Dimethyl disulphide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2016 to August 2017. The study was performed at the request of an EU national competent authority in the frame of Regulation EC 1107/2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl disulphide

EC Number:

210-871-0

EC Name:

Dimethyl disulphide

Cas Number:

624-92-0

Molecular formula:

C2H6S2

IUPAC Name:

(methyldisulfanyl)methane

Test material form:

liquid: volatile

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 7-8 weeks old
- Weight at study initiation:
221 g to 263 g for males and from 155 g to 188 g for females in the Range-Finding Phase
211 g to 272 g for males and from 166 g to 208 g for females in the Definitive Phase
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: 2 to 3 per cage by sex in clean, solid bottom cages
- Diet (ad libitum): PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal)
- Water (ad libitum): reverse osmosis treated (on site) drinking water
- Acclimation period: at least 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5–22.8
- Humidity (%): 35.7-51.5
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: vapour

Vehicle:

Air

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 500-L or 1000-L glass and stainless steel whole-body exposure chambers
- Method of holding animals in test chamber: individually in standard exposure batteries
- Source and rate of air: in-house compressed air
- Method of conditioning air: HEPA charcoal-filtered, temperature- and humidity-controlled supply air source
- System of generating vapors: Vapors of the test substance were
generated using a bubbler-type vaporization system. A 300-mL or 500-mL gas washing bottle, (Ace Glass, Inc.; Vineland, NJ) was filled with an appropriate amount of liquid test substance. Compressed nitrogen was metered into the inlet stem of the gas washing bottle and bubbled through a fritted disc. Nitrogen was metered to the gas washing bottle using a Coilhose regulator (Model No. 8802K) and controlled using a needle valve and Gilmont rotameter-type flowmeter. Concentrated vapors of the test substance were delivered to the exposure chamber inlet and diluted to the desired atmosphere concentration by mixing with the chamber supply air prior to entering the chamber.
- Temperature, humidity, pressure in air chamber: The mean temperature and mean relative humidity were to be between 19°C to 25°C and 30% to 70%, respectively.
- Air flow rate:
Range-Finder Phase: 214-221 L/min
Definitive Phase: 111-120 L/min
- Air change rate: at least 12 air changes per hour.
- Method of particle size determination: Microdust Pro 880nm aerosol monitoring system. Aerosol content was 0.0 mg/m3 for all high dose groups.
- Treatment of exhaust air: activated-carbon drum prior to passing through the facility exhaust system which consists of redundant exhaust blowers preceded by activated-charcoal and HEPA-filter units

TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography/FIG
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

3 consecutive days

Post exposure period:

2 and 4 hours (DMDS exposed groups)
18–24 hours (positive control group)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

175 other: ppm (target)

Remarks:

173 ppm (analytical)

Doses / concentrations 2

Dose / conc.:

350 other: ppm (target)

Remarks:

351 ppm (analytical)

Doses / concentrations 3

Dose / conc.:

700 other: ppm (target)

Remarks:

679 ppm (analytical)

No. of animals per sex per dose:

6

Control animals:

yes, sham-exposed

Positive control(s):

Cyclophosphamide monohydrate
- Justification for choice of positive control(s): recommended by the guideline
- Route of administration: oral, on study Days 0 and 1 (first and second days of exposure)
- Doses / concentrations: 20 mg/kg/day

Examinations

Tissues and cell types examined:

Polychromatic erythrocytes [PCEs] of bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In order to assess the potential for exposure-limiting toxicity, the high target exposure concentration for the range-finding phase (750 ppm) was selected based on the following previous inhalation studies.
• Four-hour acute inhalation toxicity study in rats: LC50 was 1310 ppm. Mortality was 0/10, 4/10, 4/10, and 9/10 animals in the 847, 1188, 1308, and 1650 ppm groups respectively;
• Four-hour acute inhalation mammalian erythrocyte micronucleus assay in rats: no mortality up to 825 ppm. No significant reductions in the ratio of PCEs to total erythrocytes;
• Four-hour acute inhalation exposure in rats: increased serum glutamic-pyruvic-transaminase immediately after exposure to 600 ppm and increased gamma glutamyltransferase immediately after 500 ppm;
• Six-hour acute neurotoxicity study in rats: mortality was 0/24, 0/24 and 1/24 animals for the 100, 200, and 750 ppm group, respectively;
• Six-hour acute inhalation toxicity study in rats: no mortalities were observed up to 600 ppm. Acute inflammation and degeneration of the transitional and olfactory epithelia and acute inflammation of the respiratory epithelium were noted at 50, 150, 300, and 600 ppm, and degeneration of the respiratory epithelium was noted at concentration of 150 ppm and higher;
• Twenty-four-hour acute inhalation toxicity study: exposure-related degeneration of the olfactory epithelium at exposure levels of 9, 12.5, and 18 ppm and a slight increase in inflammation of the respiratory and olfactory epithelia at 18 ppm;
• Five-day subacute (6 hours/day) inhalation toxicity in rats:10 no mortalities were observed up to 600 ppm. Mean absolute lung and lung/body weights were higher in the 300 and 600 ppm group females. Hyperplasia of the squamous nasal epithelium was noted in = 300 ppm group males and all test substance-exposed group females (= 50 ppm). Hyperplasia of the transitional and respiratory epithelia and degeneration and regeneration of the olfactory epithelium were noted at all test substance exposure concentrations (= 50 ppm) in both sexes.

Range-finding study
In the range finding study, vaporized test substance (DMDS) was administered at 650 anf 750 ppm via whole-body inhalation exposure for 6 hours per day for 3 consecutive days to 2 groups (Groups 2–3) of Crl:CD(SD) rats. A concurrent control group (Group 1) received filtered air on a comparable regimen. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed prior to exposure and at 0–1 hours (+0.5 hour) following exposure. Detailed physical examinations were performed within 4 days of receipt, on the day of randomization, and on Study Days 0 and 2. Individual body weights and cage food weights were recorded within 4 days of receipt and on the day of randomization (body weights only), on the day the animals were placed into treatment groups (food weights only), and daily throughout the exposure period. The animal found dead had a gross necropsy performed and the carcass was discarded without tissue collection. Clinical pathology parameters (hematology and serum chemistry) were analyzed for all surviving animals within 3 hours following exposure on Study Day 2. Gross necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. Selected tissues were collected possible future histopathology. Bone marrow was collected for evaluation of cytotoxicity from all surviving animals at the scheduled euthanasia (Study Day 2; between 2 and 4 hours following the last exposure).

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed prior to exposure and at 0–1 hours (+0.5 hour) following exposure for Groups 1–4 and at the time of dosing and at 0–2 hours (+0.25 hour) following dose administration for Groups 5–6. Detailed physical examinations were performed within 4 days of receipt, on the day of randomization, and on Study Days 0 and 2. Individual body weights were recorded within 4 days of receipt (body weights only), on the day of randomization, and on Study Days 0 and 2. Individual food weights were recorded on the day of animal placement into groups and on Study Days 0 and 2. All animals were euthanized on Study Day 2. Bone marrow was collected from 5 animals/sex/group from Groups 1–5 at the scheduled necropsy (between 2–4 hours following the last exposure or 18–24 hours following the last dose administration).

Bone marrow was collected from the first 5 of 6 animals in each sex in Groups 1–5 at the time of euthanasia from the right femur of animals anesthetized by isoflurane inhalation and euthanized by exsanguination. Five animals/sex/group from the filtered air control group (Group 1) and test substance-exposed (Groups 2–4) groups were euthanized approximately 2–4 hours following the Study Day 2 exposure, and 5 animals/sex/group from the positive control group (Group 5) were euthanized approximately 18–24 hours following the Study Day 2 dose administration.

DETAILS OF SLIDE PREPARATION:
Prior to analysis, the coded slides were stained with acridine orange (A/O) staining solution.

METHOD OF ANALYSIS:
- Bone Marrow Cytotoxicity Analysis
A total of 500 erythrocytes (TE = PCE + NCE) per animal were counted, and the PCE:TE ratios were determined.
- Micronuclei Analysis
For each slide, 500 TE/animal were counted to determine PCE:TE ratios, and 4000 PCEs/animal were scored to determine %MN-PCEs.

Evaluation criteria:

Data will not be used either in the individual animal data or for statistical evaluation if obtained from study rat with either fewer than 500 PCEs when scoring the %MN-PCEs in immature erythrocytes or a PCE/TE ratio of less than 20% of the filtered air control value.
The filtered air control group mean must lie within the historical control range. The positive control response must be higher than the filtered air control group and be consistent with historical positive control data.

Criteria for Negative Response
Cases that do not clearly fit into the positive or negative criteria may be judged equivocal. In these cases, the Individual Scientist, based on sound scientific judgment, may take additional factors into consideration in evaluating the test results. As a general rule, the biological relevance of any result will be considered first.

Statistics:

For slides prepared in Range-Finding Phase, the ratio of PCEs to total erythrocytes for the test substance-exposed groups was compared to the filtered air control group using an ANOVA.12 If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett’s test13 was used to compare each test substance-exposed group to the filtered air control group. Statistical significance was assessed at a 95% confidence level (p=0.05).
For the slides prepared in Definitive Phase, the %MN-PCEs and PCE/TE for the filtered air control and test substance-exposed groups were compared using an ANOVA.12 If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett’s test13 was used to compare each test substance-exposed group to the filtered air control group. The Cochran Armitage test, was used for the detection of dose response trends in the test substance-exposed group only. In addition, a comparison of the positive (Group 5) and filtered air control groups was made using a separate ANOVA. Statistical significance was assessed at a 95% confidence level (p=0.05).

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

animals exposed up to the maximal tolerated concentration but no bone marrow cytotoxicity

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
One female was found dead at 750 ppm immediately following exposure on Study Day 0. There were no clinical observations noted for the female found dead. There were no other test substance related effects on survival. A single female in the 750 ppm group was observed with labored respiration on Study Day 2 prior to exposure. Yellow material on the urogenital area and clear material on the ventral neck were observed in the 650 ppm group males and females. In addition, a single 650 ppm group female was observed with vocalization during handling on Study Day 0. There were no other test substance related clinical observations. Test substance-related effects on body weight were noted in all test substance-exposed groups. Significantly lower mean body weights were noted on Study Day 2 in the 650 and 750 ppm group males and females, with only the 650 ppm group females not being statistically significant. Statistically significant lower body weights were also noted in the 750 ppm group males on Study Day 1. Statistically significant mean body weight losses or lower mean body weight gains were noted in the 650 and 750 ppm group males and females throughout the entire exposure period. Test substance-related lower mean food consumption was noted in the 650 and 750 ppm group males and females throughout the exposure period. Test substance-related effects on hematology was noted in all exposed groups. Higher mean RBC, HGB, HCT, and HDW and lower mean absolute eosinophil values (statistically significant in all male groups) were noted in all test substance-exposed group males and females. Statistically significant lower mean neutrophil (percent and absolute) values and higher mean percent lymphocyte values were noted in the 650 and 750 ppm group males. In addition, significantly higher mean platelet values were noted in the 650 ppm group males and 750 ppm group males and females (statistically significant in both male groups). Statistically significant lower mean percent reticulocyte values were noted in the 750 ppm group males and nonstatistically significant lower mean absolute reticulocyte values were noted in the 750 ppm group males. None of the effects on hematology were considered to be evidence of dose-limiting toxicity that would have impacted the dose selection for Definitive Phase. Test substance-related effects on serum chemistry were noted in all exposed group males and females. Test substance-related higher mean albumin, total protein, and globulin values and lower ALT values were noted in all test-substance exposed group males and females. Higher mean creatinine (statistically significant) and lower mean triglyceride values were noted in the 750 ppm group males. In addition, statistically significant higher mean cholesterol values were noted in the 750 ppm group males and females. Test substance-related lower mean phosphorus values were noted in all test substance-exposed group males and was attributed to possible dehydration. Statistically significant lower mean SDH was noted in the 650 ppm group males but was not attributed to the test substance as the change was not present in a dose-responsive manner. None of the effects on serum chemistry were considered to be evidence of dose limiting toxicity that would have impacted the dose selection for Definitive Phase. The test substance did not produce a statistically significant decrease in the PCE:TE ratios at 650 and 750 ppm compared to the filtered air control for male and female rats. Test substance-related lower mean absolute liver weights were noted in the 650 ppm group males and 750 ppm group males and females, higher mean kidney weight relative to body weight was noted in the 750 ppm group females and higher mean relative lung weights were noted in 650 and 750 ppm group males and females. The differences were attributed to the lower final body weights in the test substance-exposed groups.
Based on the achieved exposure concentrations, the concentration of 700 ppm was selected as the high-dose in Definitive Phase, as exceeding this concentration would likely result in dose-limiting toxicity (mortality and/or moribundity).

RESULTS OF DEFINITIVE STUDY
Dimethyl disulfide did not produce a statistically significant increase in the percent mean number of micronucleated polychromatic erythrocytes (%MN-PCEs) of bone marrow compared to the vehicle control for male and female rats. No bone marrow cytotoxicity was noted in any male and female rats at any dimethyl disulfide dose level. All animals survived to the scheduled euthanasia. There were no test substance related clinical observations. Statistically significant test substance-related body weight losses or lower mean body weight gains were noted in all test substance exposed group males and in the 700 ppm group females. Test substance-related lower mean food consumption values were noted in all test substance treated group males and females. The group mean values for both %MN PCEs and PCE:TE ratios for the vehicle and positive controls were comparable to the respective historical control ranges demonstrating the sensitivity of the assay.

Any other information on results incl. tables

Table1
Range-Finding Phase. Bone Marrow Data for Male Sprague Dawley Rats Administered Dimethyl Disulfide for3 Consecutive Days (6 hrs/day)

TREATMENT	ANIMAL No.	PCEs		NCEs	PCE:TE Ratio
Filtered Air (Negative Control)	1523	205		295	0.41
	1524	281		219	0.56
	1532	161		339	0.32
	1539	234		266	0.47
	1540	280		220	0.56
Mean ± SD					0.46 ± 0.10
Dimethyl Disulfide (650 ppm)	1519	201		299	0.40
	1529	284		216	0.57
	1530	248		252	0.50
	1535	254		246	0.51
	1543	176		324	0.35
Mean ± SD					0.47 ± 0.09
Dimethyl Disulfide (750 ppm)	1525	200		300	0.40
	1526	181		319	0.36
	1531	327		173	0.65
	1541	199		301	0.40
	1542	184		316	0.37
Mean ± SD					0.44 ± 0.12
			NCE = Normochromatic Erythrocyte
TE = Total erythrocytes (PCE + NCE)			PCE = Polychromatic Erythrocyte

 

Table2
Range-Finding Phase. Bone Marrow Data for Female Sprague Dawley Rats Administered Dimethyl Disulfide for 3 Consecutive Days (6 hrs/day)

TREATMENT	ANIMAL No.	PCEs		NCEs	PCE:TE Ratio
Filtered Air (Negative Control)	1554	375		125	0.75
	1556	206		294	0.41
	1561	242		258	0.48
	1564	263		237	0.53
	1569	251		249	0.50
Mean ± SD					0.53 ± 0.13
Dimethyl Disulfide (650 ppm)	1552	127		373	0.25
	1558	286		214	0.57
	1566	293		207	0.59
	1568	197		303	0.39
	1570	125		375	0.25
Mean ± SD					0.41 ± 0.16
Dimethyl Disulfide (750 ppm)	1547	315		185	0.63
	1555	268		232	0.54
	1560	168		332	0.34
	1563	230		270	0.46
Mean ± SD					0.49 ± 0.12
			NCE = Normochromatic Erythrocyte
TE = Total erythrocytes (PCE + NCE)			PCE = Polychromatic Erythrocyte

Table3
Definitive Phase. Micronucleus Assay Data for Male Sprague Dawley Rats Administered Dimethyl Disulfide for 3 Consecutive Days (6 hrs/day)^a

TREATMENT	ANIMAL No.	MN PCEs/ 4000 PCEs	% MN-PCEs		PCEs	NCEs	PCE:TE Ratio
Filtered Air (Negative Control)	1451	0	0.00		272	228	0.54
	1459	3	0.08		241	259	0.48
	1467	4	0.10		239	261	0.48
	1475	0	0.00		299	201	0.60
	1476	0	0.00		269	231	0.54
Mean ± SD			0.04 ± 0.05				0.53 ± 0.05
Dimethyl Disulfide (175 ppm)	1445	6	0.15		190	310	0.38
	1446	0	0.00		315	185	0.63
	1450	2	0.05		255	245	0.51
	1453	4	0.10		190	310	0.38
	1463	0	0.00		256	244	0.51
Mean ± SD			0.06 ± 0.07				0.48 ± 0.11
Dimethyl Disulfide (350 ppm)	1456	1	0.03		266	234	0.53
	1460	0	0.00		202	298	0.40
	1461	2	0.05		338	162	0.68
	1473	6	0.15		144	356	0.29
	1482	3	0.08		354	146	0.71
Mean ± SD			0.06 ± 0.06				0.52 ± 0.18
Dimethyl Disulfide (700 ppm)	1448	4	0.10		151	349	0.30
	1455	5	0.13		417	83	0.83
	1457	3	0.08		247	253	0.49
	1458	0	0.00		193	307	0.39
	1478	12	0.30		114	386	0.23
Mean ± SD			0.12 ± 0.11				0.45 ± 0.24
Cyclophosphamide (20 mg/kg/day)^a	1452	33	0.83		310	190	0.62
	1468	64	1.60		186	314	0.37
	1471	129	3.23		191	309	0.38
	1472	120	3.00		86	414	0.17
	1484	113	2.83		359	141	0.72
Mean ± SD			2.30 ± 1.04*				0.45 ± 0.22
MN = Micronucleated				NCE = Normochromatic Erythrocyte
TE = Total erythrocytes (PCE + NCE)				PCE = Polychromatic Erythrocyte
*Statistically different than negative controlp= 0.05.
^aExcept for cyclophosphamide treatment; dosed for 2 consecutive days harvested approximately 18 to 24 hours after the second dose was administered

Table4
Definitive Phase. Micronucleus Assay Data for Female Sprague Dawley Rats Administered Dimethyl Disulfide for 3 Consecutive Days (6 hrs/day)^a

TREATMENT	ANIMAL No.	MN PCEs/ 4000 PCEs	% MN-PCEs		PCEs	NCEs	PCE:TE Ratio
Filtered Air (Negative Control)	1488	13	0.33		207	293	0.41
	1489	11	0.28		267	233	0.53
	1493	3	0.08		132	368	0.26
	1498	15	0.38		304	196	0.61
	1525	7	0.18		275	225	0.55
Mean ± SD			0.25 ± 0.12				0.47 ± 0.14
Dimethyl Disulfide (175 ppm)	1499	8	0.20		220	280	0.44
	1501	4	0.10		300	200	0.60
	1504	6	0.15		138	362	0.28
	1510	8	0.20		296	204	0.59
	1513	12	0.30		333	167	0.67
Mean ± SD			0.19 ± 0.07				0.51 ± 0.16
Dimethyl Disulfide (350 ppm)	1487	8	0.20		127	373	0.25
	1496	4	0.10		243	257	0.49
	1503	8	0.20		116	384	0.23
	1507	2	0.05		281	219	0.56
	1509	2	0.05		220	280	0.44
Mean ± SD			0.12 ± 0.08				0.39 ± 0.15
Dimethyl Disulfide (700 ppm)	1490	12	0.30		322	178	0.64
	1494	2	0.05		222	278	0.44
	1500	1	0.03		302	198	0.60
	1508	4	0.10		189	311	0.38
	1516	9	0.23		185	315	0.37
Mean ± SD			0.14 ± 0.12				0.49 ± 0.13
Cyclophosphamide (20 mg/kg/day)^a	1486	99	2.48		109	391	0.22
	1505	38	0.95		133	367	0.27
	1514	76	1.90		86	414	0.17
	1517	91	2.28		197	303	0.39
	1518	41	1.03		134	366	0.27
Mean ± SD			1.73 ± 0.70*				0.26 ± 0.08*
MN = Micronucleated				NCE = Normochromatic Erythrocyte
TE = Total erythrocytes (PCE + NCE)				PCE = Polychromatic Erythrocyte
*Statistically different than vehicle controlp= 0.05.
^aExcept for cyclophosphamide treatment; dosed for 2 consecutive days harvested approximately 18 to 24 hours after the second dose was administered

 

Applicant's summary and conclusion

Conclusions:

Dimethyl disulfide met the criteria for a negative response for clastogenic activity and/or disruption of the mitotic apparatus under the conditions of this assay. In the absence of medullar toxicity, indirect evidence of the bone marrow exposure was provided by the systemic toxicity, including mortality and decrease of the body weight gain, observed in the range-finding and/or definitive studies and consistent with the available acute toxicity data (Kirkpatrick, 2005; Nemec, 2005; Kirkpatrick, 2008).

Executive summary:

The potential of vaporized dimethyl disulfide (DMDS, CAS Reg. no. 624-92-0) to induce micronuclei in polychromatic erythrocytes (PCEs) in rat bone marrow was assessed when administered via whole-body inhalation to Sprague Dawley rats for 6 hours per day for 3 consecutive days. The study was performed following the OECD Testing Guidelines 474 (29 July 2016).

In the range finding study, vaporized test substance (DMDS) was administered at target concentrations of 650 and 750 ppm via whole-body inhalation exposure for 6 hours per day for 3 consecutive days to 2groups (Groups 2–3) ofCrl:CD(SD) rats. A concurrent control group (Group 1) received filtered air on a comparable regimen. Afemale in the 750 ppm group was found dead immediately following exposure on Study Day 0. There were no clinical observations noted for the female found dead. There were no other test substance-related effects on survival. A single female in the 750 ppm group was observed with labored respiration on Study Day 2 prior to exposure. Yellow and clear material around the ventral neck and urogenital area were observed in the 650 ppm group males and females. In addition, a single 650 ppm group female was observed with vocalization during handling. There were no other test substance-related clinical observations. Test substance-related effects on body weight were noted in all test substance-exposed groups. Lower body weights were noted in the 650 and 750 ppm group males and females. Body weight losses or lower body weight gains were noted in the 650 ppm group males and females throughout the exposure period. These changes in body weight correlated with decreased food consumption in the test substance-exposed groups. Test substance-related effects on hematology was noted in all exposed group males and females. Higher red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), and hemoglobin distribution width (HDW) and lower eosinophil (absolute) values were noted in all test substance-exposed groups. Lower neutrophil (percent and absolute) values and higher percent lymphocyte values were noted in the 650 and 750 ppm group males. Based on the microscopic findings noted in the 175 ppm group, it is possible that the effects on the white blood cells could be secondary to an inflammatory response in the nasal cavity at these higher concentrations. In addition, higher platelet values were noted in the 650 ppm group males and 750 ppm group males and females. Lower percent and absolute reticulocyte values were noted in the 750 ppm group males, but were not of sufficient magnitude to be indicative of bone marrow depression. Test substance-related effects in serum chemistry were noted in all exposed group males and females. Test substance-related higher albumin, total protein, and globulin values and lower alanine aminotransferase (ALT) were noted in all test substance-exposed group males and females. Higher creatinine and lower triglyceride values were noted in the 750 ppm group males. In addition, higher cholesterol values were noted in the 750 ppm group males and females. Test substance-related lower phosphorus values were noted in all test substance-exposed group males and was attributed to possible dehydration. Lower sodium dehydrogenase (SDH) was noted in the 650 ppm group males but was not attributed to the test substance as the change was not present in a dose-responsive manner. None of the effects on serum chemistry were considered to be evidence of dose-limiting toxicity that would have impacted the dose selection for Definitive Phase B. Test substance-related lower kidney and liver weights were noted in the 650 ppm group males and 750 ppm group males and females and higher lung weights relative to body weight were noted in 650 and 750 ppm group males and females. The differences were attributed to the lower final body weights in the test substance-exposed groups. The test substance was negative for bone marrow cytotoxicity in both male and female rats at 650 and 750 ppm. The concentration of 700 ppm was considered to be the maximal tolerated concentration for the definitive study.

In the definitive study, vaporized test substance (DMDS) was administered at target concentrations of 175, 350 and 700 ppm via whole-body inhalation exposure for 6 hours per day for 3 consecutive days to 3 groups (Groups 2–4) of Crl:CD(SD) rats. A concurrent control group (Group 1) received filtered air on a comparable regimen. For Group 5, cyclophosphamide monohydrate (CP) was administered once daily on Study Days 0 and 1 (first and second days of exposure) orally by gavage. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed prior to exposure and at 0–1 hours (+0.5 hour) following exposure for Groups 1–4 and at the time of dosing and at 0–2 hours (+0.25 hour) following dose administration for Group 5. Detailed physical examinations were performed within 4 days of receipt, on the day of randomization, and on Study Days 0 and 2. Individual body weights were recorded within 4 days of receipt (body weights only), on the day of randomization, and on Study Days 0 and 2. Individual food weights were recorded on the day of animal placement into groups and on Study Days 0 and 2. All animals were euthanized on Study Day 2. Bone marrow was collected from 5 animals/sex/group from Groups 1–5 at the scheduled necropsy (between 2–4 hours following the last exposure or 18–24 hours following the last dose administration).All animals survived to the scheduled necropsy. There were no test substance-related clinical observations. Test substance-related lower mean body weight gains were noted in all test substance-exposed groups, which correlated with decreased food consumption in the DMDS-exposed groups. Dimethyl disulfide did not produce a statistically significant increase in the percent mean number of micronucleated polychromatic erythrocytes (%MN-PCEs) of bone marrow compared to the vehicle control for male and female rats.  In the absence of medullar toxicity, indirect evidence of the bone marrow exposure was provided by the systemic toxicity, including mortality and decrease of the body weight gain, observed in the range-finding and/or definitive studies and consistent with the available acute toxicity data (Kirkpatrick, 2005; Nemec, 2005; Kirkpatrick, 2008).

In conclusion, male and female Crl:CD(SD) rats were exposed to DMDS via whole-body inhalation for 6 hours per day for 3 consecutive days at exposure concentrations of 175, 350, and 700 ppm. Negative response for induction micronucleated polychromatic erythrocytes (%MN-PCEs) in bone marrow was obtained.