Registration Dossier
Registration Dossier
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EC number: 200-939-8 | CAS number: 76-16-4
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Assays are available for perfluoroethane (F-116) with respect to In Vitro (Mutagenic effects - bacterial): OECD 471; Bacterial reverse mutation assay. Negative. Reliability = 1. In Vitro (Clastogenic effects - mammalian): OECD 473; Chromosome aberrations in human lymphocytes. Negative. Reliability = 1 . In vitro mutagenicity assays in mammalian cells are available for close structural analogues 1,1,1-trifluoroethane (HFC 143a) and 1,1,1,2,3,3,3-heptafluoropropane (HFC-227ea), Negative, Reliability=1
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- See attached justification below
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- It was concluded that the substance did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP compliant, near guideline study, available as unpublished report, fully adequate for assessment
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 homogenate
- Test concentrations with justification for top dose:
- Nominal concentrations:
Preliminary assay: 100000; 200000; 300000; 400000; 500000; 600000; 700000; 800000; 900000 ppm
Mutagenesis assay: 100000; 300000; 500000; 700000; 900000 ppm
IR determined concentrations:
Preliminary assay: 63,967, 147,112; 204,380, 269,853, 344,792; 378,811; 423,733; 458,673; 538,535 ppm
Mutagenesis assay: 84,765; 274,259; 408,759; 518,570; 568,484 ppm - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: hycanthone and cyclophosphamide
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- It was concluded that the substance did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 25 June 2018 - 24 July 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- See attached justification below
- Reason / purpose for cross-reference:
- read-across source
- Vehicle / solvent:
- Sterile air
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- The osmolality and pH of the substance in medium were measured by analysing samples of R10p media, dosed with either the vehicle (sterile air) or a substance formulation at 70% v/v. For medium dosed with substance at 70% v/v; no fluctuations in osmolality of the medium of more than 50 mOsmol/kg and no fluctuations in pH of more than 1.0 unit were observed compared with the vehicle control. The maximum final concentration tested in the preliminary toxicity test was 70% v/v as this is the maximum achievable concentration in the test system without detrimental effects on the cell culture.
Preliminary Toxicity Test:
No precipitate (observed by eye at the end of treatment) was observed at concentrations of 70% v/v in the absence and presence of S9 mix, respectively, following a 3-hour exposure. Exposure to the substance at concentrations from 1 to 70% v/v in the absence and presence of S9 mix resulted in no reduction in the relative suspension growth (RSG) values. Concentrations used in the main test were based upon these data.
Main Mutation Test - 3-hour Treatment in the Absence of S9 Mix:
Cultures were exposed to the substance at concentrations from 25.2 to 45.7% v/v. No precipitate was observed by eye at the end of treatment. The results of the achieved concentration analysis showed that the highest concentration was -34.7% from the nominal concentration of 70% v/v, this was considered to be unacceptable. The test was therefore terminated and an additional test was performed.
Additional main Mutation Test - 3-hour Treatment in the Absence of S9 Mix:
Cultures were exposed to the substance at concentrations from 22.3 to 70% v/v. No precipitate was observed by eye at the end of treatment. Cultures exposed to the substance at concentrations from 22.3 to 70% v/v were assessed for determination of mutation frequency. Mean relative total growth (RTG) values from 84 to 65% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity.
The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.
Main Mutation Test - 3-hour Treatment in the Presence of S9 Mix:
Cultures were exposed to the substance at concentrations from 30 to 70% v/v. No precipitate was observed by eye at the end of treatment. Cultures exposed to the substance at concentrations from 30 to 70% v/v were assessed for determination of mutation frequency. Mean RTG values from 107 to 92% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.
The positive control, benzo[a]pyrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.
Formulation Analysis:
The results of formulation analysis showed that the achieved concentrations of HFC-143a were within the target range 100% +/- 20%, with the exception of the highest concentration in the main test –S9 mix, and the lowest concentration in the additional main test –S9 mix, which were -34.7% and -25.7%, respectively. - Remarks on result:
- other: No mutagenic potential.
- Remarks:
- Negative study.
- Conclusions:
- The substance did not demonstrate mutagenic potential in this in vitro cell mutation assay in the presence or absence of a metabolic activation system, under the experimental conditions described.
- Executive summary:
The mutagenic potential of the substance was evaluated by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix) according to the most recent OECD guideline in compliance with GLP. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).
The study consisted of a preliminary toxicity test and three independent mutagenicity assays. The cells were exposed for either 3 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix. The substance was tested up to a maximum final concentration of 70% v/v in the preliminary toxicity test, in order to test up to the maximum achievable concentration in the test system without detrimental effects on the cell culture.
Following a 3-hour exposure to the substance at concentrations from 1 to 70% v/v, no reduction in the relative suspension growth (RSG) was observed in the absence or presence of S9 mix. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to test up to the maximum achievable concentration in the test system without detrimental effects on the cell culture.
Following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentration assessed for mutant frequency in the 3-hour treatment in the absence and presence of S9 mix was 70% v/v. In the both the absence and presence of S9 mix there was no significant reduction in RTG.
In all tests the concurrent vehicle and positive control were within acceptable ranges.
It was concluded that the substance did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 25 June 2018 - 24 July 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- 29 July 2016
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver was stored at -90 to -70°C.
- Test concentrations with justification for top dose:
- The highest concentration selected (70% v/v) was the maximum achievable concentration in the test system without detrimental effects on the cell culture.
- Vehicle / solvent:
- Sterile air
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- methylmethanesulfonate
- Details on test system and experimental conditions:
- L5178Y mouse lymphoma (3.7.2c) cells heterozygous at the thymidine kinase locus, TK +/- were used. Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24-hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196 to -150°C, in heat inactivated donor horse serum (HiDHS) containing 10% DMSO. Cultures were used within ten days of recovery from frozen stock. Cell stocks are periodically checked for freedom from mycoplasma contamination.
The following media were used:
R0 RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 ug/mL gentamicin.
R10p R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v.
R10p medium was used for cell culture unless otherwise specified.
Selective medium consisted of R10p containing 4 ug/mL trifluorothymidine (TFT).
Positive Controls
In the absence of S9 mix, Methyl methanesulphonate (MMS) in DMSO at concentration of 10 ug/mL (3-hour exposure).
In the presence of S9 mix, Benzo[a]pyrene (BaP) in DMSO at concentration of 1.5 ug/mL (3-hour exposure).
S9 Metabolizing System
S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver and stored at -90 to -70°C. S9 fraction (5% v/v), glucose-6-phosphate (6.9 mM), NADP (1.4 mM) in R0 was used. The co-factors were prepared, neutralised with 1N NaOH and filter sterilised before use.
The highest concentration selected (70% v/v) was the maximum achievable concentration in the test system without detrimental effects on the cell culture. Concentrations within the range of 5 to 30% was avoided due to the known lower and upper flammability levels of the test item, 7 and 19% respectively.
All concentrations cited in this report are expressed in terms of pure HFC-143a as received.
The final nominal concentrations to which cells were exposed initially are given below:
Preliminary toxicity test:: 1, 5, 30, 40, 50, 60 and 70% v/v
Mutation tests:
-S9 mix (3 hours) 30, 40, 50, 60 and 70% v/v
+S9 mix (3 hours) 30, 40, 50, 60 and 70% v/v (also assessed for determination of the mutant phenotype)
Additional Mutationtests:
-S9 mix (3 hours) 22.3*, 40, 50, 60 and 70% v/v (also assessed for determination of the mutant phenotype)
* Actual concentration
Osmolality and pH
The effects of HFC-143a on the osmolality and pH of the culture medium were measured by analyzing samples of R10p media treated with either the vehicle (sterile air) or test item at 70% v/v. Precipitate was assessed by eye at the end of the exposure period in treated R10p media-only cultures as part of the preliminary toxicity test.
Preliminary Toxicity Test Procedure: Cells were exposed to the substance for 3 hours in the absence and presence of S9 mix. For 3-hour exposures, cultures contained a total of 1.2 x 10^7 cells. The final volume of the cultures was 10 mL and the final concentration of the S9 fraction was 2% v/v. One culture was prepared for each concentration of the substance for each test condition. Vehicle controls were tested in duplicate for each test condition.
Mutation Test Procedure: 3-hour Treatment in the Absence and Presence of S9 Mix. The procedure for preparing the cell suspension was the same as for the preliminary toxicity test. Cultures contained a total of 1.2 x 10^7 cells in a final volume of 10 mL. The final concentration of the S9 fraction was 2% v/v. Duplicate cultures were prepared throughout for each concentration of substance and positive control. Quadruplicate cultures were prepared for vehicle controls. Aliquots of 100 µL of positive control were added to the relevant cultures, and then all cultures were incubated, for 3 hours at 34 to 39°C. Five dilutions of the substance were tested. - Evaluation criteria:
- Acceptance criteria for substance:
The highest concentration tested was one that allowed the maximum exposure up to 70% v/v for freely soluble compounds, or the limit of toxicity (i.e. relative total growth reduced to approximately 10 to 20% of the concurrent vehicle control) or the limit of solubility. For a toxic substance, at least 4 analysable concentrations should have been achieved which ideally spanned the toxicity range of 100 to 10% RTG.
Acceptance criteria for vehicle controls:
The mean vehicle control value for mutant frequency was between 50 to 170 x 10^-6.
The mean cloning efficiency was between 65 to 120%.
The mean suspension growth was between 8 to 32 on Day 2 following 3-hour treatments.
Obvious outliers were excluded. However, there were at least 2 vehicle control cultures remaining.
Acceptance criteria for positive controls:
Positive controls showed an absolute increase in mean total MF above the mean concurrent vehicle control MF of at least 300 x 10^-6. At least 40% of this was due to the number of small mutant colonies.
Mean RTG’s for the positive controls were greater than 10%.
There was an absence of confounding technical problems such as contamination, excessive numbers of outliers and excessive toxicity.
Criteria for Assessing Mutagenic Potential
The following criteria were applied for assessment of individual assay results using data for MF where the RTG normally exceeded 10%:
Definitions: GEF = Global Evaluation Factor. For microwell assays this is 126 x 10-6.
Providing that all acceptability criteria were fulfilled, the test item was considered to be clearly positive if, in any of the experimental conditions examined the increase in MF above the concurrent background exceeded the GEF and the increase was concentration related (i.e., there is a significant positive linear trend). The test item is then considered able to induce mutation in this test system. - Statistics:
- The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users). Statistics were only reported if the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was exceeded, and this was accompanied by a significant positive linear trend (p<0.05).
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- The osmolality and pH of the substance in medium were measured by analysing samples of R10p media, dosed with either the vehicle (sterile air) or a substance formulation at 70% v/v. For medium dosed with substance at 70% v/v; no fluctuations in osmolality of the medium of more than 50 mOsmol/kg and no fluctuations in pH of more than 1.0 unit were observed compared with the vehicle control. The maximum final concentration tested in the preliminary toxicity test was 70% v/v as this is the maximum achievable concentration in the test system without detrimental effects on the cell culture.
Preliminary Toxicity Test:
No precipitate (observed by eye at the end of treatment) was observed at concentrations of 70% v/v in the absence and presence of S9 mix, respectively, following a 3-hour exposure. Exposure to the substance at concentrations from 1 to 70% v/v in the absence and presence of S9 mix resulted in no reduction in the relative suspension growth (RSG) values. Concentrations used in the main test were based upon these data.
Main Mutation Test - 3-hour Treatment in the Absence of S9 Mix:
Cultures were exposed to the substance at concentrations from 25.2 to 45.7% v/v. No precipitate was observed by eye at the end of treatment. The results of the achieved concentration analysis showed that the highest concentration was -34.7% from the nominal concentration of 70% v/v, this was considered to be unacceptable. The test was therefore terminated and an additional test was performed.
Additional main Mutation Test - 3-hour Treatment in the Absence of S9 Mix:
Cultures were exposed to the substance at concentrations from 22.3 to 70% v/v. No precipitate was observed by eye at the end of treatment. Cultures exposed to the substance at concentrations from 22.3 to 70% v/v were assessed for determination of mutation frequency. Mean relative total growth (RTG) values from 84 to 65% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity.
The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.
Main Mutation Test - 3-hour Treatment in the Presence of S9 Mix:
Cultures were exposed to the substance at concentrations from 30 to 70% v/v. No precipitate was observed by eye at the end of treatment. Cultures exposed to the substance at concentrations from 30 to 70% v/v were assessed for determination of mutation frequency. Mean RTG values from 107 to 92% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.
The positive control, benzo[a]pyrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.
Formulation Analysis:
The results of formulation analysis showed that the achieved concentrations of HFC-143a were within the target range 100% +/- 20%, with the exception of the highest concentration in the main test –S9 mix, and the lowest concentration in the additional main test –S9 mix, which were -34.7% and -25.7%, respectively. - Remarks on result:
- other: No mutagenic potential.
- Remarks:
- Negative study.
- Conclusions:
- The substance did not demonstrate mutagenic potential in this in vitro cell mutation assay in the presence or absence of a metabolic activation system, under the experimental conditions described.
- Executive summary:
The mutagenic potential of the substance was evaluated by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix) according to the most recent OECD guideline in compliance with GLP. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).
The study consisted of a preliminary toxicity test and three independent mutagenicity assays. The cells were exposed for either 3 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix. The substance was tested up to a maximum final concentration of 70% v/v in the preliminary toxicity test, in order to test up to the maximum achievable concentration in the test system without detrimental effects on the cell culture.
Following a 3-hour exposure to the substance at concentrations from 1 to 70% v/v, no reduction in the relative suspension growth (RSG) was observed in the absence or presence of S9 mix. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to test up to the maximum achievable concentration in the test system without detrimental effects on the cell culture.
Following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentration assessed for mutant frequency in the 3-hour treatment in the absence and presence of S9 mix was 70% v/v. In the both the absence and presence of S9 mix there was no significant reduction in RTG.
In all tests the concurrent vehicle and positive control were within acceptable ranges.
It was concluded that the substance did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Remarks:
- The study was conducted according to the guideline in effect at the time of study conduct.
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- Remarks:
- The study was conducted according to the guideline dated 1997.
- Qualifier:
- according to guideline
- Guideline:
- other: EC Directive 2000/32/EC Annex 4A-B10 Number L 136
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Whole venous blood was drawn from healthy donor(s) less than 50 years old without previous chemotherapy or radiotherapy; and without recent (within the last 6 months) viral disease or X-ray exposure
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data - Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9
- Test concentrations with justification for top dose:
- Preliminary toxicity assay: 7 concentrations ranging from 1.25 to 75% v/v
Chromosome aberration concentrations: 0, 25%, 50%, 75% 100% - Vehicle / solvent:
- - Vehicle: None
- Untreated negative controls:
- yes
- Remarks:
- Ambient Air
- Negative solvent / vehicle controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: mitomycin-C (MMC) (CAS 50-07-7) -S9; cyclophosphamide (CP) (CAS 6055-19-2) +S9
- Remarks:
- Positive controls were dissolved in sterile water and were assumed to be stable.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: The HPBL cultures were initiated in labelled sterile, 15 mL centrifuge tubes by inoculating 0.5 mL of heparinized blood to 9.5 mL of complete medium (RPMI 1640 medium containing approximately 15% foetal bovine serum (FBS), 2 mM L-glutamine, 100 units penicillin/mL, and 100 μg streptomycin/mL) supplemented with PHA-M. Cultures were incubated at 37±2˚C in a humidified atmosphere of 5±2% CO2 in air.
Approximately 48 hours after culture initiation (by addition of PHA-M), the whole blood cultures were centrifuged and the culture medium was discarded and replaced with 4.5 mL of treatment medium (with or without 10% S9 mixture). Sets of duplicate cultures were then exposed to a preformulated test substance atmosphere (4 concentrations were applied), the negative control, and 2 positive control substance concentrations for each test condition. The treatment medium was RPMI 1640 medium for all testing conditions. The cells were treated for approximately 4 and 22 hours in the non-activated test condition, and for approximately 4 hours in the S9-activated test condition. After completion of the 4-hour exposure periods only, the glass tubes were vented in a fume hood and the HPBL suspension was transferred to 15-mL centrifuge tubes. The cells were collected by centrifugation, and the treatment medium replaced with complete RPMI 1640 culture medium and incubated until cell harvest. The incubations were conducted at 37±2˚C in a humidified atmosphere of 5±2% CO2 in air. The cells were arrested in metaphase at ~19 hours after treatment initiation by adding Colcemid® to the cultures at a 0.1 μg/mL final concentration in the culture media. For the 22-hour exposure period, Colcemid® was added with a syringe through the rubber septum. ~22 hours after treatment initiation, the cells were collected by centrifugation and the medium removed. The cells were treated with 0.075M KCl hypotonic buffer, fixed in methanol:glacial acetic acid (3:1 v/v), and stored frozen. To prepare slides, the cells were collected by centrifugation and resuspended in fixative. Slides were prepared by applying an aliquot of the fixed cells onto clean microscope slides and air-drying them. The slides were stained by Giemsa and permanently mounted.
Cytogenetic analyses were conducted for 3 test substance concentrations (25%, 50%, and 100%) as well as the negative and positive control in each test condition. The highest test substance concentration level that was analysed was that which caused a depression in mitotic index of greater than 50% when compared with the negative control or, if the test substance lacked toxicity, the highest concentration used in the test. The percentage of cells in metaphase per 1000 cells scored per concentration level (500 from each duplicate culture) was determined prior to coding the slides. After selection of the slides for cytogenetic analyses, the slides were coded and scored. Metaphase cells were selected for scoring based on good chromosome morphology and staining characteristics. Only metaphase cells with 46 centromeres were analysed for structural aberrations.
DURATION
- Preincubation period: 48 hr
- Exposure duration: 4 hour (-S9 & +S9) and 22 hours (-S9)
- Expression time (cells in growth medium): 19 hours after treatment initiation
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcemid®
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 200 metaphases per concentration level (100 from each duplicate culture)
DETERMINATION OF CYTOTOXICITY
- Method: 1) mitotic index greater than 50% when compared with the negative control or, if the test substance lacked toxicity, the highest concentration used in the test, 2) only metaphase cells with 46 centromeres were analysed for structural aberrations, 3) numerical aberrations, 4) chromatid-type aberrations included chromatid and isochromatid breaks and exchange figures, 5) pulverized chromosome(s) and cells, and severely damaged cells.
OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes - Evaluation criteria:
- The following conditions were used as a guide to determine a positive response:
• A statistically significant increase (p < 0.05, Fisher’s exact test) in the percentage of cells with structural aberrations was seen in one or more treatment groups relative to the negative control response.
• The observed increased frequencies were accompanied by a concentration-related increase.
• A statistically significant increase was observed at the highest concentration only.
• Note: Statistically significant values that did not exceed the historical control range for the negative/negative control may be judged as not being biologically significant.
The following condition was used as a guide to determine an equivocal response:
• Results observed in any of the assays resulted in statistically significant elevations in structural chromosome aberrations at more than one test concentration level, except the highest concentration, without demonstrating a concentration-responsive trend.
The test substance was judged negative if the following condition was met:
• There was no statistically significant increase in the percentage of cells with structural aberrations in any treatment group relative to the negative control group. - Statistics:
- Statistical analysis consisted of a Fisher’s exact test (with Bonferroni-Holm Adjustment) to compare the percentage of cells with structural or numerical aberrations (or the percentage of cells with more than one aberration, if required) in the test substance treated groups with the negative control response. A Cochran-Armitage test for concentration responsiveness was conducted only on values within a test condition only if statistically significant values, based on the Fisher’s exact test, were found. At the discretion of the study director, statistical analyses may have been conducted on the percentage of cells with numerical aberrations as well.
- Species / strain:
- lymphocytes:
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No pH change was observed for each test condition both at the beginning and end of the treatment period for both preliminary and chromosome aberration assays.
- Precipitation: No precipitation was observed for each test condition both at the beginning and end of the treatment period for both preliminary and chromosome aberration assays.
Preliminary Toxicity Assay
HPBL cells were exposed to 7 concentrations of the test substance ranging from 1.25 to 75% v/v, as well as a negative control. The cells were exposed for 4 hours in both the absence and presence of an exogenous metabolic activation system or for 22 hours in the absence of S9 activation. No substantial toxicity (greater than 50% reduction in mitotic index relative to the negative control) was observed at any concentration in each test condition.
RANGE-FINDING/SCREENING STUDIES:
No substantial toxicity (greater than 50% reduction in mitotic index relative to the negative control) was observed at any concentration in each test condition.
Since there were no effects in the preliminary toxicity assay, the highest concentration selected for the chromosome aberration assay was the highest achievable concentration, 100%. Cells were also exposed to 2 concentrations of a positive control substance as well as the negative control (ambient air) in each test condition. No substantial toxicity (greater than 50% reduction in mitotic index relative to the negative control) was observed at any concentration in each test condition. Selection of doses for microscopic analysis was therefore based on the highest concentration tested.
The chromosome aberration assay indicates no significant increase in structural and numerical chromosome aberrations in the test substance-treated groups relative to the respective negative control in both presence and absence of the S9 metabolic activation system. The percentages of aberrant cells in the test substance-treated groups were within the historical solvent control range. The results for the positive and negative controls indicate that all criteria for valid assay were met. The cytogenetic analysis findings from the individual treatment cultures in the S9-activated and non-activated 4-hour exposure groups, non-activated 22-hour exposure group are summarized by group in the table below. Based on these criteria, the negative result is justified and does not require a repeat of any portions of the study. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
Under the conditions of this study, the test substance was not found to induce structural or numerical chromosome aberrations in the in vitro mammalian chromosome aberration test in human peripheral blood lymphocytes in either the non-activated or S9-activated test systems. It was concluded that the test substance was negative in this in vitro test.
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability). - Executive summary:
The test substance was evaluated for its ability to induce structural chromosome aberrations in vitro using human peripheral blood lymphocytes (HPBL) in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9). Numerical aberrations were also recorded. To establish a concentration range for the chromosome aberration assay, a preliminary toxicity test was initially conducted. The test substance was provided as a colourless gas and diluted with ambient air in Tedlar® bags to desired concentrations. HPBL were exposed to the pre-formulated test substance atmosphere concentrations in air-tight glass tubes.
In the preliminary toxicity assay the cells were treated for 4 and 22 hours in the non-activated test condition and for 4 hours in the S9-activated test condition. All cells were harvested 22 hours after treatment initiation. The cells were exposed to 7 concentrations of the test substance ranging from 1.25 to 75% v/v, as well as a negative control (ambient air). Neither precipitation nor toxicity was observed in the preliminary toxicity assays in each test condition, therefore the highest concentration selected for the chromosome aberration assay was based on the highest achievable test substance concentration, 100%.
The concentrations chosen for the chromosome aberration assay were 25, 50, 75 and 100%. No precipitation or toxicity was observed in any test condition. Selection of doses for microscopic analysis was based on the highest concentration tested.
Cytogenetic evaluations were conducted at 25, 50, and 100% as well as the negative and positive control in each test condition. The percentage of cells with numerical or structural aberrations in the test substance-treated groups was not significantly increased above that of the negative control at any concentration (p ≥ 0.05, Fisher’s exact test).
All criteria for a valid study were met. Under the conditions of this study, the test substance was not found to induce structural or numerical chromosome aberrations in the in vitro mammalian chromosome aberration test in human peripheral blood lymphocytes in either the non-activated or S9-activated test systems. It was concluded that the test substance was negative in this in vitro test.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Remarks:
- Conducted according to guideline in effect at time of study conduct
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- no
- Remarks:
- Conducted according to guideline dated 1997
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- Remarks:
- Conducted according to guideline in effect at time of study conduct
- Qualifier:
- according to guideline
- Guideline:
- EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)
- Deviations:
- no
- Remarks:
- Conducted according to guideline in effect at time of study conduct
- Qualifier:
- according to guideline
- Guideline:
- EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
- Deviations:
- no
- Remarks:
- Conducted according to guideline in effect at time of study conduct
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- histidine (S. typhumurium)
tryptophan (E. coli) - Species / strain / cell type:
- other: TA 1535, TA 97a, TA 98, TA 100 and E. coli WP2 uvrA
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9
- Test concentrations with justification for top dose:
- Trial 1 (with activation): 0.00%, 9.13%, 19.6%, 41.5%, 65.3%, and 92.8%
Trial 1 (without activation): 0.00%, 20.3%, 42.0%, 66.6%, and 90.4%
Trial 2 (with activation): 0.00%, 9.85%, 25.3%,48.3%,74.0%, and 102.1 %
Trial 2 (without activation): 0.00%, 10.1%, 24.4%, 46.9%, 73.5%, and 102% - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Air
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- other: 2-aminoanthracene [2AA] (CAS 613-13-8; +S9: TA100, TA1535, TA97a, TA98, WP2 uvrA)
- Positive control substance:
- other: 2-nitrofluorene [2NF] (CAS 607-57-8; -S9: TA98); sodium azide [NAAZ] (CAS 26628-22-8; -S9: TA100 and TA 1535); ICR-191 Acridine (CAS 17070-45-0; -S9: TA97a); methylmethanesulfonate [MMS] (CAS 66-27-3; -S9: WP2 uvrA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
Treatments with activation were conducted by adding 0.5 mL of S9 mix and 0.1 mL of an overnight culture containing 1e8 bacteria to 2 mL of top agar [0.6% agar (w/v) and 0.6% NaCI (w/v)] supplemented with 0.05 mM L-histidine and 0.05 mM D-biotin for S. typhimurium strains. These components were mixed and poured onto a plate containing approximately 25 mL of Davis minimal agar with dextrose. Treatments without activation were identical to those with activation with the exception that the S9 mix was replaced with 0.5 mL of sterile phosphate buffered saline. For positive controls, 0.1 mL of the positive indicators were added to the above mixes for both activated and non-activated treatments.
Specially designed glass chambers (6 L) were used to expose the bacterial cultures to the test gas. A flow rate of approximately 10 L/minute for approximately 3 minutes was used to create approximately 5 volume changes within the chambers to insure homogenous concentrations. Chambers were closed and at least 3 samplings of each chamber were taken and analysed by gas chromatography to determine the initial concentration of the test substance. Chambers were placed into an incubator at ~37°C for ~48 hours. Chambers were again sampled and analysed to determine test substance concentration at the end of incubation. Chambers were flushed with at least 5 chamber volumes of filtered air and then refrigerated until counted. Plates were then removed for an evaluation of background lawns and colony formation.
DURATION
- Exposure duration: 48 hr
- Expression time (cells in growth medium): 48 hr
NUMBER OF REPLICATIONS: 3
NUMBER OF CELLS EVALUATED: 10e8
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (average number of revertants) - Evaluation criteria:
- An individual trial must have included at least five test concentrations (of which at least four must have been acceptable), a solvent control, and a positive indicator for each selectee: tester strain. Data acceptability criteria were as follows:
• The density of each overnight bacterial culture was at least 1e9 cells/mL.
• A single data point may have been rejected if contamination or excessive toxicity was seen on a treatment plate. A single data point may also have been rejected if excessive precipitate on the plate prevented accurate colony counting.
• A negative control data point may have been rejected if it fell outside the acceptable spontaneous mutation range.
• A concentration level was rejected if there were less than two data points at the treatment level or if the data point values were judged by the study director to be too divergent.
• A trial for the affected strain was rejected if the negative control was rejected or if there was no evidence of mutagenic activity on any positive indicator plate or if the tester strains failed to exhibit the appropriate phenotypes.
• Only those trials which met the criteria of acceptability were included in this report. - Statistics:
- For each tester strain, the average number of revertants and the standard deviation at each concentration with and without S9 activation were calculated.
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 97a
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Cytotoxicity as measured by a significant reduction (greater than 50%) in the number of revertants was observed at the highest concentration in Salmonella typhimurium strain T A98 and Escherichia coli strain WP2 uvrA (pKMIOl) in both Trials 1 and 2 with and without activation.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
Under the conditions of this study, no evidence of mutagenic activity was detected in either of two independent trials. In this study, the test substance was negative.
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability). - Executive summary:
The test substance was evaluated for mutagenicity in Salmonella typhimurium strains TA100, TA1535, TA97a, and TA98 and in Escherichia coli strain WP2 uvrA (pKM101) with and without an exogenous metabolic activation system (S9).
In Trial 1, cells were exposed to average concentrations of 0.00, 9.13, 19.6, 41.5, 65.3, and 92.8% with metabolic activation. Without metabolic activation, average concentrations of 0.00, 20.3, 42.0, 66.6, and 90.4% were analysed. No test substance was detected after the 48 hour incubation at the lowest targeted concentration (10%) in the treatment without activation. A leak in the chamber was suspected as the cause.
In Trial 2, cells were exposed to average concentrations of 0.00, 9.85, 25.3, 48.3, 74.0, and 102.1 % with metabolic activation. Without metabolic activation, cells were exposed to average concentrations of 0.00, 10.1, 24.4, 46.9, 73.5, and 102%. Plates were compared to negative (filtered air) controls.
Cytotoxicity as measured by a significant reduction (greater than 50%) in the number of revertants was observed at the highest concentration in Salmonella typhimurium strain TA98 and Escherichia coli strain WP2 uvrA (pKM101) in both Trials 1 and 2 with and without activation.
No mutagenic activity was observed in either Trial 1 or 2.
Referenceopen allclose all
Table 1: Preliminary toxicity test, relative suspension growth
i) In the absence of S9 mix – 3-hour exposure
| Treatment / Nominal concentration (% v/v) | Replicate ID |
Cell concentration (x105/mL) 24h/48h |
Suspension Growth Day 2 |
Relative Suspension Growth (%) |
| Vehicle Control* | A B |
4.37/12.17 5.24/10.53 |
13.30 13.80 |
100 |
HFC-143a (1) |
A | 4.80/10.72 | 12.87 | 95 |
HFC-143a (5) |
A | 4.55/11.34 | 12.90 | 95 |
HFC-143a (30) |
A | 5.28/9.87 | 13.04 | 96 |
HFC-143a (40) |
A | 5.92/9.31 | 13.79 | 102 |
| HFC-143a (50) | A | 4.69/10.50 | 12.30 | 91 |
| HFC-143a (60) | A | 4.81/10.25 | 12.32 | 91 |
| HFC-143a (70) | A | 5.14/9.97 | 12.81 | 95 |
| *Vehicle control = Sterile air | ||||
ii) In the presence of S9 mix – 3-hour exposure
| Treatment / Nominal concentration (% v/v) | Replicate ID |
Cell concentration (x105/mL) 24h/48h |
Suspension Growth Day 2 |
Relative Suspension Growth (%) |
| Vehicle Control* | A B |
5.98/11.14 5.80/10.80 |
16.64 15.65 |
100 |
HFC-143a (1) |
A | 6.12/10.91 | 16.68 | 103 |
HFC-143a (5) |
A | 6.28/11.07 | 17.36 | 108 |
HFC-143a (30) |
A | 6.10/10.89 | 16.60 | 103 |
HFC-143a (40) |
A | 5.87/11.59 | 17.01 | 105 |
| HFC-143a (50) | A | 6.26/11.15 | 17.46 | 108 |
| HFC-143a (60) | A | 5.85/11.68 | 17.09 | 106 |
| HFC-143a (70) | A | 5.92/11.49 | 17.01 | 105 |
| *Vehicle control = Sterile air | ||||
Table 2: Additional main mutation test – 3-hour treatment in the absence of S9 mix, relative total growth
| Treatment / Nominal concentration (% v/v) | Replicate ID |
Cell concentration (x105/mL) 24h/48h |
Mean Suspension Growth |
Viability Plate Count* Day 2 |
Mean Cloning Efficiency (%) |
RTG (%) |
Mean RTG (%) |
| Vehicle Control** | A B C D |
4.39/12.85 4.91/11.88 5.18/14.55 4.63/11.43 |
15.19 |
47 (192) 32 (192) 33 (192) 38 (192) |
102 | 100 | 100 |
HFC-143a (22.3#) |
A B |
3.89/11.94 4.32/12.54 |
12.57 | 39 (192) 56 (192) |
87 | 75 67 |
71 |
HFC-143a (40) |
A B |
4.92/11.58 4.40/14.10 |
14.87 | 42 (192) 55 (192) |
86 | 87 78 |
83 |
HFC-143a (50) |
A B |
4.27/13.27 4.71/13.23 |
14.88 | 43 (192) 52 (192) |
87 | 85 82 |
84 |
| HFC-143a (60) | A B |
4.47/11.58 3.63/11.47 |
11.68 | 30 (192) 39 (192) |
107 | 97 67 |
82 |
| HFC-143a (70) | A B |
4.41/11.53 3.87/11.72 |
12.03 | 52 (192) 48 (192 |
84 | 67 63 |
65 |
| MMS (10 µg/mL) | A B |
5.20/10.89 3.92/11.78 |
12.85 | 61 (192) 49 (192) |
78 | 65 64 |
65 |
* Number of non-colony bearing wells (total number of wells) ** control = Sterile air # Actual concentration reported MMS - Methyl methanesulphonate RTG - Relative Total Growth |
|||||||
Table 3: Additional main mutation test – 3-hour treatment in the absence of S9mix, mutation frequency
| Treatment / Nominal concentration (% v/v) | Replicate ID |
Mutant Plate Count* Day 2 |
Mean RTG (%) |
Mean MF (x10 -6) |
|||
| Vehicle Control** | A B C D |
160 (192) 157 (192) 159 (192) 159 (192) |
100 | 93 | |||
HFC-143a (22.3#) |
A B |
159 (192) 159 (192) |
71 |
108 | |||
HFC-143a (40) |
A B |
163 (192) 160 (192) |
83 | 101 | |||
HFC-143a (50) |
A B |
159 (192) 163 (192) |
82 | 101 | |||
| HFC-143a (60) | A B |
157 (192) 162 (192) |
82 | 86 | |||
| HFC-143a (70) | A B |
160 (192) 156 (192 |
65 |
116 | |||
| MMS (10 µg/mL) | A B |
52 (192) 55 (192) |
65 |
818 | |||
* Number of non-colony bearing wells (total number of wells) ** control = Sterile air # Actual concentration reported MMS - Methyl methanesulphonate RTG - Relative Total Growth MF - Mutant Frequency |
|||||||
Table 4: Additional main mutation test – 3-hour treatment in the absence of S9mix, colony size analysis
| Treatment / Nominal concentration (% v/v) | Replicate ID |
Small Mutant Plate Count* Day 2 |
Mean Small Colony MF (x10-6) |
Large Mutant Plate Count* Day 2 |
Mean Large Colony MF (x10-6) |
|||
| Vehicle Control** | A B C D |
175 (192) 176 (192) 176 (192) 174 (192) |
45 | 173 171 173 173 |
52 | |||
| MMS (10 µg/mL) | A B |
69 (192) 72 (192) |
641 |
146 151 |
164 | |||
* Number of non-colony bearing wells (total number of wells) ** control = Sterile air MMS - Methyl methanesulphonate RTG - Relative Total Growth MF - Mutant Frequency |
||||||||
Table 1: Preliminary toxicity test, relative suspension growth
i) In the absence of S9 mix – 3-hour exposure
| Treatment / Nominal concentration (% v/v) | Replicate ID |
Cell concentration (x105/mL) 24h/48h |
Suspension Growth Day 2 |
Relative Suspension Growth (%) |
| Vehicle Control* | A B |
4.37/12.17 5.24/10.53 |
13.30 13.80 |
100 |
HFC-143a (1) |
A | 4.80/10.72 | 12.87 | 95 |
HFC-143a (5) |
A | 4.55/11.34 | 12.90 | 95 |
HFC-143a (30) |
A | 5.28/9.87 | 13.04 | 96 |
HFC-143a (40) |
A | 5.92/9.31 | 13.79 | 102 |
| HFC-143a (50) | A | 4.69/10.50 | 12.30 | 91 |
| HFC-143a (60) | A | 4.81/10.25 | 12.32 | 91 |
| HFC-143a (70) | A | 5.14/9.97 | 12.81 | 95 |
| *Vehicle control = Sterile air | ||||
ii) In the presence of S9 mix – 3-hour exposure
| Treatment / Nominal concentration (% v/v) | Replicate ID |
Cell concentration (x105/mL) 24h/48h |
Suspension Growth Day 2 |
Relative Suspension Growth (%) |
| Vehicle Control* | A B |
5.98/11.14 5.80/10.80 |
16.64 15.65 |
100 |
HFC-143a (1) |
A | 6.12/10.91 | 16.68 | 103 |
HFC-143a (5) |
A | 6.28/11.07 | 17.36 | 108 |
HFC-143a (30) |
A | 6.10/10.89 | 16.60 | 103 |
HFC-143a (40) |
A | 5.87/11.59 | 17.01 | 105 |
| HFC-143a (50) | A | 6.26/11.15 | 17.46 | 108 |
| HFC-143a (60) | A | 5.85/11.68 | 17.09 | 106 |
| HFC-143a (70) | A | 5.92/11.49 | 17.01 | 105 |
| *Vehicle control = Sterile air | ||||
Table 2: Additional main mutation test – 3-hour treatment in the absence of S9 mix, relative total growth
| Treatment / Nominal concentration (% v/v) | Replicate ID |
Cell concentration (x105/mL) 24h/48h |
Mean Suspension Growth |
Viability Plate Count* Day 2 |
Mean Cloning Efficiency (%) |
RTG (%) |
Mean RTG (%) |
| Vehicle Control** | A B C D |
4.39/12.85 4.91/11.88 5.18/14.55 4.63/11.43 |
15.19 |
47 (192) 32 (192) 33 (192) 38 (192) |
102 | 100 | 100 |
HFC-143a (22.3#) |
A B |
3.89/11.94 4.32/12.54 |
12.57 | 39 (192) 56 (192) |
87 | 75 67 |
71 |
HFC-143a (40) |
A B |
4.92/11.58 4.40/14.10 |
14.87 | 42 (192) 55 (192) |
86 | 87 78 |
83 |
HFC-143a (50) |
A B |
4.27/13.27 4.71/13.23 |
14.88 | 43 (192) 52 (192) |
87 | 85 82 |
84 |
| HFC-143a (60) | A B |
4.47/11.58 3.63/11.47 |
11.68 | 30 (192) 39 (192) |
107 | 97 67 |
82 |
| HFC-143a (70) | A B |
4.41/11.53 3.87/11.72 |
12.03 | 52 (192) 48 (192 |
84 | 67 63 |
65 |
| MMS (10 µg/mL) | A B |
5.20/10.89 3.92/11.78 |
12.85 | 61 (192) 49 (192) |
78 | 65 64 |
65 |
* Number of non-colony bearing wells (total number of wells) ** control = Sterile air # Actual concentration reported MMS - Methyl methanesulphonate RTG - Relative Total Growth |
|||||||
Table 3: Additional main mutation test – 3-hour treatment in the absence of S9mix, mutation frequency
| Treatment / Nominal concentration (% v/v) | Replicate ID |
Mutant Plate Count* Day 2 |
Mean RTG (%) |
Mean MF (x10 -6) |
|||
| Vehicle Control** | A B C D |
160 (192) 157 (192) 159 (192) 159 (192) |
100 | 93 | |||
HFC-143a (22.3#) |
A B |
159 (192) 159 (192) |
71 |
108 | |||
HFC-143a (40) |
A B |
163 (192) 160 (192) |
83 | 101 | |||
HFC-143a (50) |
A B |
159 (192) 163 (192) |
82 | 101 | |||
| HFC-143a (60) | A B |
157 (192) 162 (192) |
82 | 86 | |||
| HFC-143a (70) | A B |
160 (192) 156 (192 |
65 |
116 | |||
| MMS (10 µg/mL) | A B |
52 (192) 55 (192) |
65 |
818 | |||
* Number of non-colony bearing wells (total number of wells) ** control = Sterile air # Actual concentration reported MMS - Methyl methanesulphonate RTG - Relative Total Growth MF - Mutant Frequency |
|||||||
Table 4: Additional main mutation test – 3-hour treatment in the absence of S9mix, colony size analysis
| Treatment / Nominal concentration (% v/v) | Replicate ID |
Small Mutant Plate Count* Day 2 |
Mean Small Colony MF (x10-6) |
Large Mutant Plate Count* Day 2 |
Mean Large Colony MF (x10-6) |
|||
| Vehicle Control** | A B C D |
175 (192) 176 (192) 176 (192) 174 (192) |
45 | 173 171 173 173 |
52 | |||
| MMS (10 µg/mL) | A B |
69 (192) 72 (192) |
641 |
146 151 |
164 | |||
* Number of non-colony bearing wells (total number of wells) ** control = Sterile air MMS - Methyl methanesulphonate RTG - Relative Total Growth MF - Mutant Frequency |
||||||||
Table - Chromosome Aberration Assay Summary |
|
|
Cells with Aberrationsb |
||||||||
Treatmenta |
S9 |
Treatment |
Mitotic |
Cells Scored |
Aberrations Per Cell |
Numerical |
Structural |
||||
(% TS) |
Activation |
Time |
Index (%) |
Numerical |
Structural |
Mean |
SD |
(%) |
(%) |
||
Negative Controlc |
-S9 |
4 |
16.8 |
200 |
200 |
0 |
0 |
0 |
0 |
||
25 |
-S9 |
4 |
14.6 |
200 |
200 |
0 |
0 |
0 |
0 |
||
50 |
-S9 |
4 |
16.1 |
200 |
200 |
0 |
0 |
0 |
0 |
||
100 |
-S9 |
4 |
13.7 |
200 |
200 |
0.010 |
0.014 |
0 |
1 |
||
MMC 0.2d |
-S9 |
4 |
11.1 |
200 |
200 |
0.135 |
0.035 |
0 |
12.0e |
||
|
|||||||||||
Negative Controlc |
+S9 |
4 |
13.0 |
200 |
200 |
0 |
0 |
0 |
0 |
||
25 |
+S9 |
4 |
9.7 |
200 |
200 |
0.01 |
0 |
0 |
1 |
||
50 |
+S9 |
4 |
10.8 |
200 |
200 |
0 |
0 |
0 |
0 |
||
100 |
+S9 |
4 |
11.3 |
200 |
200 |
0 |
0 |
0 |
0 |
||
CP 5d |
+S9 |
4 |
9.3 |
200 |
200 |
0.14 |
0.071 |
0 |
13.0e |
||
|
|||||||||||
Negative Controlc |
-S9 |
22 |
13.3 |
200 |
200 |
0.005 |
0.007 |
0 |
0.5 |
||
25 |
-S9 |
22 |
11.5 |
200 |
200 |
0.025 |
0.021 |
0 |
2.5 |
||
50 |
-S9 |
22 |
11.9 |
200 |
200 |
0.005 |
0.007 |
0 |
0.5 |
||
100 |
-S9 |
22 |
13.1 |
200 |
200 |
0.01 |
0 |
0 |
1 |
||
MMC 0.2d |
-S9 |
22 |
8.1 |
200 |
200 |
0.17 |
0 |
0 |
16.0e |
||
a – Human peripheral blood lymphocytes (HPBL) cells were treated at 37ºC b – Excluding cells with only gaps |
|||||||||||
c – Ambient Air |
|||||||||||
d – μg/mL |
|||||||||||
e - Statistically significant difference from control at p < 0.05 by Fisher's test. |
|||||||||||
TS = Test Substance |
|||||||||||
Table 1. Trial #1 A. (without Activation) Revertants per Plate |
|||||
(% R116) |
Strain T100 |
Strain T1535 |
Strain TA97a |
Strain TA98 |
WP2 uvrA (pKM101) |
Concentration |
Plates 1, 2, 3 |
Plates 1, 2, 3 |
Plates 1, 2, 3 |
Plates 1, 2, 3 |
Plates 1, 2, 3 |
0 |
174, 140, 134 |
15, 24, 28 |
119, 135, 151 |
24, 34, 33 |
156, 183, 157 |
20.3 |
126, 131, 133 |
13, 9, 18 |
135, 131, 130 |
19, 22, 25 |
122, 118, 145 |
42 |
119, 116, 131 |
19, 23, 15 |
103, 89, 109 |
21, 19, 9 |
107, 108, 102 |
66.6 |
101, 103, 103 |
9, 19, 10 |
87, 78, 99 |
10, 10, 12 |
69, 78, 66 |
90.4 |
120, 95, 106 |
15, 19, 19 |
80, 87, 71 |
8, 6, 9 |
5, 7, 4 |
Control Info |
NAAZ (2 µg/plate) |
NAAZ (2 µg/plate) |
ICR (2 µg/plate) |
2NF (25 µg/plate) |
MMS (1000 µg/plate) |
|
752, 825, 809 |
547, 563, 554 |
1827, 1995, 1883 |
1541, 1929, 1910 |
1938, 2090, 2113 |
Note: Control abbreviations are 2AA =2-aminoanthracene, 2NF=2-nitrofluorene, NAAZ=sodium azide, ICR=ICR -191 Acridine, MMS=methylmethanesulfonate. |
|||||
B. Trial #1 (with Activation) Revertants per Plate |
|||||
(% R116) |
Strain T100 |
Strain T1535 |
Strain TA97a |
Strain TA98 |
WP2 uvrA (pKM101) |
Concentration |
Plates 1, 2, 3 |
Plates 1, 2, 3 |
Plates 1, 2, 3 |
Plates 1, 2, 3 |
Plates 1, 2, 3 |
0 |
152, 167, 159 |
15, 11, 23 |
118, 125, 95 |
29, 19, 21 |
134, 147, 130 |
9.13 |
134, 136, 126 |
17, 22, 28 |
130, 116, 126 |
24, 26, 19 |
147, 144, 147 |
19.6 |
153, 129, 142 |
24, 21, 20 |
165, 99, 145 |
19, 25, 24 |
110, 138, 115 |
41.5 |
149, 104, 115 |
19, 18, 22 |
92, 131, 102 |
18, 33, 20 |
98, 81, 74 |
65.3 |
112, 88, 126 |
17, 18, 18 |
127, 133, 112 |
12, 13, 19 |
65, 51, 60 |
92.8 |
117, 95, 82 |
11, 10, 12 |
79, 81, 68 |
5, 6, 8 |
2, 4, 14 |
Control Info |
2AA (1 µg/plate) |
2AA (2 µg/plate) |
2AA (1 µg/plate) |
2AA (2 µg/plate) |
2AA (25 µg/plate) |
|
1403, 1397, 1214 |
393, 371, 440 |
889, 942, 866 |
2611, 2491, 2470 |
1841, 2226, 2071 |
Table 2. Trial #2 A. (without Activation) Revertants per Plate |
|||||
(% R116) |
Strain T100 |
Strain T1535 |
Strain TA97a |
Strain TA98 |
WP2 uvrA (pKM101) |
Concentration |
Plates 1, 2, 3 |
Plates 1, 2, 3 |
Plates 1, 2, 3 |
Plates 1, 2, 3 |
Plates 1, 2, 3 |
0 |
119, 107, 102 |
10, 12, 10 |
101, 97, 80 |
19, 21, 22 |
129, 141, 125 |
10.1 |
119, 114, 102 |
8, 9, 10 |
81, 84, 81 |
19, 16, 22 |
136, 148, 130 |
24.4 |
111, 107, 91 |
11, 13, 8 |
120, 97, 90 |
22, 21, 24 |
120, 127, 128 |
46.9 |
93, 83, 99 |
6, 13, 10 |
93, 107, 63 |
16, 19, 23 |
126, 125, 107 |
73.5 |
77, 73, 92 |
15, 11, 8 |
96, 77, 100 |
14, 18, 19 |
78, 84, 60 |
102 |
78, 73, 60 |
12, 5, 5 |
60, 82, 68 |
6, 4, 6 |
11, 4, 0 |
Control Info |
NAAZ (2 µg/plate) |
NAAZ (2 µg/plate) |
ICR 191 (2 µg/plate) |
2NF (25 µg/plate) |
MMS (1000 µg/plate) |
|
811, 827, 828 |
563, 554, 554 |
2058, 1914, 2000 |
1572, 1593, 1566 |
2326, 2289, 2103 |
B. Trial #2 (with Activation) Revertants per Plate |
|||||
(% R116) |
Strain T100 |
Strain T1535 |
Strain TA97a |
Strain TA98 |
WP2 uvrA (pKM101) |
Concentration |
Plates 1, 2, 3 |
Plates 1, 2, 3 |
Plates 1, 2, 3 |
Plates 1, 2, 3 |
Plates 1, 2, 3 |
0 |
110, 124, 115 |
10, 7, 9 |
122, 97, 105 |
41, 25, 26 |
145, 126, 142 |
9.85 |
100, 101, 116 |
6, 6, 9 |
82, 87, 81 |
25, 30, 28 |
122, 126, 127 |
25.3 |
100, 83, 87 |
9, 14, 7 |
101, 99, 87 |
22, 22, 25 |
117, 130, 125 |
48.3 |
78, 83, 92 |
7, 12, 7 |
94, 107, 117 |
30, 29, 24 |
118, 118, 104 |
74 |
87, 73, 80 |
8, 10, 8 |
77, 100, 96 |
18, 22, 17 |
57, 75, 48 |
102 |
43, 62, 64 |
8, 13, 6 |
64, 64, 54 |
8, 5, 8 |
16, 7, 0 |
Control Info |
2AA (1 µg/plate) |
2AA (2 µg/plate) |
2AA (1 µg/plate) |
2AA (2 µg/plate) |
2AA (25 µg/plate) |
|
942, 957, 1011 |
281, 376, 337 |
599, 691, 720 |
1607, 1747, 1747 |
2335, 2423, 2472 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
In Vivo (Clastogenic effects - mammalian): OECD 474; in vivo rat micronucleus study; Negative at concentrations up to 50000 ppm (282233 mg/m3). Reliability = 1.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- Remarks:
- The study was conducted according to the guideline in effect at the time of study conduct.
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
- Deviations:
- no
- Remarks:
- The study was conducted according to the guideline dated 1998.
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- rat
- Strain:
- other: Crl:CD(SD)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: Approximately 70 days
- Weight at study initiation: 317-445 grams (males) and 201-269 grams (females)
- Fasting period before study: No reported
- Housing: Individually in solid bottom caging with bedding, and enrichment as appropriate; sexes on separate racks
- Diet (e.g. ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum except when fasted
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC (68-79ºF)
- Humidity (%): 30%–70%
- Air changes (per hr): 10-13
- Photoperiod (hrs dark / hrs light): 12-hours light/12 hours dark - Route of administration:
- inhalation: gas
- Vehicle:
- - Vehicle used: inline house air
- Details on exposure:
- TYPE OF INHALATION EXPOSURE: whole body
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: A stainless steel baffle below the chamber turret inside the chambers promoted uniform chamber distribution of the test atmosphere
Exposure #s 1 to 10, stainless steel and glass (NYU style) with a nominal internal volume of 350 L
Exposure #s 11 to 71, control animals were exposed to air in the same chamber while test substance exposures were performed in stainless steel and glass (NYU style) chamber with a nominal internal volume of 150 L
- Method of holding animals in test chamber: individually placed in stainless steel, wire-mesh modules and exposed whole-body in chamber
- Source and rate of air: high pressure house line air and oxygen
- System of generating particulates/aerosols: test substance vapour and supplemental oxygen were metered into a 1-liter 3-neck mixing flask by mass flow controllers. The mixture left the mixing flask and entered the glass transfer tube where chamber air supply was added to the mixture by mass flow controllers. The gas mixture then entered the top of the exposure chamber through a turret. The air-control atmosphere was similarly generated in a different room without test substance.
- Temperature, humidity, pressure in air chamber: 21-25ºC, 45-67%, and 21% oxygen, no data on pressure
- Air flow rate: Exposure #s 1 to 10 - chamber flow 60-64 L/min; Exposure #s 11 to 20 - chamber flow 29-33 L/min
- Air change rate: 10-13 per hour
- Treatment of exhaust air: exhausted from the bottom of each chamber through MSA filters using vacuum pumps and discharged into the fume hood
TEST ATMOSPHERE
- Brief description of analytical method used: vapour concentration was directly injected into a gas chromatograph
- Samples taken from breathing zone: yes - Duration of treatment / exposure:
- Approximately 20 days of exposure
- Frequency of treatment:
- 6 hours per day, 5 days per week
- Post exposure period:
- None
- Remarks:
- Doses / Concentrations:
0, 2500, 10000, and 50000 ppm
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
0.0 ± 0.0; 2500 ± 11; 10000 ± 74; and 51000 ± 650 ppm
Basis:
analytical conc. - No. of animals per sex per dose:
- 5/sex/dose
- Control animals:
- yes, concurrent vehicle
- Tissues and cell types examined:
- RETs plus normochromatic erythrocytes [NCEs] from rat tail vein
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: In a previous acute inhalation study the inhalation LC50 in male and female rats was greater than 502000 ppm. Based on these results, exposure concentrations of 0, 2500, 10000, and 50000 ppm were selected for the current study.
TREATMENT AND SAMPLING TIMES: the evaluation was conducted by flow cytometry. Peripheral blood samples were collected by tail vein venipuncture from the 5 animals per sex per dose. There were 2 blood collections, one following the fourth exposure (test day 3), and on the last exposure day before scheduled sacrifice.
DETAILS OF SLIDE PREPARATION: Approximately 6 to 8 drops (i.e. 60-120 µL) of blood was collected from the tail vein of each animal directly into a labelled microcentrifuge tube containing 350 µL anticoagulant/diluent (anticoagulant) provided in the In Vitro Micro Flow Plus® Rat Micronucleus assay kit. The tubes were capped and inverted several times to mix the blood with the anticoagulant. The blood/anticoagulant mixture was either stored for up to 6 hours at room temperature or for up to 24 hours refrigerated, prior to fixation. The blood samples were fixed in duplicate. Approximately 180 µL of blood/anticoagulant mixture was fixed in 2 mL ultra-cold reagent grade methanol and stored below -75ºC until processed.
METHOD OF ANALYSIS: Samples were analysed by flow cytometry on a BD FACSCalibur™ running Cell Quest Pro Software. At least 20000 reticulocytes were analysed per blood sample for induction of micronuclei, and toxicity as indicated by the frequency of immature erythrocytes (%RETs) among the total (RETs plus normochromatic erythrocytes [NCEs]). The samples were analysed and evaluated using the reagents and templates provided in the In Vitro MicroFlow Plus® Rat Micronucleus assay kit. The frequency of micronucleated reticulocytes (%MN-RETs) were used as a measure of induction of aneugenic or clastogenic alterations by the test substance. Initially, only the samples from the control and high-concentration group were analysed. Samples from the lower exposure groups were also analysed from the day 3 female blood collection to confirm the findings from the high concentration group. The positive and negative control standards, provided in the assay kit, were used as a methods control. - Evaluation criteria:
- Data were evaluated using scientific judgment taking into account both statistical and biological significance. Results not meeting the indicated criteria for positive or negative findings were evaluated on a case-by-case basis by the principal investigator for the micronucleus evaluation or the study director.
The test substance was judged positive if the following conditions were met:
• A statistically significantly increase mean MN-RETs was observed at one or more concentrations of the test substance compared to the concurrent negative control values.
• An accompanying statistically significant dose-response increase in MN-RETs was observed.
The test substance was judged negative if the following conditions were met:
• No statistically significant increases in the mean MN-RETs above the concurrent negative control value occurred at any concentration of the test substance.
• The MN-RET values of the test substance-treated animals were within reasonable limits of the recent laboratory negative historical control range. - Statistics:
- Micronucleus data were evaluated using scientific judgment taking into account both statistical and biological significance. The individual animal was considered the experimental unit. For each treatment group, the mean and standard deviation of % RETs and % MN-RETs were calculated. Data were transformed prior to analysis using an arcsine square root or Freeman-Tukey function. This transformation was appropriate for proportions since the distribution of the transformed data more closely approximates a normal distribution than does the nontransformed proportion. The individual animal was considered the experimental unit for the micronucleus data. All data analyses were one-tailed and conducted at a significance level of 5%. See Table 1 below.
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Additional information on results:
- RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Negative
- Ratio of PCE/NCE (for Micronucleus assay): No statistically significant test substance-related decreases in %RET. No statistically significant increases in the frequency of MN-RETs compared to the negative control group; the negative control group exhibited a response consistent with the %MN-RETs historical control data.
- Statistical evaluation: yes - Conclusions:
- Interpretation of results : negative
No statistically significant increases in the frequency of MN-RETs compared to the negative control group were observed in any evaluated test substance treated group of male or female animals at any time point. There were no statistically significant test substance-related decreases in %RET.
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability). - Executive summary:
The purpose of this study was to determine the potential micronucleus evaluation from repeated inhalation exposure of male and female rats to 0, 2500, 10000, or 50000 ppm of the test substance.
Groups of 20 young adult male or nulliparous, non-pregnant female Crl:CD(SD) rats were exposed to the test substance 6 hours per day, 5 days per week over a one-month period. Concentrations of test substance were generated by metering the test substance into the inhalation chambers and diluting it with conditioned, filtered air, supplemented with oxygen to achieve an oxygen concentration of 20% in the high level chamber. An air control group was also evaluated using a similar generation method. Vapour concentrations of the test substance were measured by gas chromatography (GC). Temperature, humidity, and airflow were also recorded periodically during each exposure day. Body weights, clinical signs, and food consumption were recorded throughout the study. After 3 days and 28 days of exposure, blood samples were collected from 5 male and 5 female rats per group and evaluated for induction of micronuclei. The mean concentrations (± standard error of the mean) were 2500 ± 11, 10000 ± 74, and 51000 ± 647 ppm in chambers targeted at 2500, 10000, and 50000 ppm, respectively.
No statistically significant increases in the frequency of MN-RETs compared to the negative control group were observed in any evaluated test substance treated group of male or female animals at any time point. There were no statistically significant test substance-related decreases in %RET. The negative control group exhibited a response consistent with the %MN-RETs historical control data.
Reference
OTHER: These results are taken from the 28-Day Inhalation Study in Section 7.5.2.
CLINICAL SIGNS AND MORTALITY
Unscheduled deaths did not occur in the animals assigned to the Subchronic and Subchronic with Recovery Subsets. No test substance-related clinical sigs of toxicity were observed in males or females at any exposure concentration. Incidental clinical signs common to the age and strain of rat were occasionally observed during the exposure and/or recovery periods.
BODY WEIGHT AND WEIGHT GAIN
No adverse, test substance-related effects on body weight or weight gain were observed for males or females exposed to any concentration of the test substance during the exposure or recovery periods. Body weight of the 10000 ppm males was statistically significantly greater compared to the control group on test days 3 and 7. However, this statistical difference was considered to be spurious since a dose-response relationship was not evident. Weight gain for 2500 ppm females was statistically significantly lower compared to the control group during test days 7-14. However, this statistical difference was considered to be spurious since a dose-response relationship was not present.
FOOD CONSUMPTION & FOOD EFFICIENCY
No test substance-related effects or statistically significant differences in food consumption or food efficiency were observed for males or females exposed to any concentration of the test substance during the exposure of recovery periods.
OPHTHALMOSCOPIC EXAMINATION
No test substance-related ophthalmological observations occurred in males or females exposed to any concentration of the test substance.
HAEMATOLOGY
There were no exposure-related changes in any group mean haematology parameter in male or female rats at any concentration tested.
The following statistically significant difference was not considered to be related to exposure to the test substance:
• Absolute large unstained cells (ALUC) were minimally higher in female rats exposed to 50000 ppm (90% above the control). There were no other statistically significant differences in other white blood cell parameter in any subsets of male or female rats at any concentration tested. Therefore, this change was considered unrelated to exposure and non-adverse.
• Coagulation - There were no statistically significant or exposure-related changes in any group mean coagulation parameter in male or female rats at any concentration tested.
CLINICAL CHEMISTRY
There were no exposure-related changes in group mean clinical chemistry parameters in male or female rats at any concentration tested.
The following statistically significant difference was not considered to be related to exposure to the test substance:
• Blood urea nitrogen (BUN) was minimally lower in female rats exposed to 50000 ppm (18% below the control). However, there were no statistically significant differences in other kidney-related clinical pathology parameters and no exposure-related changes in renal histopathology in this group. In addition, there were no changes in BUN in any other male or female group, including the 50000 ppm females from the reproduction subset. Therefore the lower BUN in the 50000 ppm females from the subchronic subset was considered unrelated to exposure and non-adverse.
URINALYSIS
There were no statistically significant differences in group mean urinalyses parameters in male or female rats at any concentration tested.
• Plasma and Urine Fluoride - There were no statistically significant changes in plasma and urine fluoride parameters in male or female rats.
NEUROBEHAVIOUR (only done on Subchronic with Recovery Subset)
• Forelimb and Hindlimb Grip Strength - No test substance-related or statistically significant effects occurred on forelimb or hindlimb grip strength for males or females exposed to any concentration of the test substance.
• Open Field Observations - No test substance-related or statistically significant effects were observed for any behavioural parameter evaluated in males or females exposed to any concentration of the test substance. The incidences for all groups were similar to the values for the control groups.
A wound observed during the FOB at week 6 in one male in the 50,000 ppm group was considered to be incidental.
• Motor Activity - No test substance-related or statistically significant effects were observed on motor activity for males or females exposed to any concentration of the test substance. At week 4, the mean duration and number of movements for 50000 ppm females during the 2nd – 6th intervals as well as for the total session were decreased (not statistically significant) compared to control. However, these decreases, although they showed a trend, were not considered to be adverse because the decreases were not observed in males, were not statistically significant, and were within historical control. Mean values for duration and number of movements for the 2500 and 10000 ppm males and females and 50000 ppm males were similar to their respective controls.
ORGAN WEIGHTS
There were no test substance-related organ weight effects at inhalation exposures up to 50000 ppm.
All individual and mean organ weight differences were considered spurious and unrelated to test substance exposure.
• In males, two of the accessory sex organs (ASO) mean weight parameters were statistically (p < 0.05) decreased in the 2500 ppm exposure group, as compared to the respective control values. However, since there was no dose response, no microscopic correlate, and no similar finding in the Subchronic with Recovery Subset or Reproduction Subset, the statistical finding was interpreted to be spurious.
• In females, the mean absolute adrenal weight and mean absolute kidneys weight were both statistically decreased in the 2500 ppm group, as compared to the respective control values. These were also considered spurious since there was no dose response, no microscopic correlate, and no similar finding in the Subchronic Subset males, Subchronic with Recovery Subset, or Reproduction Subset.
In the Subchronic with Recovery Subset, there were no test substance-related organ weight effects.
All individual and mean organ weight differences were considered spurious and unrelated to test substance exposure.
• In males, both the mean relative (% body weight) heart weight and lung weight were statistically decreased in the 10,000 ppm exposure group compared to the respective control values. These were both considered spurious since there was no dose response, no microscopic correlate, and no similar finding in the main study, recovery females, or reproduction study subsets. A statistically significant decrease in the 50000 ppm mean absolute lung weight was also interpreted as spurious because there was no microscopic correlate and no similar finding in the Subchronic Subset, Subchronic with Recovery females, or Reproduction Subsets.
• In females, the mean relative (% brain weight) lung weight was increased in the 50000 ppm group, as compared to the respective control values. This was also considered spurious since there was no microscopic correlate and no similar finding in the Subchronic, Subchronic with Recovery males, or Reproduction Subsets.
GROSS PATHOLOGY
At the terminal sacrifice, there were no test substance-related gross observations in either sex. All gross observations were consistent with normal background lesions in rats of this age and strain.
HISTOPATHOLOGY: NON-NEOPLASTIC
There were no test substance-related microscopic findings in male or female rats in the 50000 ppm inhalation exposure group. All microscopic findings in this study were consistent with normal background lesions in rats of this age and strain.
For additional details regarding Reproductive/Developmental test refer to Section 7.8.1 (DI.K1.1Gen.RD/REPRO/DEV.R.D-19695-1422.KD) and 7.8.2 (DI.K1.DEV.RD/REPRO/DEV.R.D-19695-1422.KD)
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
The test substance did not produce mutagenicity or clastogenicity when evaluated in in vitro systems or in laboratory animals. Based on an assessment of the robust genetic toxicity data for this substance, the substance does not need to be classified for germ cell mutagenicity according the EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
