Methyl isothiocyanate

Genetic toxicity: in vivo

Administrative data

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

Study period:

2011-04-11 - 2011-10-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ICH Guidelines S2A and S2B

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl isothiocyanate

EC Number:

209-132-5

EC Name:

Methyl isothiocyanate

Cas Number:

556-61-6

Molecular formula:

C2H3NS

IUPAC Name:

isothiocyanatomethane

Test material form:

solid: crystalline

Test animals

Species:

mouse

Strain:

other: Crl:CD1(ICR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc., United States
- Age at study initiation: approx. 9 weeks
- Weight at study initiation: 27.2 - 36.0g for males; 23.2 - 29.3g for females
- Assigned to test groups randomly: yes (computerized randomization procedure based on body weight stratification)
- Fasting period before study: no data
- Housing: Animals were housed in an animal colony room during non-exposure hours. Exposures were conducted in four 2-m^3 stainless steel and glass whole-body inhalation exposure chambers.
- Diet: ad libitum, but withheld during the exposure period
- Water: ad libitum
- Acclimation period: 28 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21.2 - 21.4°C
- Humidity: 39.6 - 47.4%
- Air changes: 10 fresh air changes per hour
- Photoperiod: 12-hours dark / 12-hours light

Administration / exposure

Route of administration:

inhalation

Vehicle:

- Vehicle(s)/solvent(s) used: filtered air
- Concentration of test material in vehicle: 20, 40, 100 ppm

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE
Vapour atmospheres of methyl isothiocyanate (MITC) were generated using a single vapor generator coupled with a distribution system that delivered a controlled amount of vapor to the inlet of each test substance exposure chamber. The generator consisted of a sealable 1000-mL reaction flask that contained liquid MITC and was held at a stable temperature of approximately 40-45°C. Heated nitrogen was metered to the reaction flask and, acting as a carrier gas, transported MITC vapor from the flask to the distribution system. The distribution system consisted of a bypass valve, an air dilution stage connected to a manifold, and compressed air-powered transvector jets that pulled controlled amounts of the vapour atmosphere from the manifold. The transvector jets directed the partially diluted vapour through heated delivery lines to the chamber inlets where chamber supply air was added
to produce the targeted exposure concentrations.

METHODS FOR CHARACTERIZATION OF EXPOSURE ATMOSPHERES
- Nominal Concentrations:
Although test substance usage was documented, it was not possible to determine nominal exposure concentrations. The use of a single vapor generator and a distribution system with excess vapor exhaust makes it inappropriate to calculate nominal concentrations.
- Analyzed Exposure Concentrations:
Analyzed exposure concentrations were determined at approximately 45-minute intervals using a gas chromatograph (GC) under the control of the WINH data acquisition software application. Samples were collected from the approximate animal breathing zone of the inhalation exposure chambers via heated stainless steel sampling lines. Test atmosphere samples were collected automatically using a heated external multi-position valve. Gas sample injection onto the chromatography column occurred via an internal gas-sampling valve with sample loop. For each sample, the chromatograph was displayed and the area under the sample peak was calculated and stored. The concentration in parts per million
(ppm) was calculated using an ln-quadratic equation based on the GC calibration curve.

Temporal Stability and Homogeneity of Vapour Atmospheres
During pre-study method development trials, the stability of the MITC vapour concentrations over time and the spatial homogeneity of the MITC vapour concentrations inside the exposure chambers were evaluated for each test substance chamber. Methods and results of these evaluations are not documented in this endpoint study record.

Duration of treatment / exposure:

6-hour whole body exposure

Frequency of treatment:

single dose

Post exposure period:

48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 20, 40, 100 ppm
Basis:
other: target exposure conc.

No. of animals per sex per dose:

12 males and 12 females per dose

Control animals:

yes

Positive control(s):

Cyclophosphamide monohydrate (CPS);
- Route of administration: oral (single dose)
- Doses / concentrations: The positive control group received a single oral dose of 60 mg/kg CPS on study day 0, the dose volume was 10 mL/kg. The positive control substance formulation was prepared at a concentration of 6 mg/mL on the day of dosing (study day 0) as a weight/volume (CPS/deionized water) mixture. The formulation was stirred until homogenous and was kept on ice after preparation and throughout dosing.

Examinations

Tissues and cell types examined:

Bone marrow collection for micronucleus evaluation was performed for 5 of 6 animals/sex/group at the scheduled euthanasia (study day 1 or 2). All animals were discarded without necropsy. Bone marrow smears were prepared and the coded slides were counted for polychromatic erythrocytes (PCEs), normochromatic erythrocytes (NCEs), and MN-PCEs following the final bone marrow sample collection on study day 2

A gross necropsy was conducted on the female found dead on study day 1. The necropsy included, but was not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. The carcass was discarded without tissue collection.

Details of tissue and slide preparation:

At the time of euthanasia at approximately 24 hours post-exposure (subgroup consisting of ≤ 6 animals/sex/group) and 48 hours post-exposure (subgroup consisting of remaining 6 animals/sex/group), bone marrow was collected from the first 5 of 6 animals in each sex/subgroup from the right and left femur of animals euthanized by inhalation of carbon dioxide. Bone marrow was aspirated or flushed 2 to 3 times from the right and left femurs into a centrifuge tube using a syringe containing heat inactivated fetal bovine serum (HI FBS). The bone marrow was centrifuged and all but approximately 0.25 mL (or a volume approximately twice that of the cell pellet) of HI FBS was decanted, and the pellet was resuspended in the remaining HI FBS. Bone marrow smears were prepared by placing approximately 1 drop of cell suspension onto a minimum of 2 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 at least 20 minutes, and allowed to air dry a second time. Thereafter the slides were stored refrigerated. Prior to analysis, the coded slides were stained with acridine orange (A/O) staining solution (Hayashi et al., 1983) and placed in numerical order using the animal numbers.
Two separate evaluations were made for each slide: 1) a total of 1000 erythrocytes (both polychromatic erythrocytes [PCEs] and normochromatic erythrocytes [NCEs]) per animal were counted and the PCE:total erythrocytes [TE] ratio was determined; and 2) the number of micronucleated PCEs (MN-PCEs) from a total of 2000 PCEs was scored per animal.

Evaluation criteria:

MICRONUCLEUS EVALUATION
At the time of euthanasia at approximately 24 hours post-exposure (subgroup of ≤ 6 animals/sex/group) and 48 hours post-exposure (subgroup of remaining 6 animals/sex/group). Bone marrow was aspirated into a centrifuge tube using a syringe containing heat inactivated fetal bovine serum (HI FBS). The bone marrow was centrifuged and all but approximately 0.25 mL (or a volume approximately twice that of the cell pellet) of HI FBS was decanted, and the pellet was resuspended in the remaining HI FBS. Bone marrow smears were prepared by
placing approximately 1 drop of cell suspension onto a minimum of 2 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 at least 20 minutes, and allowed to air dry a second time. The slides were stored refrigerated until shipped at ambient temperature to Midwest BioResearch, LLC, Skokie, IL.
Prior to analysis, the coded slides were stained with acridine orange (A/O) staining solution (Hayashi et al., 1983) and placed in numerical order using the animal numbers. Two separate counts were made for each slide: 1) a total of 1000 erythrocytes, (both polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) per animal were counted and PCE/total erythrocytes (TE) determined; and 2) number of micronucleated PCEs from a total of 2000 PCEs were scored per animal. Percentage of micronucleated PCE (% MN-PCEs) and PCE:TE ratio results were compared across groups for each harvest time-point for the test substance and filter air control treatments, and for the positive control and filtered air control treatments using analysis of variance (ANOVA) (Snedecor and Cochran, 1980) with statistical significance set at a 95% confidence level (p≤0.05).
In addition, the vehicle control values were within Midwest BioResearch, LLC, historical control reference range values.

Statistics:

STATISTICAL ANALYSES
Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Statistical analyses were not conducted if the number of animals was 2 or less. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly.

STATISTICS BY WIL RESEARCH
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the vehicle control group by sex. Body weight, body weight change, and food consumption data were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the vehicle control group.

STATISTICS BY MIDWEST BIORESEARCH (MICRONUCLEUS TEST)
The percentages of micronucleated cells in PCEs and in the ratio of PCEs to TEs for the test substance-treated and vehicle control group (Group 1) were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p≤0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the vehicle control group (Group 1). In addition, the positive control and vehicle control groups were compared using a separate parametric one-way ANOVA. Statistical significance was assessed at a a 95% confidence level (p≤0.05).

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

systemic toxicity in the highest concentration tested (100 ppm)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There were no test substance-related deaths or macroscopic findings noted at the time of necropsy for the 100 ppm group female found dead prior to the scheduled euthanasia.
Test substance-related clinical observations noted in the 20, 40, and/or 100 ppm group males and females included hypoactivity, extremities and/or body cool to the touch, intermittent tremors, labored respiration and/or decreased respiration rate, partial and/or complete closure of the eyes, clear discharge from the eyes, standing posture, clear material around the mouth, and yellow material around the urogenital area and/or on the ventral trunk.
Test substance-related body weight loss and lower food consumption was noted in the 20 ppm group females and 40 and 100 ppm group males and females.
Methyl isothiocyanate (MITC) did not produce an increase in the mean number of MN-PCEs compared to the vehicle control group. No bone marrow cytotoxicity (decreases in the PCE:total erythrocytes [TE] ratio) was noted in any test substance-treated group. Therefore, MITC met the criteria for a negative response for bone marrow cytotoxicity and clastogenicity under the conditions of this assay.

Any other information on results incl. tables

ANALYSES OF EXPOSURE CONCENTRATIONS

The overall mean analyzed concentrations are summarized in the following table:

Chamber Number	Mean Conc. (ppm)	Standard Deviation	Number of Samples
1	0	0.0	8
2	20	1.2	8
3	40	2.3	8
4	98	5.3	8

SURVIVAL

There were no test substance-related effects on survival. Female no. 1690 (100 ppm group) was found dead on study day 1; however, this death was related to physical trauma (the animal was accidentally dropped to the floor) and was not related to test substance exposure. All other animals survived to the scheduled euthanasia on study day 1 or 2.

CLINICAL OBSERVATIONS

Test substance-related clinical observations noted in the 20, 40, and/or 100 ppm group males and females included hypoactivity, extremities and/or body cool to the touch, intermittent tremors, labored respiration and/or decreased respiration rate, partial and/or complete closure of the eyes, clear discharge from the eyes, standing posture, clear material around the mouth, and yellow material around the urogenital area and/or on the ventral trunk. Details of these findings (incidence or presence by group) follow.

Test substance-related clinical observations of hypoactivity, intermittent tremors, extremities and/or body cool to the touch, labored respiration, and/or decreased respiration rate were noted in a majority of the 100 ppm group males and females, as well as a 40 ppm group female (no. 1698) following the 6-hour exposure period. Intermittent tremors and labored respiration persisted to study day 1 in one female (No. 1694, 100 ppm

group). In addition, clear material around the mouth accompanied a majority of the above-mentioned clinical observations in two 100 ppm group females (No. 1718 and 1735) following the 6-hour exposure period.

Test substance-related effects on the eyes were noted with observations of partial or complete closure of the eyes in all test substance-treated group males and females during and/or after the 6-hour exposure period. In general, increases in severity (partial to complete closure) and/or frequency of occurrences were noted in a time- and dose-dependent trend.

Test substance-related observations of standing posture correlated with clinical observations of partial or complete closure of the eyes. Partial closure of the eyes persisted beyond the end of the exposure period in the 100 ppm group males and females. Following the exposure period, clear discharge from the eyes was noted in a 100 ppm group male (No. 1604) and three 100 ppm group females (Nos. 1694, 1738 and 1744); however, with the exception of one female (No. 1694), there were no effects noted on the eyes of these animals during exposure.

Test substance-related clinical observations of yellow material around the urogenital area and on the ventral trunk were noted following the exposure period in the 20, 40, and 100 ppm group males and 100 ppm group females with higher incidence than the control group. These observations were also noted on study days 1 or 2 (urogenital area only) in the 100 ppm group males (urogenital area only) and females.

On the day(s) following exposure, clinical observations of yellow material around the urogenital area and/or on the ventral neck persisted in the 100 ppm group males (urogenital area only) and females.

All other clinical findings in the test substance-exposed groups were noted with similar incidence in the vehicle control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory mice of this age and strain.

BODY WEIGHTS

Test substance-related body weight loss was noted in the 20 ppm group females and 40 and 100 ppm group males and females.

Statistically significant mean body weight loss was noted from study day 0 to 1 in the 40 and 100 ppm group males and females when compared to the vehicle control group. A small mean body weight loss was also noted in the 20 ppm group females from study day 0 to 1 when compared to the vehicle control group. Although not statistically significant, the differences in body weight in the 20 ppm group females were considered test substance-related due to concurrent effects noted in food consumption in this group.

Mean cumulative body weight loss (often statistically significant when compared to the vehicle control group) was noted from study day 0 to 2 in the 40 and 100 ppm group males and females. On study day 1 (the 24-hour post-exposure scheduled euthanasia), the mean body weights were 5.2% and 9.2% lower for the 40 and 100 ppm group males, respectively, and 4.3, 7.1%, and 12.6% lower for the 20, 40, and 100 ppm group females,

respectively, when compared to the vehicle control group. On study day 2 (the 48-hour post-exposure scheduled euthanasia), the mean body weights were 9.7% and 15.3% lower for the 40 and 100 ppm group males, respectively, and 11.4% and 13.7% lower for the 40 and 100 ppm group females, respectively, when compared to the vehicle control group.

There were no other test substance-related effects on body weight.

FOOD CONSUMPTION

Test substance-related lower food consumption was noted for the 20, 40, and 100 ppm group females and 40 and 100 ppm group males.

Lower mean food consumption (often statistically significant when compared to the vehicle control group) was noted from study day 0 to 1 and 1 to 2 for females in all exposure groups (20, 40, and 100 ppm) and for males in the 40 and 100 ppm groups.

Only 1 valid food consumption value (male No. 1619) was able to be recorded for the 100 ppm group males from study day 0 to 1. All other food consumption values for 100 ppm group males during this interval were recorded as “NA” because the food jar weights were greater at the end of the interval compared to the initial weight which indicated that the food jar had gained weight (negative food consumption) or food jar contamination had occurred. However, due to a concurrent food consumption effect in females and a subsequent decrease in food consumption from study day 1 to 2 compared to study day 0 to 1 for males and females at 100 ppm, it is considered probable that food consumption was lower in the 100 ppm group males from study day 0 to 1.

There were no other test substance-related effects on food consumption.

ANATOMIC PATHOLOGY - MACROSCOPIC EXAMINATION

There were no test substance-related macroscopic findings noted in the gross necropsy of one female (No. 1690), examined on study day 1 following accidental death.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Based on the results of this study, administration of methyl isothiocyanate to Crl:CD1(ICR) mice via a single, 6-hour inhalation exposure period resulted in a negative response for induction of bone marrow micronuclei at any concentration tested, including the highest concentration tested (100 ppm) which caused systemic toxicity.

Executive summary:

The objective of this OECD 474 guideline study (conducted in compliance with GLP) was to assess the potential of methyl isothiocyanate to induce micronuclei in polychromatic erythrocytes (PCEs) in mouse bone marrow following treatment via inhalation.

The vehicle (filtered-air) and methyl isothiocyanate were administered via inhalation as a single 6-hour whole body exposure to male and female Crl:CD1(ICR) mice; target exposure concentrations were 20, 40, and 100 ppm. The positive control substance (cyclophosphamide) was administered orally v

ia gavage as a single dose. Bone marrow was harvested approximately 24 and 48 hours after the final dose.

Prior to micronuclei analysis, the blinded slides were stained with acridine orange solution.Two separate counts were made for each slide: 1) a total of 1000 erythrocytes, (both polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) per animal were counted and PCE/total erythrocytes (TE) determined; and 2) number of micronucleated PCEs from a total of 2000 PCEs were scored per animal. Percentage of micronucleated PCE (% MN-PCEs) and PCE:TE ratio results were compared across groups for each harvest time-point for the test substance and filter air control treatments, and for the positive control and filtered air control treatments using analysis of variance (ANOVA) (Snedecor and Cochran, 1980) with statistical significance set at a 95% confidence level (p≤0.05).

Methyl isothiocyanate did not produce a statistically significant increase in the % MN-PCEs compared to the vehicle control. No statistically significant bone marrow cytotoxicity (decreases in the PCE:TE ratio) was noted in any test substance treated group. The %MN-PCEs in the positive control groups were statistically significantly higher than in the vehicle control groups. The group mean values for both %MN-PCEs and PCE:TE ratios for the vehicle and positive controls were within the respective historical control ranges. Therefore, methyl isothiocyanate met the criteria for a negative response for bone marrow cytotoxicity and clastogenicity under the conditions of this assay.

Finally, administration of methyl isothiocyanate to Crl:CD1(ICR) mice via a single, 6-hour inhalation exposure period resulted in a negative response for induction of bone marrow micronuclei at any concentration tested, including the highest concentration tested (100 ppm) which caused systemic toxicity.