I changes (CF3I is heavier than air).

TEST ATMOSPHERE MONITORING
- The nominal concentration for each exposure was determined by dividing the total amount of the test material consumed (weight of the gas cylinder determined before
and after each exposure) by the total exposure chamber airflow.
- Actual chamber concentrations were determined by infrared analyses (IR) (Miran 1A, Foxboro Analytical, South Norwalk, CT). The IR analyzer was calibrated using a closedloop calibration method with CF3I gas either just prior to the exposure or, if an existing calibration was used, the calibration was checked, via the closed loop method, just prior to exposure, and the IR calibration was rechecked after each exposure.
- The IR absorbance response, expressed in recorder chart lines, was determined for each known quantity of CF3I injected. A least-squares regression was determined using a Texas TI-60 calculator. Samples for injection into the IR analyzer were collected using a gas-tight syringe from an unused animal port. The IR settings were: pathlength, 12.75 meters; wavelength, 9.7 microns; absorbance, 0.25; response, x 1; slit, 2; and meter response, 4. Oxygen levels from the chamber exhausts were determined using an 0 2/Explosion meter (MSA Model 421).

Duration of treatment / exposure:

six hours/ day

Frequency of treatment:

3 days consecutive

Post exposure period:

All mice were observed immediately upon removal from the exposure chamber and daily during the post-exposure observation period. Necropsy was not done on any of the mice.

Dose / conc.:

25 000 ppm

Dose / conc.:

50 000 ppm

Dose / conc.:

75 000 ppm

No. of animals per sex per dose:

5 animals/sex/dose (including positive/negative control)

Control animals:

yes, concurrent vehicle

Positive control(s):

one i.p. dose of 0.4 mg/kg triethylenemelamine (TEM, CAS No. 51-18-3),

Tissues and cell types examined:

Slides of peripheral blood smears

Details of tissue and slide preparation:

Sacrifice and Slide Preparation
Slides of peripheral blood smears were made for all animals at 24 ± 3 hours after the last exposure by the following procedure: Bovine calf serum, 2-3 µL, was placed on a slide pre-cleaned with methanol. Each mouse was sacrificed by cervical dislocation and 2-3 µL of blood per slide was obtained from the mid-ventral tail vein of a mouse and placed on top of the serum. The blood was mixed with the serum and spread on the slide to produce a thin, even film, then the slide was allowed to air-dry. Three slides were prepared per mouse, and, after the slides were dry, the erythrocytes were fixed by placing the slides in absolute methanol for two minutes; then they were allowed to air-dry vertically. ManTech/RTP was responsible for disposal of the carcasses.

Staining of Slides
The slides were stained for 20 minutes in 5 % Giemsa stain in phosphate buffer containing 3 % methanol and 3 % 0.1M citric acid, rinsed by dipping them in deionized water until clear, and allowed to air dry vertically. Coverslips were attached with Permount
before the erythrocytes were analyzed at 100X, oil immersion, magnification.

Scoring of Slides
Micronuclei were scored in slides from male and female mice from CF3I exposure group, and in slides from the positive and negative control animals. The treated and control slides were divided into three identical groups. Two groups of slides were coded
by an individual not involved in the scoring or analysis, and the third group of slides was held in reserve, uncoded. Two observers were utilized, one for each set of coded slides. Each bone marrow smear was inspected under low power to observe the distribution of cells and to select an area with good cell morphology and thin, even density (without overlapping cells) for scoring. Each slide was then scored for micronuclei using oil immersion objectives. The criteria which distinguish micronuclei from artifacts have been described by Schmid (1976). Micronuclei are identified as round or oval shaped bodies found in the cytoplasm of erythrocytes. Bodies which are refractile, improperly shaped or stained, or which are not in the focal plane of the cell are not scored as micronuclei. Cells containing more than one micronucleus are scored as a single micronucleated cell.

Evaluation criteria:

- Positive: The criteria of a positive response defined in the protocol were refined to incorporate criteria defined in the literature (Margolin and Risco, 1988). Thus, a test material is considered to have elicited a positive response in the mouse erythrocyte micronucleus test if a statistically significant (p < 0.05) dose-related increase in micronuclei is observed.

- Negative: A test material is considered to have elicited a negative response if the positive control responses are in the appropriate ranges and the criterion for a positive response is not met.

Both biological and statistical significance were considered together in the evaluation of the results.

Statistics:

The results were analyzed by ManTech/Dayton using a statistical design that was a two factorial analysis of variance with Bonferroni multiple comparisons. This statistical design has two assumptions, normal residuals and equality of variances. The presence of normal residuals was tested using the Wilk-Shapiro test of normality; the equality of variances was tested by Leven's test of the equality of variances. Either natural log or square root trasformation of the data was used when tests for normality or equality of variances indicated heterogeneity. If sex was not a significant interaction in the factorial analysis, male and female data were combined.

Key result

Sex:

male/female

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Toxicity of CF3I was evidenced by dose-related depressions in weight for both sexes and by dose-related depressions in ratios of PCEs/1000 erythrocytes for female mice.
- No mice died during the study, and all mice appeared normal throughout the study.
- Significant (p < 0.05) dose-related increases in micronuclei/1000 PCEs were observed in both male and female mice of the 5.0 and 7.5 % CF3I exposure groups.

Control data
- In the negative control animals, PCE ratios were 11.1‰ for male mice and 20.8 ‰for female mice. Both ratios were within historical control ranges for the laboratory, and no physiological basis for the difference in ratios between the sexes was apparent. Appropriately low numbers of MN/1000 PCEs were observed in the negative control mice: 2.0 ‰ in male mice and 1.0‰ in female mice, values which were also within historical ranges for the laboratory.
- PCE/erythrocyte ratios were depressed and MN/PCE ratios were elevated in the positive control mice. In response to 0.4 mg/kg TEM, the PCE ratio was 2.8‰ in male mice and 3.3‰ in female mice, and high ratios of MN/1000 PCEs were observed for the positive control animals: 24.6‰ in male mice and 34.1‰ in female mice. The PCE ratios had normal residuals and equal variances among all potential effects (dose, sex, and dose by sex), and the sex of the animals influenced the effect of the positive control (p = 0.0290). The MN ratios did not have normal residuals or equal variances; therefore, the natural log transformation was used on MN ratios. The natural logs of MN ratios had normal residuals and equal variances. The interaction between the dose of TEM and the sex of the animals was not statistically significant (p = 0.0541), but the effect of TEM dose was statistically significant (p = 0.01) for both sexes

Conclusions:

Test substance was evaluated as positive in the mouse bone marrow micronucleus test.

Executive summary:

The potential of test substance to induce micronuclei in erythropoietic cells of the bone marrow was studied. Based on preliminary toxicity information, a mouse bone marrow micronucleus test of the test substance was conducted using 2.5 %, 5.0 %, and 7.5 % test substance, administered to male and female Swiss Webster mice by inhalation for six hours on each of three consecutive days. Bone marrow cells were obtained from the mice sacrificed 24 hours after the third exposure. Erythrocytes from mice exposed to the test material, and to the negative and positive controls, were evaluated for toxicity and the presence of micronuclei. The positive control, 0.4 mg triethylenemelamine (TEM)/kg (administered intraperitoneally) significantly (p<0.01) elevated the number of micronuclei in newly-formed erythrocytes (PCEs, polychromatic erythrocytes) from male and female mice.Toxicity of the test substance was evidenced by dose-related depressions in weight for both sexes and by dose-related depressions in ratios of PCEs/1000 erythrocytes for female mice. Significant (p<0.05) dose-related increases in micronuclei/1000 PCEs were observed in both male and female mice of the 5.0 and 7.5 % CF3I exposure groups. Therefore, test substance was evaluated as positive in the mouse bone marrow micronucleus test.

Reference 5

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15 Nov 2017 to 21 Nov Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

other: CByB6F1-Tg(HRAS)2Jic (wild type) and CByB6F1/TgrasH2 hemizygous (transgenic)

Details on species / strain selection:

The CByB6F1-Tg(HRAS)2Jic wild type mouse was chosen as the animal model for this study because it has the same genetic background as the CByB6F1/TgrasH2 hemizygous transgenic (Tgras H2) mouse, except that it lacks the Tg element and is a preferred strain by regulatory agencies for range-finding studies to set doses for the 26-week TgrasH2 carcinogenicity assays. The Tgras H2 mouse is recommended for genotoxic and non-genotoxic carcinogen identification and will be used in a future 6-month carcinogenicity study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Biosciences, Inc., Germantown, NY
- Age at study initiation: 8 weeks
- Weight at study initiation: wild type mice 16.8 to 26.7 g, transgenic mice: 19.7 to 30.3 g
- Assigned to test groups randomly: yes, by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately (by strain)
- Housing: animals were group housed (2 to 3 animals of the same sex and strain) until randomization. Following randomization, animals were housed individually in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve. Enrichment devices were provided to all animals as appropriate.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (meal) ad libitum throughout the study, except during the inhalation exposure periods
- Water: Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system.
- Acclimation period: at least 5 days

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

IN-LIFE DATES: From: 15 Nov 2017 To: 21 Nov 2017

Route of administration:

inhalation: gas

Vehicle:

- Vehicle used: air (filtered)

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

INHALATION EXPOSURE METHODS
Filtered air and the test substance were administered as a daily 6-hour and approximately 20-minute (to accommodate the T99 period), whole body inhalation exposure for 5 consecutive days; the 5 chambers were staggered by 5–22 minutes to accommodate body temperature measurements following exposure. The filtered-air control exposures were conducted in a 1000-L stainless steel and glass whole-body exposure chamber. Test substance exposures were conducted in four 500-L stainless-steel and glass whole-body chambers. One chamber was dedicated to each of the 5 exposure levels. The chambers were operated under dynamic conditions, at a slight negative pressure, and with a minimum of 12 air changes per hour. Air supplied to the chambers was provided from the Inhalation Department breathing quality, in-house HEPA- and charcoal-filtered, temperature- and humidity controlled supply air source. All chambers exhaust was directed to the facility exhaust system, which consisted of redundant exhaust blowers preceded by activated-charcoal and HEPA-filtration units. All animals were housed in a normal animal colony room during nonexposure periods. Prior to each exposure, the animals were transferred to exposure caging, transported to the exposure room, exposed for the requisite duration, and then returned to their home cages. During the exposure period, animals had access to water, but food was withheld. The mean temperature and mean relative humidity of the exposure atmospheres were to be between 73°F ± 5°F (23°C ± 3°C) and 50 ± 20%, respectively. Oxygen content of the exposure atmospheres was measured during the method development phase and was 20.9% for all groups.

EXPOSURE ATMOSPHERE GENERATION METHODS
CONTROL EXPOSURE SYSTEM
The control exposure system (Group 1) was operated as follows. Humidified supply air was delivered to the chamber from the facility humidified supply air source and metered using a rotameter-type flowmeter.

TEST SUBSTANCE EXPOSURE SYSTEMS
Test substance was generated by releasing the test substance gas (1,000,000 ppm) from the original cylinder. One cylinder was used to generate to Chambers 2 and 3 and another cylinder was used to generate to Chambers 4 and 5. The cylinder outlet pressure was controlled using a regulator equipped with a pressure gauge on each test substance cylinder. From the cylinder the test substance gas was delivered to a stainless steel manifold where it was distributed to the chambers. The manifold pressure was monitored using a Dwyer Digital Pressure Gauge. The test substance gas from the manifold was controlled using a needle valve and monitored using a rotameter-type flowmeter for Chambers 2 and 3 and needle valve in-line with an Omega mass flowmeter for Chambers 4 and 5. Test substance gas was directed to the chamber inlet, where it mixed with Inhalation Department dilution supply air to achieve the desired atmosphere concentration.

METHODS FOR CHARACTERISATION OF EXPOSURE ATMOSPHERES
NOMIAL EXPOSURE CONCENTRATIONS
Nominal exposure concentrations were not calculated due to the configuration of the exposure atmosphere generation systems where a manifold system was used to deliver test substance between 4 exposure chambers. Documentation of daily test substance usage was performed by calculating the total amount of test substance consumed during each exposure. The amount of test substance consumed during each exposure was defined as the difference between the weight of the test substance cylinder prior to each exposure and the weight of the cylinder after each exposure.

ANALYSED EXPOSURE CONCENTRATIONS
Exposure atmospheres were sampled and analysed at approximately 45-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. Sampling and analyses was performed as follows. An external multi-position valve 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 ln-quadratic equation based on the GC calibration curve was used to calculate the measured concentration in ppm.

PURITY ANALYSIS
A sample of the test substance was collected from a primary test substance cylinder prior to initial use. The percent concentration of the test substance was determined by the Analytical Chemistry Department of the test facility using a gas chromatographic method with detection by flame ionization.

STABILITY ANALYSIS
A sample of the test substance was collected from a primary test substance cylinder following the last animal exposure. The percent concentration of CF3I was determined in a similar manner as the purity analysis. The stability of the test substance was determined by the Analytical Chemistry Department of the test facility by comparing the end of use percent concentrations to the time zero (pre-use) values.

Duration of treatment / exposure:

5 consecutive days

Frequency of treatment:

6 hours/day

Dose / conc.:

2 500 ppm (nominal)

Remarks:

Group 2

Dose / conc.:

5 000 ppm (nominal)

Remarks:

Group 3

Dose / conc.:

10 000 ppm (nominal)

Remarks:

Group 4

Dose / conc.:

20 000 ppm (nominal)

Remarks:

Group 5

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control slides (stock slides) were prepared in a previous study at the test facility. In that study, the positive control (cyclophosphamide monohydrate, 60 mg/kg) was administered by oral gavage and bone marrow was harvested approximately 24 hours after the dose was administered.

Tissues and cell types examined:

Bone marrow (wild type mice only): polychromatic and normochromatic erythrocytes

Details of tissue and slide preparation:

For wild type mice only, bone marrow was collected from all animals at the time of euthanasia, following blood collection, from both femurs of animals euthanized by inhalation of carbon dioxide. Positive control slides (stock slides) were prepared in a previous study. In that study, the positive control (cyclophosphamide monohydrate, 60 mg/kg) was administered by oral gavage and bone marrow was harvested approximately 24 hours after the dose was administered. Bone marrow was aspirated or flushed 2 to 3 times from both femurs into a centrifuge tube using a syringe containing heat inactivated fetal bovine serum (HI FBS). The bone marrow was centrifuged and all but approximately 0.25 mL (or a volume approximately twice that of the cell pellet) of HI FBS was decanted, and the pellet was resuspended in the remaining HI FBS. Bone marrow smears were prepared by placing approximately 1 drop of cell suspension onto a minimum of 4 appropriately labeled, clean microscope slides. Each slide was coded so that the treatment group would not be revealed during subsequent analysis. The slides were air dried, fixed in 100% methanol for approximately 20 minutes, and allowed to air dry a second time. The slides were stored and shipped at ambient temperature to the test facility in Montreal for analysis. Prior to analysis, the coded slides were stained with acridine orange staining solution. Two separate evaluations were made for each slide: 1) a total of at least 500 erythrocytes (both polychromatic erythrocytes [PCEs] and normochromatic erythrocytes [NCEs]) per animal were counted and the PCE:total erythrocytes [TE] ratio was determined; and 2) the number of micronucleated PCEs from a total of 4000 PCEs was scored per animal.

Statistics:

Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables.
For statistics conducted for micronucleus evaluation and for in-life procedures and observations, see 'Any other information on materials and methods incl. tables'.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Animals treated with the test substance did not show any substantial decreases in the polychromatic erythrocyte and normochromatic erythrocyte ratio, indicating that the test substance did not cause bone marrow toxicity following the administration of 5 consecutive days by 6-hour whole-body exposures to male and female mice. Moreover, all test substance-treated group mean values were within the historical vehicle/negative control range of the test facility. Male and female rats treated with the test substance did show statistically significant increases in the highest treated males (20,000 ppm) for the percent mean number of micronucleated polychromatic erythrocytes (%MN-PCEs) (0.300%) compared to the control group (filtered air: 0.200%). However, the statistical analysis did not show a trend increase throughout the groups. Moreover, group mean values for all other male lower groups, as well as all female groups, were within the historical negative control range of the test facility and did not show statistically significant increases compared to the control group (filtered air: 0.200%). Following closer evaluation of the individual data and discussion with the Study Director and Sponsor, it was suspected that 1 or more individual values could have caused an artefactual mean group increase and might not be biologically relevant. Therefore, the micronucleus data for males (Groups 1 to 5), were submitted to outlier assessment and a single male was found to be an outlier value and was removed from the statistical re-analysis. Following the re-analysis, excluding the outlier value, male mice treated with the test substance did not show statistically significant increases in the highest treated males (20,000 ppm) for the %MN-PCEs (0.238%) compared to the control group (filtered air: 0.200%). Therefore, the test substance had no clastogenic effects nor did it cause disruption of the mitotic apparatus through micronuclei formation in bone marrow immature erythrocytes of male and female mice, when treated by 6-hour whole-body exposures of the test substance at dose levels of 2500, 5000, 10,000, and 20,000 ppm, for 5 consecutive days. Thus, the test substance was considered not to be genotoxic in this in vivo test when considering the biological relevance of the results.

Characterization of Exposure Atmospheres

Results of the characterization of exposure atmospheres are summarized below.

 

Actual Exposure Concentrations

Daily mean atmosphere concentrations for each exposure system are presented in Table 1.

 

Table 1. Overall Mean Exposure Concentrations

Exposure System:	1	2	3	4	5
Target Concentration (ppm)	0	2500	5000	10,000	20,000
Mean Concentration (ppm)	0	2538	5007	9916	20,137
Standard Deviation	0.0	52.4	96.0	370.0	445.1
Total Number of Exposures	6	6	6	6	6

 

Analyses of Test Substance Purity and Stability

The analysed test substance collected after completion of exposure was determined to be 100% of the pre-use value which was within the Test facilities SOP acceptance criteria (the end of use value was not < 90% of the pre-use value).

 

Mortality

All animals survived until scheduled necropsy.

 

Observations

There were no test substance-related clinical observations in either mouse strain.

 

Body Weights

Body weights were unaffected by test substance administration. However, statistically significant higher mean body weight gains were noted in the 20,000 ppm group wild type and transgenic males from Day 2 through 4 and Day 0 through 2, respectively, compared to the control group. In addition, statistically significant higher mean cumulative body weight gains were also noted in the 20,000 ppm group transgenic males from Day 0 through 2 and Day 0 through 5 compared to the control group. Although these differences in body weight gain and cumulative body weight gain were possibly test substance-related, they were not considered toxicologically significant based on the direction of the change and the lack of effects on mean body weights. Statistically significant lower mean body weight gains were noted in the 2500 ppm group wild type females from Day 0 through 2 compared to the control group. Although these differences in body weight gain and cumulative body weight gain were possibly test substance-related, they were not considered toxicologically significant based on the transient nature of the difference compared to the control group and/or the lack of a dose-dependent response.

 

Body Temperature

There were no test substance-related effects on body temperatures in either mouse strain. However, a statistically significant higher mean body temperature was noted in the 20,000 ppm group wild type females on Day 0 compared to the control group. This difference was not considered test substance-related, because the difference appeared to result from a low mean for the control group on Day 0 only.

Conclusions:

Based on the results of this study, test substance exposure by whole-body inhalation to CByB6F1-Tg(HRAS)2Jic wild type and CByB6RF1/TgrasH2 Hemizygous transgenic mice at exposure concentration levels of 2500, 5000, 10,000, and 20,000 ppm for 5 consecutive days resulted in no toxicologically significant effects on clinical observations, body temperature, or body weight and in a negative response for induction of bone marrow micronuclei at exposure concentration levels up to 20,000 ppm.

Executive summary:

The objective of this GLP compliant OECD 474 study was to evaluate the potential toxicity of the test substance when administered by whole-body inhalation for 6 hours per day for 5 consecutive days to CByB6F1-Tg(HRAS)2Jic wild type and CByB6F1/TgrasH2 Hemizygous transgenic mice. Five animals per sex per strain were exposed to concentrations of 2500, 5000, 10,000 and 20,000 ppm. The results of this study will be used to support the selection of exposure concentrations for a 4-week range-finding study in wild type mice and a 26-week inhalation carcinogenicity study in Tg.rasH2 mice. In addition, blood and bone marrow samples were collected from the wild type mice for evaluation of micronuclei in bone marrow and other possible future micronucleus evaluations. Animals were dosed for 6 hours per day for 5 consecutive days via whole-body inhalation. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, body temperature, and micronucleus parameters (wild type mice only). All animals survived to the scheduled necropsy. There were no test substance-related clinical observations or effects on body weight or body temperature. The test substance did not show a substantial decreases in the polychromatic erythrocytes and normochromatic erythrocyte ratio. Therefore, the test substance did not cause bone marrow toxicity. Based on the results of this study, test substance exposure by whole-body inhalation to CByB6F1-Tg(HRAS)2Jic wild type and CByB6RF1/TgrasH2 Hemizygous transgenic mice at exposure concentration levels of 2500, 5000, 10,000, and 20,000 ppm for 5 consecutive days resulted in no toxicologically significant effects on clinical observations, body temperature, or body weight and in a negative response for induction of bone marrow micronuclei at exposure concentration levels up to 20,000 ppm.

Reference 6

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:

11 Jul 2005 to 24 Aug 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

TNO Nutrition and Food Research, 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 liquidified gas
CAS no.: 2314-97-8
Purity: 99.99 wt%
Batch number: BU-173
Storage conditions: ambient temperature
Expiry date: 30 June 2006

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 considered suitable for this type of study, rich in background data and commonly used in in-vivo genotoxicity tests.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 4-6 weeks old
- Weight at study initiation: ranged from 189.0 to 235.4 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 the exposure period. 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 the exposure period. Tap-water was supplied in polypropylene bottles which were cleaned weekly and filled as needed. Tap-water 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: 3 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 to 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: gas

Vehicle:

air

Details on exposure:

- Exposure apparatus: Animals were exposed to the test atmosphere in nose-only exposure units. Each unit consisted of a cylindrical column, surrounded by a transparent hood. Entrance of the test atmosphere was at the bottom of the central column, and the exhaust was at the top. The column had a volume of ca.70 L. The column had two rodent tube section with 40 ports for animal exposure in each section. Empty ports were used for test atmosphere sampling and/or measurement of oxygen concentration, temperature and relative humidity.
- 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. By securing a positive pressure in the central column and a slightly negative pressure m the outer hood, which encloses the entire animal holder, dilution of test atmosphere by air leaking from the animals' thorax to the nose was avoided.
- Generation of test atmosphere: To generate the test atmosphere, evaporated test substance was mixed with clean humidified air before entering the inhalation chamber. Because of the relatively high concentration of the test material in the test atmosphere, oxygen was added as necessary to ensure an oxygen concentration of at least 19 %. The flows were controlled by mass flow control units (Bronkhorst, Ruurlo, The Netherlands), which were calibrated for the gas used with a volumetric flow meter (DryCal, Bios International Corporation, Butler, NJ, USA).
- Temperature, relative humidity and oxygen: The temperature and relative humidity of the test atmospheres were recorded ten times during exposure at regular intervals (about twice per hour during the 4 h exposure period) using an RH/T device (TESTO 610, TESTO GmbH & Co, Lenzkirch, Schwarzwald, Germany). The oxygen concentration was checked once during exposure (Oxygen analyser type PMA10, M&C Products Analysentechnik GmbH, Ratingen-Lintorf, Germany).

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

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.:

20 000 ppm

Remarks:

corresponding to actual concentration of 20135 ± 97 ppm

Dose / conc.:

80 000 ppm

Remarks:

corresponding to actual concentration of 80576 ± 347 ppm

Dose / conc.:

200 000 ppm

Remarks:

corresponding to actual concentration of 201052 ± 973 ppm

No. of animals per sex per dose:

- 5 males / group, including positive control group
- 10 males for negative control and high dose group (5 animals for 24h killing time and 5 animals for 48 hour killing time)
- 4 additional males were added to the high dose group in order to replace any mortality.

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 cells

Details of tissue and slide preparation:

Bone marrow sampling:
Two hours prior to sacrifice, animals were injected intraperitoneally with colchicine (Sigma-Aldrich Chemie BV, The Netherlands) at a concentration of 4 mg/kg bw (dosing volume 10 mL/kg bw; stock concentration 0.4 mg/mL) to accumulate cells in the metaphase stage of their cell-cycle. For each individual rat the dosing volume was related to the body weight. At ca. 24 hours of exposure. 5 rats of treatment groups A, B, C and E and 3 surviving animals of group D were euthanized. At ca. 48 hours after exposure, 5 rats of treatment group A and 3 surviving rats of treatment group D were euthanized. After sacrifice of rats, 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:
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 numbers of cells with structural chromosomal aberrations or a clear increase in the number of cells with structural chromosomal aberration 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 concetrations 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.

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 were analysed statistically by a non-parametric Kruskal-WaIIis Anova, followed by Mann-Whitney U-tests when Kruskal-Wallis Anova yielded a significant effect (p < 0.05). When only two groups are present, Mann-Whitney U-tests were applied.
- At time point 24 hours, data from groups A, B, C and D were subjected to Kruskal-Wallis one way Anova.If Kruskal-Wallis Anova yielded a significant effect (p < 0.05) it was followed by Mann-Whitney U-tests (2-sided). Mann-Whitney U-tests were applied to the negative control group A versus treatment groups B, C and D. The positive control group E and the negative control group A were compared using Mann-Whitney U-tests.
- At time point 48 hours, data from groups A and D were subjected to Mann-Whitney U-tests
- 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:

- Clinical signs:
In treatment group D (200000 ppm), 8 rats (D66, D68, D70, D76, D78 and three reserve rats) of a total of 14 rats were found dead within half an hour after the start of exposure. Exposure was therefore discontinued after 35 minutes. The six survivors showed trembling but recovered without further mortality and were dividedbetween the 24 hours and 48 hours sacrifice time points.
In treatment goup C (80000 ppm), all rats showed trembling and piloerection after exposure. The trembling gradually subsided until it could no longer be seen.
Clinical signs were not seen in groups A (control) or B (20000 ppm)

- Microscopic evaluation of the bone marrow slides:
At the sacrifice time of 24 hours, the mean mitotic index of the lowest exposure level used (20000 ppm) was not reduced when compared to the mean mitotic index of the negative control (clean air) rats. The mean mitotic indices of the two highest exposure level used (80000 ppm and 200000 ppm) were reduced to 84 % and 76 %, respectively of that of the mean mitotic index of the negative control (clean air) rats. The statistical test results indicated there were no statistically significant differences compared to the negative controls.
At the sacrifice time of 48 hours, the mean mitotic index of the highest exposure level used (group D; 200000 ppm) was reduced to 74 % of that of the mean mitotic index of the negative control (clean air) rats. The statistical test results showed that the mean mitotic index of group D differed significantly (* < 0.05) from the mean mitotic index of the negative control group A. This indicates that the test substance reached the bone marrow and that treatment with this test substance resulted in cytotoxicity to the bone marrow cells.
At both sacrifice times, 24 hours and 48 hours after exposure, the test substance did not induce a statistically significant increase in the number of cells with structural chromosomal aberrations at all exposure levels (20000, 80000 and 200000 ppm) analysed, when compared to the concurrent negative (clean air) control values.
At the sacrifice time of 24 hours, the results of rats treated with the positive control substance mitomycin C (group E) showed the expected statistically significant effects (**p < 0.01) on the percentage of cells with structural chromosomal aberrations (including and excluding cells with only gaps) and the mitotic index (**p < 0.01), when compared to the negative control (clean air) group A. The latter results demonstrated the validity of the test system.

Conclusions:

It is concluded that test substance 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. This indicates that exposure to test substance by inhalation did not induce clastogenicity to the bone marrow cells of male rats.

Executive summary:

The test substance, administered by inhalation, was examined for its potential to induce structural chromosomal aberrations in bone marrow cells of male Wistar Crl:[WI] WU BR rats according to OECD 475. 5 male animals/ dose per killing time were treated for a period of 4 hours with three exposure levels, 200000, 80000 and 20000 ppm of the test substance. In addiditon 4 spare animals were introduced in the high dose group to compensate for mortality and a positive control group ( Mitomycin C) was included to show the validity of the assay. The animals were exposed and sacrificed on four consecutive days. The exposure of the rats to the highest exposure level (200000 ppm) was stopped after 35 minutes, because 8 rats of 14 rats died as a result of this exposure level and the surviving animals showed reversible clinical signs. At both sacrifice times, 24h and 48h (latter timepoint control and high dose only) after exposure, no dose level of the test substance induced a statistically significant increase in the number of cells with structural chromosomal aberrations, when compared to the concurrent negative (clean air) control values. Exposure to test substance did not result in clastogenicity to bone marrow cells of rats.

Reference 7

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.

Reference 8

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 12 May 1981

Deviations:

no

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:

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

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 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

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

Dose / conc.:

20 000 ppm

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)

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

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

CF₃I has been demonstrated to induce gene mutations in two strains of S. typhimurium (TA98 and TA1537) and one strain of E.coli (WP2 uvrA). These results confirm the findings reported by Dodd et al. (1997a). Some differences in mutation profile are noted between our findings and those reported by Dodd et al. (1997a), for example in our study no biologically relevant and reproducible increases in revertants were observed in S. typhimurium strains TA100 and TA1535, whereas these strains gave clear positive results in the previous evaluation. However, Dodd et al. (1997a) do not report the source or purity of the CF₃I gas used. Furthermore, the bacterial plates were exposed to the CF₃I-environment for 48 hours, compared to 24 hours as reported here. These differences are likely to have contributed to the additional mutagenicity reported by Dodd et al. (1997a). 

The mutagenic effects observed in bacteria are not seen in mammalian cells. Dodd et al. (1997a) reported CF₃I to be negative in a mammalian cell gene mutation test in. They exposed L5178Y tk^+/- cells to concentrations of CF₃I (up to 51.8%) for four hours in the absence and presence of rat liver S9 exogenous activation. Acceptable levels of cytotoxicity were achieved at the highest concentrations tested (approximately 20% relative total growth) and there was no increase in mutant frequency at any concentration tested. In the UDS assay reported here, male Wistar rats were exposed by nose-only inhalation, at concentrations 1000, 2000 or 4000 ppm CF₃I for 6 hours/day, 5 days/week for 4 weeks. No evidence of UDS was observed at any dose level. Finally, in the OECD TG 488 compliant transgenic rodent gene mutation assay reported here, male Big Blue® F344 rats were exposed to CF₃I at concentrations of 1250, 2500 or 5000 ppm by nose-only inhalation for 6 hours/day, over 28 consecutive days. No increases in mutant frequency were observed in the lung, liver or bone marrow.  

The exposure concentrations used in the Big Blue® F344 rat study are acknowledged to be considerably lower than exposures cited for other in vivo genotoxicity assessments of CF₃I. However, exposures greater than 5000 ppm in the Big Blue® F344 rats were excessively toxic, resulting in morbidity and a severe drop in body temperature. Three separate laboratories have performed inhalation genotoxicity assessments on CF₃I: the acute and report dose exposures reported by Dodd et al. (1997a and b), the acute and repeat dose exposures for chromosome aberration and/or UDS assessments and finally the repeat dose exposure for the transgenic rodent gene mutation assay. All three groups found that the required test atmospheres could be generated by dilution of CF₃I (from a gas cylinder) with air, with nominal values closely matching analytical values. For the studies by Dodd et al. (1997a and b), test substance analysis in air was conducted by an infra-red method, although different path lengths were used depending on the study, the absorption path lengths described would seem to imply measurement at some distance from the breathing zone. The method used during the chromosome aberration and UDS studies measured total carbon content by flame ionisation, whereas in the transgenic rodent gene mutation study, the actual chemical content of air was measured by a GC method from samples taken at the breathing zone where the nose of a rat would be located. However, all three methods resulted in analytical results that were in close agreement with the nominal exposure concentrations; and therefore, it is unlikely the apparent difference in toxicity is a result of analytical method or atmosphere generation. Despite the differences in actual dose levels, similar observations in treated animals were seen in all repeat dose studies. It is considered that the transgenic rodent gene mutation study was conducted to an appropriate and valid maximum tolerated dose that, combined with the dose route, ensured exposure to the three tissues examined for mutation, i.e. lung, liver and bone marrow.

Overall the available gene mutation data indicate that CF₃I is uniquely genotoxic to bacteria as mutations were not detected in either L5178Y mouse lymphoma cells or rat lung, liver or bone marrow. The reason for this bacterial specific effect is unknown.

Chromosomal effects, identified as an increase in micronuclei in immature bone marrow erythrocytes, have been observed in rats and mice. Dodd et al. (1997a) exposed Swiss Webster mice by nose-only inhalation to CF₃I at 25000, 50000 or 75000 ppm for 6 hour/day for 3 consecutive days; blood samples were taken approximately 24 hours after the last exposure. All mice appeared normal after treatment, however, animals exposed to 5 or 7.5% CF3I lost substantially more weight than the controls during the in-life portion of the study indicating treatment-induced non-specific stress. Bone marrow toxicity was observed in females only at all exposure concentrations and increases in micronuclei were observed in male and female mice at 50000 and 75000 ppm. Repeat dose exposures of rats were reported by Dodd et al. (1997b). F344 rats were exposed to concentrations up to 80000 ppm for 2 hours/day, 5 days/week via nose-only inhalation. Bone marrow samples were assessed for micronuclei after 4 and 13 weeks of exposure. Rats at all dose levels were reported to show a dose-related increase in activity during exposure, then to be lethargic after exposure. There was also a significant loss in body weight, in rats exposed to 40000 or 80000 ppm CF₃I. Increases in micronuclei together with concomitant bone marrow toxicity were observed after 4 weeks exposure at 40000 and 80000 ppm CF₃I and at 20000, 40000 and 80000 ppm CF₃I after 13 weeks exposure. Two studies have reported no evidence of micronucleus induction in rats or mice. In a single generation reproductive toxicity study (Dodd et al. (1999) Sprague Dawley rats were exposed to concentrations of up to 20000 ppm in air for 6 hours/day, 5 days/week for either 7 or 14 weeks using whole body inhalation exposure. No clinical symptoms were seen, however, females in the high exposure group (20000 ppm) lost significant body weight compared to the controls. No bone marrow toxicity or increases in micronucleus frequency were observed at any dose level. In the 5-day inhalation dose ranging finding study reported here, male and female CByB6F1-Tg(HRAS)2Jic wild type mice were exposed to CF₃I concentrations of up to 20000 ppm in air for 6 hour/day for 5 consecutive days again using a whole-body exposure and again no biologically relevant increases in micronuclei in either male or female animals were observed.

The US National Research Council review (2004) of CF₃I considered the Dodd et al. (1999) evaluation to be flawed due to the low concentrations used for exposure and lack of bone marrow cytotoxicity observed. They concluded that this study should not be viewed as having equal weight with the other micronucleus studies. A similar criticism could be made of the in vivo micronucleus data reported here, which also employed a maximum exposure of 20000 ppm. At this concentration, increases in micronuclei have been observed in the rats, but only after exposure at 5 days/week for a total of 13 weeks (Dodd et al., 1997b). Following the recommendations of the US National Research Council review (2004), we have reported here the results of acute and repeat dose rat bone marrow chromosome aberration studies. CF₃I was negative for aberrations in male Wistar rats, exposed via nose-only inhalation for either 4 hours at 20000 or 80000 ppm and or for 6 hours/day, 5 days/week for 4 weeks at 10000, 20000 or 40000 ppm. However, a decrease in mitotic index was observed in both studies that confirmed the exposures used did result in bone marrow exposure to CF₃I. The clear negative results for the two bone marrow chromosome aberrations tests, suggests an indirect mechanism may be responsible for the positive micronucleus induction previously reported in rodents.

The in vivo micronucleus assay in rodents, using either bone marrow or peripheral blood, is widely used as part of most genotoxicity test batteries for regulatory human health assessments following exposure to exogenous substances. The assay detects agents that induce chromosome damage or chromosome loss and it is considered to have moderate sensitivity for the detection of carcinogens; false negative results are usually associated with a lack of adequate exposure of the bone marrow to produce a detectable response. However, its specificity is high with a low rate of false positive results. In 2005 IWGT used published literature and proprietary data (gathered from its membership by completion of a questionnaire) to identify substances that were uniquely positive in vivo (Tweats et al., 2007b). As part of their review, they identified a sub-set of these in vivo positive substances that were considered false positives, i.e. the substances induced micronuclei via indirect, non-DNA reactive mechanisms. One of the most data-rich mechanisms was the induction of hypothermia (Tweats et al., 2007a) and the IWGT reviewed publications and/or company-held data for two phenothiazine neuroleptic drugs (chlorpromazine and reserpine), E-5824 (a triazole pyridine developed as an atypical antipsychotic drug), phenol and a coded developmental CNS drug. These substances all induce increases in micronuclei above background levels but only at dose levels that induced a concomitant decrease in core body temperature. Furthermore, except for phenol, no induction of micronuclei was observed in animals treated at the same dose level but held under conditions that enabled them to maintain normal core body temperature (e.g. in rooms with elevated ambient temperature or by the use of heat pads under the cages). No such “recovery” experiment was reported for phenol, however, Gollapudi et al. (2006) and Spencer et al. (2007) investigated the interaction of a non-hypothermic dose of phenol with environmental hypothermia. Mice held at 12°C for 7 hours under restraint, with and without phenol treatment, were found to have increases in micronuclei were observed in hypothermic mice. Mice exposed to non-hypothermic doses of phenol and maintained under normal conditions showed no micronuclei. No interaction between phenol and hypothermia was concluded, but the consequences of hypothermia as increasing micronucleus formation was confirmed. Based on in vitro investigations of the effects of non-physiological temperatures on dividing cell, Tweats et al. (2007a) summarise that hypothermia-induced micronuclei are a consequence of the disruption of chromosome binding to the mitotic spindle, resulting in loss of chromosomes during cell division. Since the IWGT review, Asanami & Shimono (2009) have reported on the species differences in hypothermia-induced micronuclei between rats and mice following intraperitoneal administration of haloperidol, a butyrophenone derivative psychotropic drug. Haloperidol-induced hypothermia was described as moderate to deep in mice but only mild in rats, which was consistent with the reported species differences for chlorpromazine and reserpine (Asanami & Shimono, 1997; Asanami & Shimono, 2000; Asanami et al., 1998). This correlated with a statistically significant increase in micronuclei in mice but no induction of micronuclei was apparent in rats. This is again consistent with the effects seen with chlorpromazine and reserpine, where micronuclei were observed in rats with chlorpromazine at a frequency lower than that seen in mice, but reserpine did not induce micronuclei in rats (Asanami & Shimono, 2000). Size analysis of the micronuclei was interpreted by the authors as being indicative of aneugenicity (i.e. chromosome loss) and evidence that haloperidol acts by disruption of chromosome binding to the mitotic spindle. 

With the exception of the transgenic gene mutation assay, no treatment related effects on body temperature were recorded for any of the rodent genotoxicity studies on CF₃I. However, recording of body temperature is not a common procedure in these studies and recording of potential observations indicative of body temperature effects, such as cold extremities or animals feeling cool to touch, would not be possible during standard inhalation treatments where the animals cannot be handled during exposure. In the transgenic rodent gene mutation study reported here reduced body temperature associated with CF₃I exposure was documented at high (≥10000 ppm) exposures, with the most marked change observed at 40000 ppm. The exposure-related decreases in body temperature observed in the transgenic rats follows Haber’s rule (proportional to dose*time) throughout the 6 hour exposure period. This same concentration has been reported to induce micronuclei in mice and mice after acute and/or repeated exposure. This implies that doses in excess of 20000 ppm over 6 hours would result in a significant hypothermia in rats. Furthermore, a more profound effect might be anticipated in mice due to the much greater surface area:bodyweight ratio of mice. This would correlate with the micronucleus findings reported for CF₃I as well as the negative chromosome aberration results and it therefore seems probable that micronuclei associated with CF₃I also occur under conditions of hypothermia. The results of the in vitro micronucleus test in human peripheral blood lymphocytes exposed to CF₃I that are reported here provide further evidence that CF₃I-induced micronuclei in rodents are a secondary consequence of dose-related physiological changes rather than the direct interaction of CF₃I with DNA. 

In summary, although CF₃I is mutagenic in bacteria it is not mutagenic to mammalian cells exposed in vitro or in vivo. At high exposures (>20000 ppm) CF₃I induces micronuclei in immature erythrocytes of exposed rats and mice. However, there is convincing evidence that this is a consequence of chemically-induced hypothermia rather than any direct DNA-damaging mechanism. Additional work is planned to investigate this hypothesis further. On the basis of the available higher-tier in vivo toxicity studies the hazard classification of CF₃I as a Cat. 2 Mutagen would not be warranted any longer in accordance with the applicable hazard classification rules under the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (UN GHS) and the European Union Regulation No 1272/2008 on Classification, Labelling and Packaging of substances and mixtures (EU CLP).

Additional information

In vitro studies: bacterial systems (Key study)

The gaseous test substance was tested to evaluate its mutagenic potential according to OECD TG 471 and in compliance with GLP by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

In the initial toxicity-mutation assay, the dose levels tested were 1.4, 2.8, 6.9, 14, 21, 28 and 37 mmol/L. No precipitate was observed. Toxicity, as a reduction in revertant count, was observed at 28 and/or at 37 mmol/L with tester strains TA100 and TA1535 in the presence and absence of S9 activation. Positive mutagenic responses (2.6- through 20.3- fold, maximum increases) were observed with tester strains TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation and TA1535 in the absence of S9 activation. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 37 mmol/L. In the confirmatory mutagenicity assay, the dose levels tested were 1.4, 2.8, 6.9, 14, 21, 28 and 37 mmol/L. No precipitate was observed. Toxicity, as a reduction in revertant count, was observed at 28 and/or at 37mmol/L with tester strains TA100 and TA1535 in the presence and absence of S9 activation. Positive mutagenic responses (3.3- through 21.1- fold, maximum increases) were observed with tester strains TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation.

These results indicate that the test substance was positive for the ability to induce reverse mutations at selected loci of some strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

 

In vitro studies: bacterial systems (Supporting study 2001)

The potential to induce mutagenicity in bacteria was assessed in another GLP study performed according to Guidelines for Screening Mutagenicity Testing Of Chemicals genetic toxicity in vitro, Japan (Japan Bioassay Research, 2001). The bacterial strains exposed to the test substance were S. typhimurium TA 1535, TA 1537, TA 98 and TA 100. The test substance was diluted in air. A Tedlar bag technique was used in this assay. All dose groups (with and without S9 mixture) was set up in duplicates. 0.1 mL of culture was added to top agar which was held with 0.5 mL of S9 mixture or incubation buffer. The contents were mixed and poured onto the surface of a minimum glucose agar. After the top agar solidified, the plates with the lid on the bottom were placed in a Tedlar bag. The bag was then sealed and evacuated. The contents of the Tedlar bag containing desired concentration were transferred into the evacuated bag. The bag with the plates and chemical was then incubated at 37 °C for 24 hours. After 24 hours of exposure, plates were removed from the bag and incubated for additional 24 hours under yellow light. Regular plate incorporation method was used for positive control. The number of revertants per dish was counted.It was found thatthe number of revertant colonies per plate exposed to E. coli WP2 uvrA with and without metabolic activation, Salmonella typhimurium TA98 and TA1537 without metabolic activation induced more than double compared to the negative control (just air exposure). Therefore, under this test conditions, the substance was judged as positive in this mutagenesis assay.

 

In vitro studies: bacterial systems (Supporting study 1995)

The potential to induce mutagenicity in bacteria was assessed in a study performed in compliance with GLP criteria and similar to OECD 471 (Genesys Research, 1995). The test substance was tested in a preliminary test, which was repeated because of excessive toxicity. In the second preliminary test (in TA 100) over 5 concentrations ranging from 1,253 to 94,873 ppm, and toxicity as indicated by a slight reduction in the density of the background lawn was observed only at the highest concentration tested, without and with activation. In this assay, a clear concentration-related increase in TA100 revertant colonies was observed over all concentrations tested, with the increase essentially as high as that of the positive controls at 94,873 ppm CF3I, in both the absence and presence of activation. In the main mutagenicity assay, the bacterial strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1538 were exposed to test substance concentrations between 1 to 85.9 ppm, in the presence and absence of a metabolic activation system (liver S9 mix from Aroclor 1254 induced rats). Test substance was found to be highly mutagenic, inducing frameshift and, particularly, base-pair mutations in Salmonella typhimurium, without and with activation. Therefore, test substance was positive in the Salmonella typhimurium histidine (his) reversion mutagenesis assay.

 

In vitro studies: mammalian cell gene mutation test

In a GLP compliant study similar to OECD 476 the potential of test substance to produce a increase in mutant frequency in the mouse lymphoma cell assay (L5178Y, TK+/-) was investigated (Genesys Research, 1995). The test substance was initially tested in two preliminary concentration range-finding assays with concentrations ranging from 55000 to 900000 ppm. The test substance appeared to be soluble in cell culture media and concentration-related increases in toxicity were obtained in each assay. In the mutagenesis assay the concentrations of test substance ranged from 125000 to 1000000 ppm conducted in the absence and presence of metabolic activation. For the mutation test the following analytical concentrations were assessed: 79610, 176884, 305887, 425606, 454184, 517777 and 496814 ppm. In this assay, the negative control cloning efficiency and spontaneous mutation frequency met the criteria for acceptability, and positive control mutant frequencies were within the historical ranges for the laboratory, and when tested to the maximum concentration that could be obtained, test substance was negative in the presence of toxicity. Therefore, test substance did not induce gene or chromosomal mutations in mammalian cells in vitro.

 

In vitro studies: chromosome aberration study

An in vitro chromosome aberration assay in Chinese Hamster Lung (CHL)/IU was performed (Japan Bioassay research centre, 2001). The cells were exposed to 20 %, 40 %, 60 % and 80 % of test gas concentration with and without metabolic activation (S-9 mix). Two tests were performed; a short term treatment test and a continuous treatment test (24- and 48-hours). The test concentrations for the main tests were based on preliminary chromosomal aberration tests. As positive controls benzo(a)pyrene, mitomycin C, Methyl chloride and vinyl chloride were used.

In case of short term treatment without S9, 3.9 % and 10.7 % frequencies of polyploid cells were observed at 60 and 80 % concentration of test material. The frequencies of structural aberration cells were less than 5 %. In case of treatment with S9, both frequencies of polyploid cells and structural aberration cells were less than 5 %. D20 (without S9) of induced polyploid cells was beyond 100 % (180 %). In case of treatment without S9, cell proliferation rates were 96, 84, 69 and 53 % at test material concentration 20, 40, 60 and 80% respectively. In case of treatment with S9, cell proliferation rates were 102, 103, 94 and 75 % at test material concentration 20, 40, 60 and 80 % respectively.

In case of 24-hour treatment, 6.5 % frequencies of polyploid cell were observed at 60 % test material concentration. Frequencies of structural chromosomal aberration were less than 5 %. In case of 48-hour treatment, 7.4 and 11.5 % frequencies of polyploid cells were observed at 40 % and 50 % concentration respectively. The frequencies of structural chromosomal aberration were less than 5 %. D20 to induce polyploid cells for 48-hour treatment was determined as 99 %. Cell proliferation rates were 98, 68, 64, and 42 % at the concentration of 20, 40, 60 and 80 % respectively for 24-hour treatment. As for 48-hour treatment, the rates were 83, 71, 41 and 30 % at the concentration of 20, 30, 40 and 50 %.

It was concluded that short term treatment without S9 and 48-hour continuous treatment test gave more than 10% of frequencies of polyploid cells. The criteria of positive result are more than 10%. Under this test conditions, chromosomal aberration induced property to CHL/IU cells of this test substance was judged as positive.

 

In vitro studies: micronucleus assay

The test substance was tested according to OECD TG 487 and in compliance with GLP to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. HPBL cells were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Acetone was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 µg/mL, which was the limit dose for this assay. Cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at 2000 µg/mL in the non-activated and S9-activated 4-hour exposure groups and at doses ≥ 600 µg/mL in the non-activated 24-hour exposure group. Based upon these results, the doses chosen for the micronucleus assay ranged from 25 to 2000 µg/mL for the non-activated and S9-activated 4-hour exposure groups; and from 5.0 to 800 µg/mL for the non-activated 24-hour exposure group. In the micronucleus assay, cytotoxicity (≥ 50% CBPI relative to the vehicle control) was observed at doses ≥ 1600 µg/mL in the S9-activated 4-hour exposure group and at doses ≥ 500 µg/mL in the non-activated 24-hour exposure group. In the non-activated 4-hour exposure group, there was a shift in cytotoxicity profile (compared to the preliminary assay, which showed >50% CBPI at the same concentration), with 29% cytotoxicity observed at 2000 µg/mL. The doses selected for evaluations of micronuclei were 250, 1000, and 2000 µg/mL for the non-activated 4-hour exposure group; 250, 1000, and 1600 µg/mL for the S9-activated 4-hour exposure group; and 25, 250, and 500 µg/mL for the non-activated 24-hour exposure group. No significant or dose-dependent increases in micronuclei induction were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

These results indicate, under the study design, the test substance was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

 

In vivo studies: Mutation assay

A Transgenic Rodent (TGR) mutation assay (BioReliance 2017), was carried out according to OECD Test Guideline TG 488 and GLP regulations. The test substance was administered via nose-only inhalation exposure for 6 hours per day to male F344 rats. Initially, the study was conducted at exposure concentrations of 0, 10,000, 20,000 and 40,000 ppm (Groups 1-4, respectively; 6 males/group). Due to overt signs of toxicity and a severe decrease in body temperatures after Day 0 exposure (prior to exposure on Day 1), Groups 3 and 4 were euthanized in extremis. Groups 1 and 2 did not receive any further exposures. This was deemed Phase 1 of the study. The study was restarted with new group assignments, at exposure concentrations of 2500, 5000 and 10,000 ppm (Groups 2-4, respectively). Due to overt signs of toxicity and a severe decrease in body temperatures during the first week of exposure to 10,000 ppm, Group 4 was euthanized in extremis on Day 5.

During the final Phase, test substance was administered for 28 consecutive days at target exposure concentrations of 1250, 2500 and 5000 ppm (Groups 2, 3 and 4, respectively). The control group (Group 1) was exposed to humidified, filtered air on a comparable regimen. All animals survived the in-life exposure phase and all animals were euthanized on Test Site Day 30 (Test Facility Day 31), 3 days following the final exposure with the exception of two animals in Group 4. These two animals died shortly after blood collection on Test Site Day 28, the day after the last exposure. In order to minimize the in situ degradation of the DNA, all surviving animals were euthanized by CO2 inhalation and necropsied; the liver, lungs and testes were removed and weighed (for prediction of the possible number of DNA extractions from a tissue). Bone marrow was collected from one femur, pelleted by centrifugation and the supernatant decanted. All tissues were immediately flash frozen in liquid nitrogen, and stored at approximately -70 °C.

Exposure of male Fisher 344 Big Blue rats to test substance using the above described regimen for Phase 3 resulted in no test substance-related mortality. Based on Phase 1 and 2 findings, the 5000 ppm level was determined to be the highest dose that would be tolerated over a 28-day exposure period, using 7 days per week dosing for 6 hours per day. However, test substance-related clinical findings (most notably, cool extremities and thin body condition), lower body weights, food consumption, body temperatures, and organ weights were noted in the 2500 and 5000 ppm groups. There were no test substance-related effects noted for the 1250 ppm group. In conclusion, treatment with test substance did not cause statistically elevated mutant frequencies at the cII gene in bone marrow, liver and lungs of Big Blue male rats under the conditions of testing. Therefore, test substance is considered not mutagenic in this TGR mutation assay, under the conditions of testing.

 

In vivo studies: Micronucleus assay as part of a sub-chronic repeated dose toxicity study

As part of a sub-chronic (90 day) repeated dose toxicity study incl. a 4 week interim sacrifice a erythrocyte micronucleus assay was performed (Dodd 1997). In total fifteen male and 15 female rats were exposed 2 hr/day, 5 days/week, for up to 13 weeks (65 exposures over 90 days) to 0 (air control), 2, 4, or 8% test substance. Prior to initiation of the exposures, rats were acclimated to the nose-only chamber restraint system, breathing air only, 2 hr/day for 1 week. Bone marrow cells were collected from the femur and smears were prepared from five rats/sex/group at both the 4- and 13-week animal terminations to investigate the mutagenic potential of the substance via induction of micronuclei in bone marrow polychromatic erythrocytes. Increases in the frequency of micronucleated bone marrow polychromatic erythrocytes were observed in rats of all three groups. Therefore the substance was considered positive under the experimental conditions.

 

In vivo studies: Micronucleus assay as part of a Reproductive toxicity screening test

An erythrocyte micronucleus assay was performed as part of a combined repeated dose and reproductive toxicity screening study was performed in Sprague-Dawley Crl:CD(SD)BR rats (detailed description in Section 7.8.1) (Dodd 1999). In total the study had 16 male and 16 female rats/group to yield at least 12 pregnant females at term. The animals were assigned to one of four groups (0, 0.2, 0.7, and 2.0%) and exposed to substance vapor for 4 wk (6 h/day, 5 days/wk) prior to mating. An additional six male and three female rats were assigned to a fifth group only to serve as positive controls in the micronuclei assay (Single dose of cyclophosphamide (7.5 mg/kg) intraperitoneally 24 hrs prior to termination.). Animals were exposed 6 h/day, 7 days/wk, during the mating, gestation, and lactation phases of the study. However, dams were not exposed from gestation day 21 through lactation day 4 to allow for parturition and early lactation. The remaining 8 male rats/group and 16 female rats/group were terminated over a 5-day period subsequent to when the last female rat on study reached lactation day 21. Animals were killed by CO2 inhalation overdose followed by exsanguination. Bone marrow cells were collected from the femur and smears were prepared from all rats. Positive control rats were administered a single dose of cyclophosphamide (7.5 mg/kg) intraperitoneally 24 hrs prior to termination. Slides were stained by the Giemsa/May-Greenwald method and observed microscopically at 100x. The frequency of micronucleated cells was evaluated by random observation of 1000 polychromatic erythrocytes (PCE) per sample. The ratio between PCE and normochromatic erythrocytes (NCE) was determined by scoring approximately 1000 erythrocytes as an indicator of toxicity of the test agent. Micronuclei scores for male and female rats exposed to CF3I for 7 or 14 wk were similar to micronuclei scores in the control animals. In the current investigation, CF3I was negative in the in vivo micronucleus test since there was no increase in micronuclei frequency in the bone marrow erythrocytes of rats exposed to the test substance.

 

In vivo studies: Micronucleus assay in mice

The potential of test substance to induce micronuclei in erythropoietic cells of the bone marrow (Genesys Research, 1995) was studied. Based on preliminary toxicity information, a mouse bone marrow micronucleus test of the test substance was conducted using 2.5 %, 5.0 %, and 7.5 % test substance, administered to male and female Swiss Webster mice by inhalation for six hours on each of three consecutive days. Bone marrow cells were obtained from the mice sacrificed 24 hours after the third exposure. Erythrocytes from mice exposed to the test material, and to the negative and positive controls, were evaluated for toxicity and the presence of micronuclei. The positive control, 0.4 mg triethylenemelamine (TEM)/kg (administered intraperitoneally) significantly (p<0.01) elevated the number of micronuclei in newly-formed erythrocytes (PCEs, polychromatic erythrocytes) from male and female mice.Toxicity of the test substance was evidenced by dose-related depressions in weight for both sexes and by dose-related depressions in ratios of PCEs/1000 erythrocytes for female mice. Significant (p<0.05) dose-related increases in micronuclei/1000 PCEs were observed in both male and female mice of the 5.0 and 7.5 % CF3I exposure groups. Therefore, test substance was evaluated as positive in the mouse bone marrow micronucleus test.

 

In vivo studies: Micronucleus assay in wild type and rasH2 Transgenic mice

The objective of this GLP compliant OECD 474 study (Charles River, 2018) was to evaluate the potential toxicity of the test substance when administered by whole-body inhalation for 6 hours per day for 5 consecutive days to CByB6F1-Tg(HRAS)2Jic wild type and CByB6F1/TgrasH2 Hemizygous transgenic mice. Five animals per sex per strain were exposed to concentrations of 2500, 5000, 10,000 and 20,000 ppm. The results of this study will be used to support the selection of exposure concentrations for a 4-week range-finding study in wild type mice and a 26-week inhalation carcinogenicity study in Tg.rasH2 mice. In addition, blood and bone marrow samples were collected from the wild type mice for evaluation of micronuclei in bone marrow and other possible future micronucleus evaluations. Animals were dosed for 6 hours per day for 5 consecutive days via whole-body inhalation. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, body temperature, and micronucleus parameters (wild type mice only). All animals survived to the scheduled necropsy. There were no test substance-related clinical observations or effects on body weight or body temperature. The test substance did not show a substantial decreases in the polychromatic erythrocytes and normochromatic erythrocyte ratio. Therefore, the test substance did not cause bone marrow toxicity. Based on the results of this study, test substance exposure by whole-body inhalation to CByB6F1-Tg(HRAS)2Jic wild type and CByB6RF1/TgrasH2 Hemizygous transgenic mice at exposure concentration levels of 2500, 5000, 10,000, and 20,000 ppm for 5 consecutive days resulted in no toxicologically significant effects on clinical observations, body temperature, or body weight and in a negative response for induction of bone marrow micronuclei at exposure concentration levels up to 20,000 ppm.

 

In vivo studies: Sub-acute inhalation toxicity study including a chromosomal aberration assay in male animals (OECD 475)

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 (TNO 2007, detailed description in 7.5.2) 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.

 

In vivo studies: Chromosomal aberration (OECD 475)

The test substance, administered by inhalation, was examined for its potential to induce structural chromosomal aberrations in bone marrow cells of male Wistar Crl:[WI] WU BR rats according to OECD 475 (TNO 2006). 5 male animals/ dose per killing time were treated for a period of 4 hours with three exposure levels, 200000, 80000 and 20000 ppm of the test substance. In addiditon 4 spare animals were introduced in the high dose group to compensate for mortality and a positive control group ( Mitomycin C) was included to show the validity of the assay. The animals were exposed and sacrificed on four consecutive days. The exposure of the rats to the highest exposure level (200000 ppm) was stopped after 35 minutes, because 8 rats of 14 rats died as a result of this exposure level and the surviving animals showed reversible clinical signs. At both sacrifice times, 24h and 48h (latter timepoint control and high dose only) after exposure, no dose level of the test substance induced a statistically significant increase in the number of cells with structural chromosomal aberrations, when compared to the concurrent negative (clean air) control values. Exposure to test substance did not result in clastogenicity to bone marrow cells of rats.

 

In vivo studies: Unscheduled DNA Synthesis (UDS) (Key study)

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 (TNO 2007, detailed description in 7.5.2) 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.

 

Overall conclusion

In summary, although CF3I is mutagenic in bacteria it is not mutagenic to mammalian cells exposed in vitro or in vivo. At high exposures (>20000 ppm) CF3I induces micronuclei in immature erythrocytes of exposed rats and mice. However, there is convincing evidence that this is a consequence of chemically-induced hypothermia rather than any direct DNA-damaging mechanism. Additional work is planned to investigate this hypothesis further. On the basis of the available higher-tier in vivo toxicity studies the hazard classification of CF3I as a Cat. 2 Mutagen would not be warranted any longer in accordance with the applicable hazard classification rules under the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (UN GHS) and the European Union Regulation No 1272/2008 on Classification, Labelling and Packaging of substances and mixtures (EU CLP).

Justification for classification or non-classification

On the basis of the available higher-tier in vivo toxicity studies the hazard classification of CF₃I as a Cat. 2 Mutagen would not be warranted any longer in accordance with the applicable hazard classification rules under the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (UN GHS) and the European Union Regulation No 1272/2008 on Classification, Labelling and Packaging of substances and mixtures (EU CLP).