Butanal, 4-(heptyloxy)-3-methyl-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

The test substance, TM 09-217, was assessed according to internationally recognized guidelines in four in vitro tests and one in vivo test: • An in vitro gene mutation study in bacteria (Ames test) • An in vitro mammalian cytogenicity study • An in vitro micronucleus test • An in vitro gene mutation study in mammalian cells • An in vivo bone marrow chromosome aberration study in rats No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains tested with any dose of the test item, either with or without metabolic activation in the Ames test. In the chromosome aberration test, the test substance was assessed as positive without metabolic activation following 21 hours exposure. Under all other experimental conditions in this in vitro cytogenetic test system, TM 09-217 has shown no evidence of causing an increase in the frequency of structural chromosome aberrations. The test substance did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes in the absence of S9 mix in the in vitro micronucleus study. The test substance was positive for mutagenic potential with metabolic activation in an in vitro gene mutation study in mammalian cells. The test substance with a single intraperitoneal administration at doses up to and including a dose of 2000 mg/kg did not induce a significant increase in the frequency of cells with structural or numerical aberrations in the bone marrow of male ICR mice. Therefore, it was concluded to be negative in the mouse chromosome aberration assay.

Link to relevant study records

Reference

Endpoint:

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

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: GLP compliant guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Species:

mouse

Strain:

ICR

Sex:

male

Details on test animals or test system and environmental conditions:

Animal Receipt and Acclimation
Virus antibody-free (VAF) mice were obtained from a supplier that monitored mice for evidence of ectoparasites, endoparasites, pathogenic bacteria, mycoplasmas, and appropriate murine viruses and were acclimatized for 5 or 7 days after receipt. At BioReliance, mice were observed each day for signs of illness and other conditions of poor health. All mice were judged to be healthy prior to utilization in the study.

Animal Welfare Provisions and Animal Care
This study is not duplicative or unnecessary. The number of mice and the procedures and experimental design used for this study have been reviewed and were approved by the BioReliance Institutional Animal Care and Use Committee #8 and #9. All procedures involving mice performed at BioReliance follow the specifications recommended in The Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, D.C., 2011).
Animals were housed in an AAALAC-accredited facility with a controlled environment of 50 ± 20% relative humidity, 72 ± 3°F temperature and a 12 hour light/dark cycle. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air every hour.
Mice of the same sex were housed up to five per rodent Micro-Barrier cage. Cages were placed on the racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system. The purpose of this system was to supply uninterrupted positive air to each individual rodent Micro-Barrier cage and to capture the effluent air from each cage and re-filter the air (HEPA) prior to introducing the air back into the cage.
Heat-treated Sani-Chip hardwood chips were used for bedding to absorb liquids (P.J. Murphy Forest Products, Montville, NJ). Bedding was analyzed by the Manufacturer for any contaminants.
Animals were allowed free access to tap water, which meets U.S. EPA drinking water standards [water source is Washington Suburban Sanitary Commission (WSSC) Potomac Plant]. Drinking water was monitored at least annually for levels of specified microorganisms, pesticides, heavy metals, alkalinity and halogens.
A certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet) was provided ad libitum. The food was analyzed by the manufacturer for the concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates and specified nutrients.

Route of administration:

intraperitoneal

Vehicle:

Corn oil was used to deliver TM 09-217c to the test system based on the solubility of the test substance, compatibility with the test system and route of administration. Corn oil (CAS number: 8001-30-7, lot number: MKBD6671, expiration date: 24 September 2013) was obtained from Sigma-Aldrich. Corn oil is characterized as per the Certificate of Analysis on file with the Testing Facility.

Details on exposure:

The test substance dose formulations were prepared fresh for each phase of the study prior to dose administration. All formulations at concentrations, 3.125, 6.25, 12.5, 25, 35, 50, 62.5, 75, and 100 mg/ml in the DRF assay, and all formulations at concentrations 25, 50 and 100 mg/mL in the definitive study were prepared by combining an appropriate amount of test substance with an appropriate volume of the vehicle. Each formulation was vortexed for 1 minute and stirred for a total of 20 minutes using a stir bar/plate. All formulations appeared as yellow solutions and were stirred prior to and during dose administration.

All dose formulations were administered as single intraperitoneal injections (IP) at a dose volume of 20 mL/kg body weight. Intraperitoneal administration has been routinely used, and is widely-accepted route of administration for use in the mammalian bone marrow chromosome aberration assay.
In order to arrest the cells in metaphase, all main assay animals received a dose of 2 mg/kg of colchicine at ~ 3hours prior to bone marrow collection. Neat colchicine was diluted in Hank’s Balanced Salt solution (HBSS) at a concentration of 0.5 mg/mL and was intraperitoneally administered to the animals at a dose volume of 4 mL/kg body weight.
All mice in the experimental groups were weighed immediately prior to dose administration, and the administered volume was based on individual body weight.

Duration of treatment / exposure:

All dose formulations were administered as single intraperitoneal injections (IP) at a dose volume of 20 mL/kg body weight.

Frequency of treatment:

single IP injection

Post exposure period:

three days

Remarks:

Doses / Concentrations:
Dose range finding assay: 62.5, 125, 250, 500, 700, 1000, 1250, 1500, or 2000 mg/kg. Definitive study:500, 1000 or 2000 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

Dose range finding assay: 3 mice/sex/group
Definitive study: 5 mice/group (only males), additional 21 mice/group as satellite groups were assigned for the toxicolinetic portion of the study

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate (CP, CAS number: 6055-19-2, lot number: 120M1253V, expiration date: 31 December 2013), obtained from Sigma – Aldrich, were used as positive control substance.

An aqueous dosing formulation of CP, at a concentration of 2.5 mg/mL, was prepared fresh on the day of dose administration. An appropriate amount of CP was dissolved in an appropriate volume of sterile water for injection (B. Braun Medical, CAS number: 7732-18-5, lot number: J12003, expiration date: September 2013). The accuracy of the preparation and stability of the CP formulation was demonstrated by acceptable results that met the criteria for a valid test.

5 mice were used in the positive control group.

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

Animals were administered colchicine intraperitoneally at ~ 3hours prior to scheduled bone marrow collection. At 18 ±0.5 hr or 42±0.5 hr post dose administration, and following colchicine treatment, mice were sacrificed by carbon dioxide asphyxiation (verified by toe pinch reflex) in the same sequence used for dosing. The femurs were exposed, cut just above the knee and bone marrow was aspirated into a syringe containing a small volume of HBSS. The bone marrow cell suspensions were transferred into appropriately labeled tubes containing 3 to 5 mL of HBSS. The tubes with bone marrow were kept on wet ice until centrifugation. The cells were collected by centrifugation (100 x g; 5-10 minutes; refrigerated centrifuge set at 4°C), re-suspended in 5 mL of warm (37°±1°C) hypotonic solution of 0.065M KCl, and then incubated for 10±1 minutes in water bath at 37°C±1°C to swell the cells. Following this incubation period, a few drops of fixative (methanol: acetic acid, 3:1 v/v) was added and the cells collected by centrifugation (100 x g; 5-10 minutes; refrigerated centrifuge). Following centrifugation, the supernatant was discarded and cell pellets were re-suspended. Fresh fixative (5 mL) was added to each tube, the tubes were centrifuged, supernatant was discarded and cell pellets were re-suspended. Following another change of fixative, centrifugation and re-suspension of cells, fresh fixative was added and the tubes were capped and stored overnight at 2°C to 8°C. To prepare slides, the cells were collected by centrifugation (100 x g; 5-10 minutes; refrigerated centrifuge) and re-suspended to opalescence in fresh fixative. Two to four drops of fixed cells were dropped onto a wet slide and air-dried. Each slide was identified by the study number and animal number. Two slides were prepared from each animal, air dried, stained with Giemsa stain and permanently mounted.

Slides were coded using a random number table by an individual not involved with the scoring process. Metaphase spreads (cells) were examined using a light microscope and under oil immersion (1000X) without prior knowledge of treatment groups.
The Mitotic Index (MI) was determined for each animal, across all treatment groups, and at both collection time points. The MI was determined as the percentage of cells in mitosis based upon 1000 cells counted per animal. The mean MI was calculated for each treatment group (including positive and negative control groups) and served as a parameter of cytotoxicity and inhibition of cell division.
A minimum of 100 metaphase spreads containing 40 chromosomes, were examined from each animal and scored for chromatid-type and chromosome-type aberrations. Fewer cells (50 metaphase spreads) were scored per animal in the cyclophosphamide group when a higher percentage of aberrations, i.e. >10%, is observed during the scoring.
The following structural aberrations were recorded:
Chromatid-type aberrations: chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials and complex rearrangements.
Chromosome-type aberrations: chromosome breaks and exchange figures such as dicentric chromosomes and rings.
Fragments observed with an exchange figure were not scored as an aberration but not considered part of the incomplete exchange.
Pulverized chromosome(s), pulverized cells and severely damaged cells (≥ 10 aberrations) were also recorded.
Chromatid and isochromatid gaps were recorded but not included in the analysis.
The XY coordinates for each cell with structural aberrations were recorded using a calibrated microscope stage.
The percent polyploid and endoreduplicated cells were determined per 100 metaphase cells per animal.

Evaluation criteria:

All conclusions are based on the scientific judgment of generated data; however as a guide to the interpretation of the data, the following criteria are taken into consideration:
• The test substance is considered to induce a positive response if the percent of aberrant cells is significantly increased at one or more dose levels relative to the vehicle (negative) control at any sampling time (Fisher's exact test; p< 0.05).
• A dose-dependent increase is also taken into consideration in the determination of a test substance positive response.
• Values that are statistically significant but do not exceed the range of historical negative or vehicle controls (fall within or just outside the range) may be judged as not biologically significant or relevant, therefore, the test substance will be considered negative for the induction of chromosome aberrations in bone marrow cells.
• The test substance is judged negative if no statistically significant increase in the percent of aberrant cells is observed in the test substance groups relative to the concurrent negative (vehicle) control groups at any of the sampling times.

Statistics:

Statistical analysis of the percentage of aberrant cells was performed using the Fisher's exact test (p< 0.05). The Fisher's exact test is used for pairwise comparisons of the percentage of aberrant cells between each test substance group and negative control group. In the event of a positive Fisher's exact test, the Cochran-Armitage test is used to evaluate the significance of a dose-response.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Clinical Signs
No mortality was observed. All mice in the control substance groups and all mice in the 500 mg/kg treatment group appeared normal during the study period. Piloerection was observed in the 1000 and 2000 mg/kg treatment groups. In addition, lethargy was noted in the 2000 mg/kg treatment group

Bone Marrow Evaluation
Microscopic evaluation of bone marrow cells indicated the following:
Reductions in the Mitotic Index (MI) of 45%, 26%, and 21% were observed in the 500, 1000, and 2000 mg/kg treatment groups, respectively at 18 hour post-dose relative to the vehicle control group. A 53% reduction in the MI was noted in the 2000 mg/kg treatment group at 42 hours post-dose.
No statistically significant increases in the number of cells with structural aberrations compared to the vehicle control were noted in any of the test substance treatment groups (Fisher’s exact test; p> 0.05). In contrast, CP induced a statistically significant increase in the number of structurally -aberrant cells compared to the vehicle control (Fisher’s exact test; p< 0.05). No numerical aberrations were observed in any of the test substance groups or the control groups.

Dose Formulation Analysis
Aliquots of vehicle control and test substance formulations used in the dose administration were analyzed by BioReliance analytical chemistry department. The analyses were performed by gas chromatography (GC) using a method validated under BioReliance Study Number AD39TS.GCGTCHEM.BTL. The analysis results indicated the following:
No test substance was detected in the vehicle control sample.
All test substance formulations met the protocol specified acceptance criteria of 85.0% -115.0% of target concentrations and ≤5.0% relative standard deviation (%RSD).
Test substance formulations from the low and high concentrations were held at room temperature for 3.5 hours and re-analyzed to assess the formulation stability. The analysis results indicated formulations were stable at room temperature at least 3 hours.
Based on these results, all formulations were accurately prepared and were stable for the purpose of this study.

Plasma Analysis

Plasma samples collected from the vehicle control group animals at 18 hours post dose, and from the test substance treatment group animals at 15 minutes, 30 minutes, 1, 2, 4, 8, and 18 hours post-dose were analyzed by the Sponsor using a validated LC-MS/MS method [A162.DR (LC/MS/MS Analysis of Clossonate (RD 28636, CID 38047) in Mouse Plasma]. The analysis results are indicated as follows: Plasma sample from one of the vehicle control treated animals (animal number# 41) was free of interferences. Plasma concentrations of the test substance treated animals at different time intervals are summarized in the following table:

Group/Treatment	Average TM 09-217c Concentrations (ng/mL) Post Dose
	15 min	30 min	1 hr	2 hr	4 hr	8 hr	18 hr
vehicle control	NS	NS	NS	NS	NS	NS	*
500 mg/kg	9	11	12	19	11	1	0
1000 mg/kg	16	20	21	39	21	15	5
2000 mg/kg	40	39	49	51	48	18	23

NS = Not sampled; * data from one animal (animal #41) free of TM 09-217c.

Toxicokinetic Evaluation

Mean plasma concentrations (bio-analytical data) of TM 09-217c were used to determine toxicokinetic parameters using Microsoft Office Professional, Excel 2010. The mean toxicokinetic parameters such as Cmax (maximum plasma concentration), Tmax (time at which Cmax was observed). AUC0-thr (the area under the concentration vs time curve), estimated total systemic clearance (CLsys) were calculated. The mean toxicokinetic parameters are presented in the following Table:

Dose (mg/kg)	Cmax (ng/mL)	Tmax (hr)	AUC(0-t) (ng/mL·hr)	CL(sys) (mL/kg/min)
500	18.7	1.7	83.3	101197
1000	39.3	2.0	280.5	60389
2000	54	4.7	521.8	7 0666

Cmax (maximum plasma concentration); Tmax (time at which Cmax was observed)

AUC0-t (the area under the concentration vs time curve)

CL(sys) estimated total systemic clearance

The results indicate an apparent increase in Cmax values trended toward linearity with increases in the relative ratio of dose of 1:2.2:4 and values of 1:2.1:2.9. The systemic exposure was greater than predicted with relative ratio increases in AUC(0-18hr) of 1:3.4:6.3, which also followed the CLsys observed and the suggestion of greater clearance at the low dose relative to the mid and high dose groups.

Conclusions:

Interpretation of results (migrated information): negative
Under the described conditions of the study, a single intraperitoneal administration of TM 09-217c at doses up to and including a dose of 2000 mg/kg did not induce a significant increase in the frequency of cells with structural or numerical aberrations in the bone marrow of male ICR mice. Therefore, TM 09-217c was concluded to be negative in the mouse chromosome aberration assay.

Executive summary:

The test substance, TM 09-217c, was evaluated for its genotoxic potential as measured by its ability to induce structural and/or numerical chromosome aberrations in mouse bone marrow of male ICR mice, according to OECD test guideline 475. with a single intraperitoneal administration of TM 09-217c at doses up to and including a dose of 2000 mg/kg did not induce a significant increase in the frequency of cells with structural or numerical aberrations in the bone marrow of male ICR mice. Therefore, TM 09-217c was concluded to be negative in the mouse chromosome aberration assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Ames test: The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9 -mix. The dose levels were selected based on observed cytotoxicity in all strains (5000 ug/pl for TA98, TA100, TA1535 and E. coli WP2uvrA, and 1500 ug/pl for TA 1537). Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in any S. typhimurium tester strains (TA1535, TA1537, TA98, TA100) or the E. coli strain (WP2 uvrA), both in the absence and presence of S9-metabolic activation. These results were confirmed in independently pre-incubation experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

In vitro chromosome aberration test in human lymphocytes: The ability of test substance TM 09 -217 to induce chromosomal aberration in vitro was evaluated according with OECD 473 (1997) and according to GLP principles. The study comprised of two independent tests. In the first test, a three hour treatment was used in both the absence and presence of S9 mix. In the second test, a 21 hour continuous treatment was used in the absence of S9 mix, and a three hour treatment using an increased S9 concentration (5 % v/v) was used in the presence of S9 mix. Justification for concentration selection was based on cytotoxicity which was assessed by the mitotic index for all cultures treated with the test substance. In the second test, the test substance TM 09 -217 produced a statistically significant increase in the frequency of cells with structural chromosome aberrations in the absence of S9 mix following a 21 hr continues exposure at concentration of 60 ug/ml (the high concentration) only compared to the solvent control. Under all other experimental conditions in this in vitro cytogenetic test system, TM 09-217 did not produce a statistically significant increase in the frequency of cells with structural chromosome aberrations.

In vitro mutation test using mouse Lymphoma: The potential mutagenicity was evaluated according with OECD 476 (1997) and according to GLP principles. The study consisted of a preliminary toxicity test and two main tests comprising three independent mutagenicity assays. The cells were exposed for either 3 hr or 24 hr in the absence of S9 or 3 hr in the presence of S9. Relative suspension growth (RSG) and relative total growth (RTG) was used to determine the toxicity. The concentrations assessed for determination of mutation frequency in the main test span the complete toxicity range of approximately 10 to 100% RTG. Adequate negative and positive controls were included. This study concluded that at concentrations of 100 and 120 μg/mL the test substance demonstrated mutagenic potential following metabolic activation in this in vitro cell mutation assay. However the test substance did not demonstrate mutagenic potential in the absence of metabolic activation following 3 or 24 hour exposure in this in vitro cell mutation assay, under the experimental conditions described.

In vitro micronucleus test in human lymphocytes: The potential of TM 09-217 to cause an increase in the induction of micronuclei in cultured human lymphocytes in vitro was accessed according to OECD 487 (2010) and according to GLP principles. The study comprised two tests, a 3-hour treatment time in both the absence and the presence of S9 mix and a 20-hour treatment time in the absence of S9 mix only. Vehicle and positive control cultures were included in all test conditions. The highest test concentration selected for determination of induction of micronuclei was intended to be that causing 55 +/-5% cytotoxicity compared with the concurrent vehicle controls. The cytotoxicity was assessed by the cytokinesis-block proliferative index (CBPI). When the test substance was administered for 3 hours in both the absence and presence of S9 mix, and administered for 20 hours in the absence of S9 mix, there was no evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described.

Mouse bone marrow chromosome aberration assay: The genotoxic potential of the test substance TM 09 -217 was evaluated in moue bone marrow of male ICR mice by measuring its ability to induce structural and/or numerical chromosome aberrations according OECD 475 and GLP principles. The study was conducted in two phases: a dose range finding study, that evaluated the toxicity of the test substance, and a definitive study that evaluated the genotoxic potential of the test substance. In the absence of mortality and severe signs of toxicity in the range-finding study and no apparent differences between the sexes, only male animals were used in the definitive assay. The highest dose of 2000 mg/kg and two lower doses at 500 and 1000 mg/kg were tested. The vehicle and positive control substances were tested concurrently. Piloerection and lethargy were noted in the high dose group. Reductions in the Mitotic Index (MI) of 45%, 26%, and 21% were observed in the 500, 1000, and 2000 mg/kg treatment groups, respectively at 18 hour post-dose relative to the vehicle control. A 53% reduction in the MI was noted in the 2000 mg/kg treatment group at 42hours post-dose. Under the described conditions of this study, a single intraperitoneal administration of TM 09-217c at doses up to and including a dose of 2000 mg/kg did not induce a significant increase in the frequency of cells with structural or numerical aberrations in the bone marrow of male ICR mice (compared to the untreated control). Therefore, TM 09-217c was concluded to be negative in the mouse chromosome aberration assay.

Justification for selection of genetic toxicity endpoint
Four in vitro mutagenicity/genotoxicity studies and one in vivo genotoxicity study are available which together cover adequately the genotoxicity endpoint. All studies were conducted according to internationally recognised guidelines.

Justification for classification or non-classification

Based on the negative result in the bone marrow chromosome aberration in vivo genotoxicity tests, and weight of evidence on the in vitro genotoxicity battery tests, classification of the test substance TM 09 -217 for genotoxicity is not warranted in accordance with EU Directive 67/548 and EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.