Propanenitrile, 2,3,3,3-tetrafluoro-2-(trifluoromethyl)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro studies and one in vivo study have been conducted on this substance. The results of the studies were:

in vitro:

OECD 471: Negative with and without metabolic activation.

OECD 473: Equivocal

OECD 490: Positive in the absence of metabolic activation

in vivo:

OECD 474 and 489 (Combined Assay): Negative

Link to relevant study records

Reference

Reference 1

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: Study was conducted in compliance with OECD GLP regulations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella thyphimurium strains: Histidine operon, Escherichia coli: thyptophan operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Arclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

0.0781, 0.156, 0.313, 0.625, 1.25, 2.50, 5.00, 10.0, 20.0, and 40.0 % v/v

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Air

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Strain Specific

Remarks:

TA100, TA1535, WP2 uvrA: Chloroethane, TA1537: ICR-191 acridine, TA98: 2-nitrofluorene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Incubation in a bag containing the test article at the appropriate concentration.

DURATION
- Preincubation period: None
- Exposure duration: 2 days
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): NA
- Fixation time (start of exposure up to fixation or harvest of cells): 2 days

SELECTION AGENT (mutation assays): None

NUMBER OF CELLS EVALUATED: All

DETERMINATION OF CYTOTOXICITY
- Method: A concentration was considered cytotoxic if it caused a >50% reduction in the mean number of revertants per plate as compared to the mean vehicle control accompanied by an abrupt concentration-dependent drop in the mean number of revertants or a significant reduction in the background lawn

Evaluation criteria:

The test substance was considered positive for mutagenicity if it induced an increase of revertants per plate with increasing concentration. The increases should be at least two times the vehicle control substance background frequency for strains with high spontaneous levels (TA100) and three times for those with low spontaneous levels (TA1537, TA98, TA1535, and WP2 uvrA). Increases should be seen in at least two or more successive concentrations or the response should be repeatable at a single concentration.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A rangefinding assay was conducted at concentrations of 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30% v/v. Cytotoxicity was observed at concentrations at and above 3% in TA100 with and without metabolic activation and WP2 uvrA with metabolic activation and at 10% or greater in WP2 uvrA without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: Controls were consistent with historical data.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of the test, the test article was not mutagenic in the Bacterial Reverse Mutation Assay in either the presence or absence of metabolic activation.

Executive summary:

The mutagenic potential of the test article was evaluated in the Bacterial Reverse Mutation Assay with S. typhimurium strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2 uvrA in the presence and absence of a metabolic activation system (S9-mix rat liver induced by Arclor 1254). The study was performed in compliance with OECD GLP. The test method was based on OECD No. 471 and ICH S2A and S2B.  Plates were exposed to the test substance via incubation in a sealed bag containing the test article at the appropriate concentration. Strains TA100, TA1535 and WP2 uvrA were exposed to chloroethane as a positive control. A dose range-finding test was performed with concentration up to 30% v/v test article in the absence and presence of metabolic activation in strains TA100 and WP2uvrA. Based on the results of the dose range finding test, the test article was tested at concentrations of 0 (control) 0.0781, 0.156, 0.313, 0.625, 1.25, 2.50, 5.00 and 10.0% v/v test article in TA1537 and TA100 with metabolic activation and at 10 % v/v in TA100 and WP2 uvrA without metabolic activation and TA1535 with and without metabolic activation. A confirmatory assay was performed at 0.0781, 0.156, 0.313, 0.625, 1.25, 2.50, 5.00, 10.0, 20.0, and 40.0 % v/v in strains TA98, TA1537 and WP2 uvrA. All treatments were performed in triplicate. No increase in revertant colonies was observed in either the presence or absence of metabolic activation in any strain tested. All criteria for a valid test were met as described in the protocol. Based on the results of the test, the test article was not mutagenic in the Bacterial Reverse Mutation Assay in either the presence or absence of metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2016

Deviations:

no

Remarks:

No deviations occurred that impacted the integrity of the study.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
3M Company, Lot 16
- Expiration date of the lot/batch:
31 March, 2019
- Purity test date:
31 March, 2017


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Ambient temperature (15-25°C)
- Solubility and stability of the test substance in the solvent/vehicle:
Since the test substance is a colorless gas , cells in culture flasks were exposed in modular incubator chambers (Billups-Rothenburg, USA) to various concentrations. The atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test substance supplemented with N2. The highest concentration theoretically achievable was therefore 76% (v/v). Additional concentrations of 60, 40, 20 and 10% (v/v) (all ± 10%) were used in the chambers to expose the cells. Air (19% O2, 5% CO2, 76% N2) without the test substance was used as negative control.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
No data

FORM AS APPLIED IN THE TEST: Gas

Species / strain / cell type:

mammalian cell line, other: See Remarks

Remarks:

Human peripheral blood lymphocytes.

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Young healthy, non-smoking individual (37 years old) with no known recent exposures to genotoxic chemicals or radiation.
- Suitability of cells: Suitable per OECD 473
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable: 37, one donor
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The medium for culturing the human peripheral blood lymphocytes consisted of RPMI 1640 medium (with HEPES and Glutamax), supplemented with heat-inactivated (30 min, 56ºC) fetal calf serum (20%), penicillin (100 U/ml medium), streptomycin (100 μg/ml medium) and phytohemagglutinin (2.4 μg/ml).

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

10, 20, 40, 60, 76% test article (highest attainable concentration).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: None

Untreated negative controls:

yes

Remarks:

Air

Negative solvent / vehicle controls:

no

True negative controls:

yes

Remarks:

Air

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: Since the test substance is a colorless gas, cells in culture flasks were exposed in modular incubator chambers (Billups-Rothenburg, USA) to various concentrations. The atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test substance supplemented with N2. The highest concentration achievable was therefore 76% (v/v%)

DURATION
- Preincubation period: 48 hours
- Exposure duration: Pulse treatment groups (+/- S9): 4 hours, Continuous treatment (- S9): 24 hours.
- Expression time (cells in growth medium): 72-74 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24-26 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the total incubation period, the cells were harvested by low speed centrifugation, treated for 15 min at 37 ºC with a hypotonic solution (0.075 M KCl), fixed three times with a freshly prepared 3:1 (v/v) mixture of methanol and glacial acetic acid and processed for chromosome preparations. Two slides were prepared from each selected culture. The slides were stained in a 2% solution of Giemsa, rinsed in water, air-dried and mounted with a coverslip. The slides were coded by a qualified person not involved in scoring the slides, to enable “blind” scoring.

NUMBER OF CELLS EVALUATED: A number of 1000 stimulated lymphocytes (500 cells per slide and two slides per culture) were examined in each culture to determine the percentage of cells in mitosis (mitotic index). Based on the results of the mitotic index scoring and the observations made with respect to the number and quality of the metaphases, at least three concentrations of the test substance together with the negative control (clean air) and positive controls were selected for chromosomal aberration analysis.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): For each treatment group, 300 well-spread metaphases per concentration (150 metaphases per culture and 75 metaphases per slide), each containing 46 centromeres, were analyzed by microscopic examination for chromatid-type aberrations (gaps, breaks, fragments, interchanges), chromosome-type aberrations (gaps, breaks, minutes, rings, dicentrics), according to the criteria recommended by Savage (1975). If heavily damaged cells or cells with numerical aberrations (such as endoreduplicated cells or polyploid cells) were observed, these cells were recorded but not counted and included in the 300 analyzed cells. The Vernier readings of all aberrant metaphases were recorded. See also Annex 3 of this report for the definition of chromosomal aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Rationale for test conditions:

Per OECD 473

Evaluation criteria:

The study was considered valid if the positive controls demonstrated a statistically significantly increase in the number of aberrant cells (compatible to the historical data of the test facility) and the solvent controls were within the historical range.
The number of metaphases containing one or more aberrations of the test substance treated groups were compared with those of the concurrent solvent controls using Fisher's exact test (one-sided). The difference was considered statistically significant when the p-value of the Fisher’s exact test was less than 0.05.
The response was considered positive if all of the following criteria are met:
- at least one of the test concentrations exhibits a statistically significant increase
compared to the concurrent negative control.
- the increase is dose-related in at least in one experimental condition when
evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical solvent control data.
A response was considered negative if all of the following criteria are met:
- none of the test concentrations exhibits a statistically significant increase
compared to the concurrent negative control.
- there is no dose-related increase when evaluated with an appropriate trend test
- all results are inside the distribution of the historical negative control data.
A test substance was considered equivocal if the response was neither positive or negative even after further investigation.
Statistical methods were used as an aid in evaluating the test results. Both biological relevance and statistical analysis were considered in evaluation of the response. Biological relevance was evaluated by comparison of the test results with the test facility’s historical range of the solvent control.

Key result

Species / strain:

other: See Remarks

Remarks:

Human Peripheral Blood Lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Pulse treatment with and without metabolic activation

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: See Remarks

Remarks:

Human Peripheral Blood Lymphocytes

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

Continuous treatment

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Severe cytotoxicity occurred resulting in an insufficient number of concentrations suitable for microscopic evaluation so the acceptability criterium was not fulfilled.

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: To exclude any effect of the pH and the osmolality, the pH and osmolality were determined in RPMI culture medium exposed for 4 h to the test substance in modular incubator chambers. Only the top concentration of 76% (v/v%) MTDID 28136 (in a mixture of O2 and CO2) was used. The measured pH and osmolality values were comparable to the concurrent control
- Effects of osmolality: To exclude any effect of the pH and the osmolality, the pH and osmolality were determined in RPMI culture medium exposed for 4 h to the test substance in modular incubator chambers. Only the top concentration of 76% (v/v%) MTDID 28136 (in a mixture of O2 and CO2) was used. The measured pH and osmolality values were comparable to the concurrent control

Remarks on result:

not determinable

Conclusions:

No significant or dose-dependent increases in structural or numerical aberrations were observed in the pulse treatment group with or without metabolic activation. In the continuous treatment group without metabolic activation, MTDID 28136 induced severe cytotoxicity at the three highest concentrations resulting in an insufficient number of concentrations suitable for microscopic evaluation, so the acceptability criterium was not fulfilled. It cannot be determined if MTDID 28136 is clastogenic.

Executive summary:

The clastogenic potential of MTDID 28136 was evaluated in human peripheral blood lymphocytes.  The study was conducted according to OECD 473 in compliance with OECD GLP. The cells were exposed to the test article via a gas atmosphere generated in the chamber that consisted of 19% O2, 5% CO2 and 76% test material (v/v). This was the highest achievable airborne concentration of MTDID 28136. The chamber was supplemented with N2 to achieve the lower test material concentrations of 60%, 40%, 20%, and 10% (v/v) MTDID 28136. The study was separated into a pulse exposure group (with and without metabolic activation) and a continuous exposure group (without metabolic activation). The treatment/recovery time was 4/20 hours for the pulse group and 24/2 hours for the continuous group. In the pulse treatment group, no cytotoxicity was observed after 4 hour exposure at all concentrations with and without metabolic activation. No significant or dose-dependent increases in structural or numerical aberrations were observed in the pulse treatment group with or without metabolic activation. In the continuous treatment group without metabolic activation, MTDID 28136 induced severe cytotoxicity at the three highest concentrations resulting in an insufficient number of concentrations suitable for microscopic evaluation, so the acceptability criterium was not fulfilled. It cannot be determined if MTDID 28136 is clastogenic.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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:

2016

Deviations:

no

Remarks:

No deviations occurred that negatively impacted the integrity of the study.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M Company, Lot 16
- Expiration date of the lot/batch: 31 March, 2019
- Purity test date: 31 March, 2017


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature (15-25°C)
- Solubility and stability of the test substance in the solvent/vehicle: Since the test substance is a colorless gas , cells in culture flasks were exposed in modular incubator chambers (Billups-Rothenburg, USA) to various concentrations. The atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test substance supplemented with N2. The highest concentration theoretically achievable was therefore 76% (v/v). Additional concentrations of 60, 40, 20 and 10% (v/v) (all ± 10%) were used in the chambers to expose the cells. Air (19% O2, 5% CO2, 76% N2) without the test substance was used as negative control.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data

FORM AS APPLIED IN THE TEST: Gas

Target gene:

thymidine kinase (TK) locus on chromosome 11

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. J. Cole, MRC Cell Mutation Unit, University of Sussex, United Kingdom
- Suitability of cells: Recommended per OECD 490
- Cell cycle length, doubling time or proliferation index: 10-12.8 hours
- Modal number of chromosomes: 40
- Normal (negative control) cell cycle time: No data

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: L5178Y cells were grown in growth medium consisting of RPMI 1640 medium (with HEPES and L-Glutamine) supplemented with heat-inactivated horse serum (10% v/v for growing in flasks, and 20% for growing in microtiter plates), sodium pyruvate and penicillin/streptomycin. The cells were cultured in a humidified incubator at ca 37 °C in air containing ca 5% CO2.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
- Periodically 'cleansed' against high spontaneous background: No data

Metabolic activation:

with and without

Metabolic activation system:

The S9 liver homogenate

Test concentrations with justification for top dose:

Since the test substance is a colorless gas , cells in culture flasks were exposed in modular incubator chambers (Billups-Rothenburg, USA) to various concentrations. The atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test substance supplemented with N2. The highest concentration theoretically achievable was therefore 76% (v/v). Additional concentrations of 60, 40, 20 and 10% (v/v) (all ± 10%) were used in the chambers to expose the cells.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: None

Untreated negative controls:

yes

Remarks:

Air

Negative solvent / vehicle controls:

no

True negative controls:

yes

Remarks:

Air

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION:Since the test substance is a colorless gas , cells in culture flasks were exposed in modular incubator chambers (Billups-Rothenburg, USA) to various concentrations. The atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test substance supplemented with N2. The highest concentration theoretically achievable was therefore 76% (v/v). Additional concentrations of 60, 40, 20 and 10% (v/v) (all ± 10%) were used in the chambers to expose the cells.

The test atmosphere was generated using Mass Flow Controllers for O2/CO2/N2 and gaseous MTDID 28136. The MFC’s were flow-calibrated prior to the experiment to determine the settings leading to the target concentrations of each compound, based on a target total flow of the mixture of 2.5 liter per minute. To ensure the stability of the test compound mixture, the liquid test substance was extracted from the cylinder, and was allowed to evaporate before entering the MFC.

For the exposure, the MFC’s were used at the settings calibrated. The resulting gas mixture was lead to the container/incubator for 10 minutes. Assuming the mixing of the gasses inside the container/incubator is ideal, flushing the container during 10 minutes would lead to an end concentration of >99% of the target concentration (T99 = 4.6 * V / Flow, V (volume incubator)= 5,3 L, Flow = 2.5 L/min, hence T99 = 9.75 min).
Directly after flushing the container/incubator, a sample of the atmosphere inside the container was taken using the gas-tight syringe (Hamilton gastight 5 ml) and injected into a gas sample bag filled with 10 Ln of air. The concentration of the diluted test atmosphere in the sample bag was measured with the photoacoustic infrared analyzer. The response of the analyzer was recorded with a chart recorder.
After exposure (4 or 24 hours later) the atmosphere inside the container was measured again using the method described to ensure that the container/incubator was not leaking.

- Cell density at seeding (if applicable): 6,000,000 in 5 mL growth medium

DURATION
- Preincubation period: Five days prior to treatment, the cells were generated from a frozen stock culture from 6 November 2009 by seeding them in sterile, screw-capped tissue culture flasks (about 10,000,000 cells per flask: area ± 75 cm²) containing 50 ml culture medium (with 10% horse serum).
- Exposure duration: 4 hours or 24 hours
- Expression time (cells in growth medium): 5-6 days
- Selection time (if incubation with a selection agent): 10-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15-18 days

SELECTION AGENT (mutation assays): Trifluorothymidine (TMT)

NUMBER OF CELLS EVALUATED: At least four cultures were selected for gene mutation analysis. The frequency of TFT-resistant mutants was determined after ca. 48 hours after treatment. The cell suspensions were diluted to a density of ca. 10,000 cells per ml in growth medium (with 20% horse serum) containing 4 μg TFT per ml. Portions (200 μl) of each dilution were transferred to each well of two 96-wells microtiter plates. The plates were incubated for 10-12 days at ca. 37 C and ca. 5% CO2 in a humidified incubator.

DETERMINATION OF CYTOTOXICITY
- Method: Relative suspension growth and Relative total growth
- Any supplementary information relevant to cytotoxicity: The cytotoxicity of the test substance was determined by counting the cells after exposure and by measuring the relative suspension growth (RSG) and the relative total growth (RTG) of the cells.

Rationale for test conditions:

Per OECD 490 and the gaseous state of the test article.

Evaluation criteria:

The following criteria were used to validate the data obtained:
a) the average cloning efficiency of the negative controls should not be less than 65% or more than 120%;
b) the average suspension growth of the negative controls should be between 8 and 32 (4 hours treatments) and between 32 and 180 (24 hours treatment, if applicable);
c) the average mutant frequency of the negative controls should fall within the range of 50-170 TFT-resistant mutants per 1,000,000 clonable cells;
d) The mutant frequency of the positive controls should meet at least one of the two following acceptance criteria:
1) The positive control should demonstrate an absolute increase in total MF, that is, an increase above the spontaneous background MF [an induced MF (IMF)] of at least 300 X 10-6. At least 40% of the IMF should be reflected in the small colony MF.
2) The positive control has an increase in the small colony MF of at least 150 X 10-6 above that seen in the concurrent untreated/solvent control (a small colony IMF of 150 X 10-6).
The upper limit of cytotoxicity observed in the positive control culture should be the same as of the experimental cultures. The RTG value of one of the data points should be aimed to be between 10 and 20%. Providing that all acceptability criteria were fulfilled, a test substance was considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the Global Evaluation Factor. The test substance was then considered able to induce mutation in this test system.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

4 hour exposure

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

4 hour exposure

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Tested up to maximum attainable concentration (76%)

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: To exclude the possibility that this is a false-positive effect due to the test substance affecting pH and/or osmolality of the culture medium, the pH and osmolality were determined in cell culture medium exposed for 4 h to the test substance in modular incubator chambers. Only the top concentration of 76% (v/v%) MTDID 28136 (in a mixture of O2 and CO2) was used. The measured pH and osmolality values were comparable to the concurrent control
- Effects of osmolality: To exclude the possibility that this is a false-positive effect due to the test substance affecting pH and/or osmolality of the culture medium, the pH and osmolality were determined in cell culture medium exposed for 4 h to the test substance in modular incubator chambers. Only the top concentration of 76% (v/v%) MTDID 28136 (in a mixture of O2 and CO2) was used. The measured pH and osmolality values were comparable to the concurrent control
- Water solubility: 0.272 mg/L
- Precipitation: Slight flocculation was observed at the high doses.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Mutation frequency: 4 hour -S9: 556 +/- 134, 4 hour +S9: 818 +/- 219, 24 hour -S9: 1195 +/- 320
- Negative (solvent/vehicle) historical control data: 4 hour -S9: 73 +/- 20, 4 hour +S9: 67 +/- 15, 24 hour -S9: 59 +/- 14

Conclusions:

MTDID 28136 tested positive for mutagenicity in this assay in the absence of metabolic activation following a 4-hour exposure.

Executive summary:

The mammalian gene mutation potential of MTDID 28136 was evaluated in the Mouse lymphoma assay. The study was conducted according to OECD 490 in compliance with OECD GLP.  MTDID 28136 was exposed to the cells through a gas atmosphere in the chamber that consisted of 19% O2, 5% CO2 and 76% test material (v/v), the highest achievable airborne concentration of MTDID 28136. The chamber was supplemented with N2 to achieve the lower test material concentrations of 60%, 40%, 20%, and 10% (v/v) MTDID 28136. Exposure time with metabolic activation was 4 hours and times without metabolic activation were 4 hours and 24 hours. In the 4-hour exposure with metabolic activation, no increase in mutant frequencies were observed. Positive mutagenic responses were noted in the absence of metabolic activation in the 4 hour exposure with a dose-dependent response and no marked cytotoxicty. At 24 hours, the two highest concentrations of MTDID 28136 (60% and 76%) induced >90% cytotoxicity which invalidated this test group for mutagenicity evaluation. MTDID 28136 tested positive for mutagenicity in this assay in the absence of metabolic activation following a 4-hour exposure.

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

genetic toxicity in vivo, other

Remarks:

Combined in vivo micronucleus assay and Comet assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Remarks:

No deviations occurred that negatively impacted the integrity of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

no

Remarks:

No deviations occurred that negatively impacted the integrity of the study.

GLP compliance:

yes

Type of assay:

other: Combined in vivo Micronucleus and Comet Assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
3M Company, Lot 16
- Expiration date of the lot/batch:
March 2020
- Purity test date:
31 March, 2017


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Ambient temperature (15-25°C)
- Solubility and stability of the test substance in the solvent/vehicle:
Since the test substance is a colorless gas , cells in culture flasks were exposed in modular incubator chambers (Billups-Rothenburg, USA) to various concentrations. The atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test substance supplemented with N2. The highest concentration theoretically achievable was therefore 76% (v/v). Additional concentrations of 60, 40, 20 and 10% (v/v) (all ± 10%) were used in the chambers to expose the cells. Air (19% O2, 5% CO2, 76% N2) without the test substance was used as negative control.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
No data

FORM AS APPLIED IN THE TEST: Gas

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Crl:CD(SD) rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity testing by regulatory agencies.
The total number of animals used in this study was based on OECD Guidelines 474 and 489. Group size at the initiation of the study (up to 6/sex/group) was chosen to provide a minimum of 5 analyzable samples/sex/group for each endpoint. Because no difference in systemic toxicity was noted between males and females in Phase 1, only males were used for Phase 2.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at study initiation: 8 weeks old
- Weight at study initiation: 167-282 g
- Assigned to test groups randomly: Yes
- Fasting period before study: None
- Housing: On arrival, animals were group housed (2 to 3 animals of the same sex) until randomization. Following randomization, animals were group housed (2 animals of the same sex and same dosing group together) in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve. Animals were separated during designated procedures/activities. Each cage was clearly labeled with a cage card indicating study number, group number, cage number, dosage level/exposure concentration, animal number(s), and sex. Cages were arranged on the racks in group order.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet 5CR4 meal was provided ad libitum throughout the study, except during designated procedures including acclimation to nose-only restraint and during inhalation exposure periods. The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Water (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system, except during acclimation to nose-only restraint and inhalation exposure periods.
- Acclimation period: 4 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: 19 January, 2019 To: 21 May, 2019

Route of administration:

inhalation: gas

Vehicle:

Air

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposures were conducted using (7.9-L) stainless steel, nose-only systems with grommets in exposure ports to engage animal holding tubes for Phase 1 and 0.74-L 12-port module CH technologies flow-past (directed-flow) nose-only exposure system for Phase 2.
- Method of holding animals in test chamber: Animal holding tubes
- Source and rate of air: Air supplied to the nose-only systems 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 conditioning air:
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber: The mean temperature and mean relative humidity of the exposure atmospheres were 22 ± 3ºC and 50 ± 20%, respectively. Oxygen content of the exposure atmospheres was measured during the method development phase and was 20.9% for all groups for both phases of the study.
- 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
- Brief description of analytical method used: Analyzed concentrations of MTDID 28136 in the exposure atmospheres was determined using a gas chromatograph (GC) equipped with a Flame Ionization Detector (FID). Samples were collected from the approximate animal-breathing zone of the exposure system.
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

Phase I: 6 hours/day for 3 days
Phase II: 6 hours/day for 2 days

Frequency of treatment:

Daily

Post exposure period:

None

Dose / conc.:

0 ppm

Remarks:

Phase I Air control

Dose / conc.:

381 ppm (analytical)

Remarks:

Phase I

Dose / conc.:

753 ppm (analytical)

Remarks:

Phase I

Dose / conc.:

1 501 ppm (analytical)

Remarks:

Phase I

Dose / conc.:

0 ppm

Remarks:

Phase II Air Control

Dose / conc.:

375 ppm (analytical)

Remarks:

Phase II

Dose / conc.:

741 ppm (analytical)

Remarks:

Phase II

Dose / conc.:

1 494 ppm (analytical)

Remarks:

Phase II

No. of animals per sex per dose:

Phase I: 6/sex/dose
Phase II: 6 males/dose

Control animals:

yes, concurrent no treatment

Positive control(s):

ositive control groups of 20 mg/kg cyclophosposphamide (CP; Micronucleus assay positive control by oral gavage) and 200 mg/kg ethyl methanesulfonate (EMS; Comet assay positive control by oral gavage) were utilized for this study.
- Justification for choice of positive control(s): Per OECD guidelines
- Route of administration: Oral gavage
- Doses / concentrations: cyclophosphamide: 20 mg/kg, ehtyl methanesulfonate: 200 mg/kg

Tissues and cell types examined:

Micronucleus: Bone marrow
Comet: nasal cavity, lung, liver, and kidney tissues

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The doses were selected based on a previously conducted inhalation study showing effects in the nose at concentrations of 250 and 550 ppm.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): For Phase 1, filtered air (control) and test substance atmospheres were administered to Groups 1–4 as 6-hour, nose-only inhalation exposures once daily for 3 consecutive days. For Group 5, CP was administered via oral gavage on Days 1 and 2, and EMS was administered via oral gavage on Days 2 and 3.

For Phase 2, filtered air (control) and test substance atmospheres were administered to Groups 1–4 as 6-hour, nose-only inhalation exposures once daily for 2 consecutive days. For Group 5, EMS was administered via oral gavage on Days 1 and 2.

Tissues were collected 2-4 hours following sacrifice.

DETAILS OF SLIDE PREPARATION:

Micronucleus assay: Blood samples were collected from all animals at approximately 1–3 hours following the last exposure or dose. Blood (approximately 0.5 mL) was collected via the jugular vein into tubes containing K2EDTA and samples from 5 animals/sex/group. Of the 6 samples/sex/group available, 5 samples in LTSS were washed with ice cold 1% FBS solution and maintained on wet ice. The cells were then pelleted by centrifugation, and the supernatant was poured off leaving a small amount of supernatant with the pellet. The cells were re-suspended and 20 μL of suspension were added to 80 μL of staining solution containing RNase, FITC-conjugated anti-CD 71 antibodies and PE-conjugated anti-CD 61 antibodies. The samples were incubated at 2°C to 8°C for 30 minutes, re-suspended, then incubated at room temperature for an additional 30 minutes. DNA staining solution (propidium iodide; 0.3 to 2 mL) was added and then the samples were placed on wet ice for at least 5 minutes prior to the flow cytometric analysis.

Bone marrow was collected from the first 5 animals/sex/group at the time of euthanasia from the right femur of animals anesthetized by inhalation of isoflurane and euthanized by exsanguination. Five animals/sex/group in the negative control (Group 1) and test substance-treated groups were euthanized approximately 2–4 hours following the last exposure (Groups 2–4) or second dose of EMS (Group 5), and the nasal cavity was collected (Groups 1–4) or discarded without tissue collection (Group 5). Bone marrow was aspirated or flushed 2 to 3 times from the right femur 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 re-suspended 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.

Comet assay: Surviving animals were anesthetized on Day 3 (Phase 1) or on Day 2 (Phase 2), between 2 to 4 hours after the final exposure, by isoflurane inhalation followed by exsanguination (to complete euthanasia). Immediately following euthanasia, kidney, liver, lung and nasal cells
samples were collected for the Comet assay from 5 animals/sex/group. Sections of kidney, liver, lung and nasal tissue were placed in 3 mL chilled mincing solution (Hanks’ balanced salt solution with EDTA and DMSO), then minced with fine scissors to release the cells. The cell suspensions were strained into pre-labeled conical polypropylene tubes through a cell strainer and were kept on wet ice during preparation of the multi-well slides.

From each cell suspension, a 2.5 μL aliquot was mixed with 75 μL of low melting agarose. The cell/agarose suspension was applied to microscope multi-well slides. Commercially purchased (Trevigen®) pre-treated, multi-well slides were used, and these slides have 20 individual circular
areas, referred to as wells. The multi-well slides were kept at 2-8°C for at least 15 minutes to allow the gel to solidify. Multi-well slides were identified with a random code that reflects the study number, group, animal number, and organ/tissue. At least two 20-well slides were prepared
per animal per tissue. Three wells were used in scoring and the other wells were designated as a backup. Following solidification of agarose, the multi-well slides were placed in jars containing lysis solution. Following solidification of agarose, the multi-well slides were submerged in a cold solution composed of a commercially available lysis solution supplemented with 10% DMSO, on the day of use. The multi-well slides were kept in this solution at least overnight at 2-8°C. After cell lysis, slides/wells were washed with neutralization buffer (0.4 M tris hydroxymethyl
aminomethane in purified water, pH ~7.5) and placed in the electrophoresis chamber. The chamber reservoirs were slowly filled with alkaline buffer, composed of 300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water; the pH was >13. All multi-well slides remained in the buffer for 20 minutes at 2-10°C, protected from light, allowing DNA to unwind.

METHOD OF ANALYSIS:

Micronucleus assay: The frequency of micronucleated reticulocytes in peripheral blood was analyzed after flow cytometer calibration using Malaria infected biostandard and negative control standards provided in the Litron kit. Up to 20,000 RETs per animal, when possible, were analyzed.

Statistical analysis was performed on the micronucleus frequency (%MnRET) and %RET using the animal as the unit. The mean and standard deviation of %MnRET and %RET was presented for each treatment group.

Comet Assay: Electrophoresis was conducted for 30 minutes at 0.7 V/cm, at 2-12°C and protected from light. The electrophoresis time was constant for all multi-well slides. After completion of electrophoresis, the multi-well slides were removed from the electrophoresis
chamber and washed with neutralization buffer for at least 10 minutes. The multi-well slides (gels) were then dehydrated with 200-proof ethanol for at least 5 minutes, then air dried for at least 2 hours and stored at room temperature with desiccant. These multi-well slides were shipped
at ambient temperature to BioReliance by overnight shipment; upon receipt, the slides were logged in by the Test Site’s repository. Multi-well slides were stained with a DNA stain (i.e., Sybr-gold) prior to scoring. The stain solution was prepared by diluting 1 μL of Sybr-gold stain in 15 mL of 1xTBE (tris-boric acid EDTA buffer solution)

Scoring: Three wells per organ/animal were used. Fifty randomly selected, non-overlapping cells per slide/well were scored, resulting in a total of 150 cells (when possible) evaluated per animal for DNA damage, using the fully validated automated scoring system Comet Assay IV from
Perceptive Instruments Ltd. (UK).

The following endpoints of DNA damage were assessed and measured:

- Comet Tail Migration; defined as the distance from the perimeter of the Comet head to the last visible point in the tail.
- % Tail DNA; (also known as % tail intensity or % DNA in tail); defined as the percentage of DNA fragments present in the tail.
- Tail Moment (also known as Olive Tail moment); defined as the product of the amount of DNA in the tail and the tail length [(% Tail DNA x Tail Length)/100; Olive et al.1990)].

Each slide/well was also examined for indications of cytotoxicity. The rough estimate of the percentage of “clouds” was determined by scanning 150 cells per animal, when possible (percentage of “clouds” was calculated by adding the total number of clouds for all multi-well slides scored, dividing by the total number of cells scored and multiplying by 100). The “clouds”, also known as “hedgehogs”, are a morphological indication of highly damaged cells often associated with severe genotoxicity, necrosis or apoptosis. A “cloud” is produced when almost the entire cell DNA is in the tail of the comet and the head is reduced in size, almost nonexistent (Collins, 2004). “Clouds” with visible gaps between the nuclei and the comet tail were excluded from comet image analysis.

Evaluation criteria:

Micronucleus Assay: A target of 20,000 RETs/animal was analyzed for the presence of micronuclei (MnRETs). The proportion of reticulocytes to total number of cells scored (%RETs) was determined for each animal and treatment group. The %RETs served as a parameter of the test substance cytotoxicity in peripheral blood. A reduction in the RET proportions to less than 5% of the control value was considered excessively toxic and the animal data was excluded from
evaluation.
Negative Controls
The group mean frequency of MnRETs should ideally be within the 95% control limits of the distribution of the historical negative control database. If the concurrent Filtered Air control fall outside the 95% control limits, they may be acceptable as long as these data are not extreme outliers (indicative of experimental or human error).
Positive Controls
The frequency of MnRETs for the scoring controls must be significantly greater than the concurrent control (p ≤ 0.05).

Comet Assay: Negative controls: For each tissue analyzed, the DNA damage (% Tail DNA) in the negative control group was expected to be within the historical vehicle/negative control range for that tissue.
Positive Controls: The group mean for the % Tail DNA must be significantly greater than the concurrent negative control (p < 0.05), and the response should be comparable with those observed in the historical positive control data base.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were
conducted using two sided tests and are reported at the 1% and 5% levels.

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

Micronucleus assay

Toxicity:

yes

Remarks:

Test substance-related body weight losses were noted in the 753 and 1501 ppm group males and 381, 753, and 1501 ppm group females during the exposure period (Days 1-3).

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

Phase I Comet Assay

Toxicity:

yes

Remarks:

Test substance-related body weight losses were noted in the 753 and 1501 ppm group males and 381, 753, and 1501 ppm group females during the exposure period (Days 1-3).

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not valid

Remarks:

See Remarks on Result

Remarks on result:

other:

Remarks:

With respect to the Comet assay for Phase 1 of the study, MTDID 28136 was evaluated as negative (non-DNA damaging) in male liver cells only. For the remaining tissues tested, the assay did not meet all the acceptance criteria (there was no EMS-induced positive response in the nasal cavity, lung, or kidney); therefore, it was considered invalid for these tissues and the Comet assay was repeated in Phase 2 of this study.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Phase II Comet Assay with valid positive controls across all tissues evaluated.

Toxicity:

yes

Remarks:

A test substance-related body weight loss was noted in the 1494 ppm group during the exposure period (Days 1-2).

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

All valid assay criteria were met (EMS positive controls produced the expected response) for all of the tissues tested.

Conclusions:

Based on the results of this study, exposure of male and female rats to MTDID 28136 via nose-only inhalation for 6 hours/day for up to 3 days at target exposure concentrations of 375, 750, and 1500 ppm resulted in a negative response for induction of bone marrow micronuclei formation and a negative response for induction of DNA damage in the nasal cavity, lung, liver, and kidney tissues.

Executive summary:

The potential for MTDID 28136 to induce micronuclei or cause DNA damage in rat liver, lung, kidney and nasal tissue was evaluated in Sprague Dawley Rats. The study was conducted according to OECD 474 and 489 in compliance with OECD GLP.  Rats were administered filtered air (0 ppm control) or MTDID 28136 via nose-only inhalation exposures at target concentrations of 375, 750, and 1500 ppm. Positive control groups of 20 mg/kg cyclophosposphamide (CP; Micronucleus assay positive control by oral gavage) and 200 mg/kg ethyl methanesulfonate (EMS; Comet assay positive control by oral gavage) were utilized for this study. Due to failure of the EMS positive control, this study was performed in two phases. For Phase 1, MTDID 28136 or filtered air was administered to male and female rats (N= 6/sex/exposure concentration) once daily for 3 consecutive days, for 6 hours/day. The mean exposure concentration levels of MTDID 28136 for Phase 1 were 0, 381, 753, and 1501 ppm. Due to the lack of response in the EMS positive control group for the comet assay, a second phase was conducted for only the comet endpoint. For Phase 2, 6 males/group were exposed to 0, 375, 741, and 1494 ppm MTDID 28136 6 hours/day for 2 consecutive days. For the positive control, EMS was again administered via oral gavage at 200 mg/kg. Target tissues analyzed by the Comet assay included the nasal cavity, lung, liver, and kidney. In phase 1, all animals survived to the scheduled euthanasia. There were no test substance-related clinical observations noted at any exposure concentration. Test substance-related body weight losses were noted in the 753 and 1501 ppm group males and 381, 753, and 1501 ppm group females during the exposure period (Days 1-3). For phase 1, there were no significant increase in the number of micronuclei in the test substance-exposed rats compared to the filtered air controls in both males and females. The filtered air control values were compatible with the expected range of percent micronucleated reticulocytes (%MnRETs). There was a statistically significant increase in MnRETs in the CP positive control group as compared to the concurrent control group. All criteria for a valid assay were met. Under the conditions of this study, the administration of MTDID 28136 at exposure concentrations up to and including 1501 ppm was concluded to be negative in the micronucleus assay. With respect to the Comet assay for Phase 1 of the study, MTDID 28136 was evaluated as negative (non-DNA damaging) in male liver cells only. For the remaining tissues tested, the assay did not meet all the acceptance criteria (there was no EMS-induced positive response in the nasal cavity, lung, or kidney); therefore, it was considered invalid for these tissues and the Comet assay was repeated in Phase 2 of this study. For phase 2 of this study, it was determined that there was no sex difference in toxicity, and only male rats were used. Male rats were exposed to 0, 375, 741, and 1494 ppm MTDID 28136 for 6 hours/day for two days. All animals survived to the scheduled euthanasia. There were no clinical observations noted for males at any exposure concentration. A test substance-related body weight loss was noted in the 1494 ppm group during the exposure period (Days 1-2). Test substance-related lower food consumption was also noted for 375, 741, and 1494 ppm groups during the exposure period (Days 1-2); however, these differences did not occur in an exposure-related manner. For Phase 2, MTDID 28136 was evaluated as negative (non-DNA damaging) with the in vivo alkaline Comet Assay of the nasal tissue, lung, liver, and kidney tissues. All valid assay criteria were met (EMS positive controls produced the expected response) for all of the tissues tested. Based on the results of this study, exposure of male and female rats to MTDID 28136 via nose-only inhalation for 6 hours/day for up to 3 days at target exposure concentrations of 375, 750, and 1500 ppm resulted in a negative response for induction of bone marrow micronuclei formation and a negative response for induction of DNA damage in the nasal cavity, lung, liver, and kidney tissues.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The mutagenic potential of the test article was evaluated in the Bacterial Reverse Mutation Assay with S. typhimurium strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2 uvrA in the presence and absence of a metabolic activation system (S9-mix rat liver induced by Arclor 1254). The study was performed in compliance with OECD GLP. The test method was based on OECD No. 471 and ICH S2A and S2B. Plates were exposed to the test substance via incubation in a sealed bag containing the test article at the appropriate concentration. Strains TA100, TA1535 and WP2 uvrA were exposed to chloroethane as a positive control. A dose range-finding test was performed with concentration up to 30% v/v test article in the absence and presence of metabolic activation in strains TA100 and WP2uvrA. Based on the results of the dose range finding test, the test article was tested at concentrations of 0 (control) 0.0781, 0.156, 0.313, 0.625, 1.25, 2.50, 5.00 and 10.0% v/v test article in TA1537 and TA100 with metabolic activation and at 10 % v/v in TA100 and WP2 uvrA without metabolic activation and TA1535 with and without metabolic activation. A confirmatory assay was performed at 0.0781, 0.156, 0.313, 0.625, 1.25, 2.50, 5.00, 10.0, 20.0, and 40.0 % v/v in strains TA98, TA1537 and WP2 uvrA. All treatments were performed in triplicate. No increase in revertant colonies was observed in either the presence or absence of metabolic activation in any strain tested. All criteria for a valid test were met as described in the protocol. Based on the results of the test, the test article was not mutagenic in the Bacterial Reverse Mutation Assay in either the presence or absence of metabolic activation.

The clastogenic potential of MTDID 28136 was evaluated in human peripheral blood lymphocytes.  The study was conducted according to OECD 473 in compliance with OECD GLP. The cells were exposed to the test article via a gas atmosphere generated in the chamber that consisted of 19% O2, 5% CO2 and 76% test material (v/v). This was the highest achievable airborne concentration of MTDID 28136. The chamber was supplemented with N2 to achieve the lower test material concentrations of 60%, 40%, 20%, and 10% (v/v) MTDID 28136. The study was separated into a pulse exposure group (with and without metabolic activation) and a continuous exposure group (without metabolic activation). The treatment/recovery time was 4/20 hours for the pulse group and 24/2 hours for the continuous group. In the pulse treatment group, no cytotoxicity was observed after 4 hour exposure at all concentrations with and without metabolic activation. No significant or dose-dependent increases in structural or numerical aberrations were observed in the pulse treatment group with or without metabolic activation. In the continuous treatment group without metabolic activation, MTDID 28136 induced severe cytotoxicity at the three highest concentrations resulting in an insufficient number of concentrations suitable for microscopic evaluation, so the acceptability criterium was not fulfilled. It cannot be determined if MTDID 28136 is clastogenic.

The mammalian gene mutation potential of MTDID 28136 was evaluated in the Mouse lymphoma assay. The study was conducted according to OECD 490 in compliance with OECD GLP.  MTDID 28136 was exposed to the cells through a gas atmosphere in the chamber that consisted of 19% O2, 5% CO2 and 76% test material (v/v), the highest achievable airborne concentration of MTDID 28136. The chamber was supplemented with N2 to achieve the lower test material concentrations of 60%, 40%, 20%, and 10% (v/v) MTDID 28136. Exposure time with metabolic activation was 4 hours and times without metabolic activation were 4 hours and 24 hours. In the 4-hour exposure with metabolic activation, no increase in mutant frequencies were observed. Positive mutagenic responses were noted in the absence of metabolic activation in the 4 hour exposure with a dose-dependent response and no marked cytotoxicty. At 24 hours, the two highest concentrations of MTDID 28136 (60% and 76%) induced >90% cytotoxicity which invalidated this test group for mutagenicity evaluation. MTDID 28136 tested positive for mutagenicity in this assay in the absence of metabolic activation following a 4-hour exposure.

The potential for MTDID 28136 to induce micronuclei or cause DNA damage in rat liver, lung, kidney and nasal tissue was evaluated in Sprague Dawley Rats. The study was conducted according to OECD 474 and 489 in compliance with OECD GLP.  Rats were administered filtered air (0 ppm control) or MTDID 28136 via nose-only inhalation exposures at target concentrations of 375, 750, and 1500 ppm. Positive control groups of 20 mg/kg cyclophosposphamide (CP; Micronucleus assay positive control by oral gavage) and 200 mg/kg ethyl methanesulfonate (EMS; Comet assay positive control by oral gavage) were utilized for this study. Due to failure of the EMS positive control, this study was performed in two phases. For Phase 1, MTDID 28136 or filtered air was administered to male and female rats (N= 6/sex/exposure concentration) once daily for 3 consecutive days, for 6 hours/day. The mean exposure concentration levels of MTDID 28136 for Phase 1 were 0, 381, 753, and 1501 ppm. Due to the lack of response in the EMS positive control group for the comet assay, a second phase was conducted for only the comet endpoint. For Phase 2, 6 males/group were exposed to 0, 375, 741, and 1494 ppm MTDID 28136 6 hours/day for 2 consecutive days. For the positive control, EMS was again administered via oral gavage at 200 mg/kg. Target tissues analyzed by the Comet assay included the nasal cavity, lung, liver, and kidney. In phase 1, all animals survived to the scheduled euthanasia. There were no test substance-related clinical observations noted at any exposure concentration. Test substance-related body weight losses were noted in the 753 and 1501 ppm group males and 381, 753, and 1501 ppm group females during the exposure period (Days 1-3). For phase 1, there were no significant increase in the number of micronuclei in the test substance-exposed rats compared to the filtered air controls in both males and females. The filtered air control values were compatible with the expected range of percent micronucleated reticulocytes (%MnRETs). There was a statistically significant increase in MnRETs in the CP positive control group as compared to the concurrent control group. All criteria for a valid assay were met. Under the conditions of this study, the administration of MTDID 28136 at exposure concentrations up to and including 1501 ppm was concluded to be negative in the micronucleus assay. With respect to the Comet assay for Phase 1 of the study, MTDID 28136 was evaluated as negative (non-DNA damaging) in male liver cells only. For the remaining tissues tested, the assay did not meet all the acceptance criteria (there was no EMS-induced positive response in the nasal cavity, lung, or kidney); therefore, it was considered invalid for these tissues and the Comet assay was repeated in Phase 2 of this study. For phase 2 of this study, it was determined that there was no sex difference in toxicity, and only male rats were used. Male rats were exposed to 0, 375, 741, and 1494 ppm MTDID 28136 for 6 hours/day for two days. All animals survived to the scheduled euthanasia. There were no clinical observations noted for males at any exposure concentration. A test substance-related body weight loss was noted in the 1494 ppm group during the exposure period (Days 1-2). Test substance-related lower food consumption was also noted for 375, 741, and 1494 ppm groups during the exposure period (Days 1-2); however, these differences did not occur in an exposure-related manner. For Phase 2, MTDID 28136 was evaluated as negative (non-DNA damaging) with the in vivo alkaline Comet Assay of the nasal tissue, lung, liver, and kidney tissues. All valid assay criteria were met (EMS positive controls produced the expected response) for all of the tissues tested. Based on the results of this study, exposure of male and female rats to MTDID 28136 via nose-only inhalation for 6 hours/day for up to 3 days at target exposure concentrations of 375, 750, and 1500 ppm resulted in a negative response for induction of bone marrow micronuclei formation and a negative response for induction of DNA damage in the nasal cavity, lung, liver, and kidney tissues.

Justification for classification or non-classification

Based on the negative results from the in vivo OECD 474 and 489 studies, this substance is not classified for genetic toxicity according to GHS.