Dimethyl disulphide

Immunotoxicity

Description of key information

A GLP-compliant 90-day inhalation immunotoxicity study in rats is available on dimethyl disulphide (Collins, 1994). Lower lymphocyte counts were noted for the DMDS exposed groups. However, the finding was not dose responsive and not treatment-related as the only statistically significant difference was noted for the low exposed group. The lower lymphocyte counts appear to be a result of higher than historical control value for the control group and the method used for determining the total lymphocytes. Evaluation of the percentage of lymphocytes to leucocytes showed that all DMDS exposed groups were similar to control and the historical control from the animal supplier. Based on the evaluation above, it was concluded that DMDS is not immunotoxic.

 

Key value for chemical safety assessment

Effect on immunotoxicity: via oral route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on immunotoxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

immunotoxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

This study was conducted to determine the inhalation immunotoxicity of Dimethyl disulfide (DMDS) in 3 treated and one control groups of male rats following administration 6 hours/day, 5 days/week over a thirteen-week period and followed by a four-week recovery period . The animals used formed part of a general toxicity study reported in IUCLID section 7.5.3 (Collins, 1992). 

GLP compliance:

yes

Remarks:

for components of the study conducted at Hazelton UK and BIBRA.

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ORGANISMS:
- Source: Charles River UK Ltd., Margate
- Age at reception: 4-6 weeks
- Weight at reception: 120-140 g
- Weight at the start of the treatment: 185-256 g
- Acclimatation period: 14 days

HOUSING
The animals were housed in group of 5 in suspended stainless steel cages.

FOOD and WATER
- Food: SZQC rat and Mouse Maintenance Diet No. 1 ad libitum excepted  during exposure
- Water: filtered tap water, ad libitum excepted during exposure

ENVIRONMENTAL CONDITIONS
- Temperature : 19-25°C
- Relative humidity : 40-70%
- Light/dark cycle : 12h/12h
- Ventilation : 15 air changes/hour

Route of administration:

other: whole-body inhalation exposure to vapour

Vehicle:

unchanged (no vehicle)

Details on exposure:

Five horizontal flow, recirculating exposure chambers were used. Each was made of stainless steel with perspex (Plexiglas) doors and a fan to mix the atmospheres by recirculation. The compressed air supply was from a clean, dry, filtered source. The total volume of the animals did not exceed 5% of the volume of the test chamber. The four concentrations of test article vapour were produced by passing metered flows of air through sintered glass frits immersed in separate containers of test article. The resulting outputs of vapour were introduced to the diluent air inlet duct of each test chamber. Mixing, within the duct and recirculation system, ensured the production of homogeneous atmospheres for animal exposure. The chambers were ventilated at a rate of at least 12 air changes per hour. Air flows were monitored continuously and recorded twice hourly during exposure. The exhaust streams were purified with activated charcoal and vented to the outside of the building.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

* Measured concentration Samples for analysis were withdrawn from the exposure chambers twice hourly through sample lines leading from each chamber through a sampling valve into a total hydrocarbon analyser. The analysis was performed with an Analysis Automation total hydrocarbon analyser type 523 Detector with a Flame ionisation detector (FID)
* Nominal concentration The total weight of test article used and total volume of diluent air were measured for each exposure.
Target / Nominal / Analytical concentrations:
0
10 / 20 / 10.17 ppm
50 / 81 / 50.25 ppm
250 / 373 / 246.59 ppm

A simslin II dust monitor was used pre-dose, and during the study at week 1, 4, 8, and 12, at each exposuire levels to confirm all the test article was in a vapour phase.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
10, 50 and 250 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

20

Control animals:

yes, sham-exposed

Details on study design:

- Post-exposure recovery period in satellite groups: yes, 4 weeks

Observations and clinical examinations performed and frequency:

The animal observations (mortality, clinical condition, body weight, food consumption, et c... made in connection with the general toxicity study are presented in section 7.5.3. (Collins, 1992). The procedures associated with the inmunotoxicity study are described below.

HAEMATOLOGY
Blood samples were obtained for the haematological studies from the ten male main study animals in 0, 10, 50 and 250 ppm groups at week 12 and the corresponding recovery males at week 17 for the following examinations:
. total white cells
. total lymphocytes
. pan B-cells
. pan T-cells
. T helper cells
. T suppressor cells

Sacrifice and pathology:

MYELOGRAMS
A femoral bone marrow smear was taken from all animals at necropsy and examined by a haematologist and full myelograms performed.

ORGAN WEIGHTS:
Popliteal, submandibular and mesenteric lymph nodes, thymus.

GROSS PATHOLOGY: Yes
Reported in section 7.5.3 (Collins, 1992)

HISTOPATHOLOGY: Yes
Lymph nodes (popliteal, mesenteric and submandibular), mid colon lymphoid tissue, spleen, thymus.

Humoral immunity examinations:

SERUM IMUNOGLOBULIN STUDIES
Whole blood samples were obtained from the aforementioned main study animals at necropsyl for the determination of IgG and IgM titres.

Positive control:

None

Statistics:

- ANOVA, Regression and Dunnett's: Immunoglobins (IgG, IgM), Mylograms, Necropsy body weight (terminal kill), Organ Weights (relative and absolute) terminal kill - Popliteal Lymph Node, Mandibular Lymph Node, Mesenteric Lymph Node, Thymus, Adrenals, Spleen, Brain (absolute only).
- Kruskal Wallis, Terpstra-Jonckheere, Wilcoxon Rank Sum Test : Organ weights (relative and absolute) - terminal kill - Brain (relative only).
- Unpaired t test (two-tailed) and Mann-Whitner U test: lymphocytes and lymphocytes sub-populations.
- Histophatology: the statistical analysis was achieved according to the test-t In order to compare the means values betwen the control group and the treated groups and the regression coefficient analysis between the dose and the different measured values.

Details on results:

HAEMATOLOGY
The treated groups showed lymphocyte counts at week 12 that were reduced compared with the control group (Table 1). The reduction was greatest in the low dose group where the reduction was approximately 20% and did achieve statistical significance. The reductions in absolute lymphocyte counts were primarily attributable to a specific reduction in T-suppressor cells of 20 to 40% and were inversely proportional to dose level; the differences compared with the concurrent control group were statistically significant for the low and intermediate dose group but not for the high dose group.

The reduction in T-suppressor cells persisted to the end of the recovery period at week 17 (Table 2) and statistically significant differences in absolute cell counts compared with the concurrent control group were present in all treated groups. There were no other statistically significant differences.

There was no evidence of any treatment-related changes in T-helper cells or B-cells at either time point.

SERUM IMMUNOGLOBULIN ANALYSIS
There were no statistically significant differences from the control group and no evidence of any treatment-related differences in serum concentrations of IgG or IgM in the samples taken at the terminal kill at week 14.

Groups n Concentration of Protein mg/L
IgG IgM
Control 10 6646 ± 1185 1229 ± 175
10 ppm 9 6664 ± 1550 1223 ± 116
50 ppm 10 6167 ± 1825 1242 ± 238
250 ppm 10 6678 ± 1687 1150 ± 124

MYELOGRAMS
The only treatment-related change in the myelograms taken at necropsy was a trend towards decreased lymphocytes in the treated groups. However, the routine examination of bone marrow conducted as part of the sub-chronique toxicity study (Collins, 1992, section 7.5.3) had not revealed any unusual abnormalities.

ORGAN WEIGHTS
There were no organ weight differences except for minor changes attributable to non-specific stress or reduced body weights in the high dose group.

IMNUNOHISTOPATHOLOGY
QUALITATIVE PATHOLOGY
Two cases of thymic atrophy were observed: one minimal in a high dose animal, the other moderate in a control animal.
Most of the mandibular lymph nodes exhibited inflammatory reaction revealed by various degrees of sinus histiocytosis (SH) and/or medullary plasmacytosis (MP). Hemosiderosis, generally minimal, was present in some of them. The distribution of inflammatory reactions was as follows:
Group...............Minimal.............Moderate..........Marked.............Very marked or severe
........................SH..MP..............SH..MP...............SH..MP..............SH..MP
control..............2.....3................7.....2.................0....0.................0....0
10 ppm.............1.....0................6.....2.................3....2.................0....0
50 ppm.............0.....0..............10.....7.................0....2.................0....0
250 ppm...........2.... 0................4.....1.................3....3.................0....1

Inflammatory reactions seemed to be more frequent and more severe in the treated groups compared with the control group.
This inflammation may result from nasal injury associated with inhalation of the test article.
In addition, in one high dose animal, a cortical and paracortical atrophy of the mandibular lymph made was observed.
Inflanmatory lesions (mainly sinus histiocytosis) were also observed in the mesenteric lymph mode and had the following distribution in the various groups:
Group........... Minimal...........Moderate...........Marked.........Very marked or severe
control..............1.......................9........................0........................0
10 ppm.............0.......................5........................5........................0
50 ppm.............0.......................e........................2........................0
250 ppm...........6.................. ....4.......................0.........................0

There was no evidence of a treatment-related effect.

Inflammatory reaction was also observed in the popliteal lymph nodes especially sinus histiocytosis and less frequently medullary plasmacytosis. Sinus histiocytosis had the following distribution in the various groups:

Group............Minimal............Moderate...........Marked..........Very marked or severe
control..............2........................8.......................0........................0
10 ppm.............0........................6.......................4........................0
50 ppm.............0........................8.......................2........................0
250 ppm...........4........................6.......................0........................0

There was no evidence of a treatment-related change. Hemasiderosis of both red and white splenic pulp was a frequent finding but is commonly observed in rodent spleen. Various levels of atrophy of the white pulp were observed in some animals. Lymphoid atrophy could be best characterized in the peri-arterial lymphoid sheath and had the following distribution in the various groups:

Group............Minimal...........Moderate............Marked..........Very marked or severe
control..............1.......................1........................0........................0
10 ppm.............0.......................4........................0........................0
50 ppm.............0.......................2........................0........................0
250 ppm...........0.......................8........................0........................0

The distribution suggested a treatment-related change.

Mid-colon
The gut associated lymphoid tissue was missing in must of the samples.
Modification of gut associated lymphoid tissue was not observed by one of the pathologists. The other observed moderate hypertrophy of gut associated lymphoid tissue in the low dose group (1/4), the intermediate group (3/5) and the high dose group (1/5).
These results were considered to be insignificant.

HISTOMETRY OF SPLEEN AND STATISTICAL ANALYSIS
The histometric evaluation of total surface of spleen cross section. white pulp surface, periarteriolar lymphoid sheath surface, and marginal zone surface was performed together with the statistical analysis. Pairwise statistical comparison of the control group and treated group showed:
- no significant difference between control group and the 10 ppm group.
- significant decrease of the spleen total cross section surface in the 50 ppm group without significant peri-arterial lymphoid sheath, marginal zone, red pulp.
- significant decrease of the spleen total cross section surface. white pulp. peri-arterial lymphoid sheath and marginal zone surfaces in the 150 ppm group, without significant difference for red pulp surface.
Regression analysis compared the site of the various measured components between the different groups. These results confirm the observations obtained by microscopic examination of the spleen sections and confirms that a significant atrophy of the lymphoid structure of the spleen is present in 250 ppm group.
For the white pulp, the periarteriolar lymphoid sheaths and the marginal zone, the slope value of the regression line is significantly negative demonstrating a reduction in the various lymphoid compartments of the spleen in the treated groups, with a dose-effect relationship.
The slope value of the regression line is almost zero for the red pulp (not statistical effect of treatment) and non-significant for the whole surface

Dose descriptor:

NOAEC

Effect level:

250 ppm (analytical)

Sex:

male/female

Basis for effect level:

other: No dose responsive and no treatment-related effects on the immunologic parameters

Table 1 : Absolute white blood cell (WBC) counts, % of lymphocytes, absolute lymphocytes counts, percentage of CD4+ T-helper, CD8+ T-suppressor and SIg-kappa+ B-cells and absolute numbers of T-cells, CD4+ T-helper cells, CD8+ T-suppressor cells and SIg-kappa+ B-cells at week 12.

Mean ± sd	Control	10 ppm	50 ppm	250 ppm
n	10	10	10	10
Total WBC count (10^9/L)	13.4 ± 30.	10.9 ± 1.9	13.1 ± 3.6	12.4 ±2.1
% lymphocytes	86.1 ± 3.3	82.2 ±8.4	83.9 ± 10.2	84.8 ± 4.02
Absolute lymphocyte count (10e9/L)	11.5 ± 2.7	9.1 ± 2.2*	11.0 ± 3.3	10.5 ± 1.8
% CD4+	46.1 ±3.5	49.9 ± 4.1*	48.9 ± 3.8	45.0 ± 3.9
% CD8+	30.1 ± 5.8	23.4 ± 3.3*	23.2 ± 4.8*	27.4 ± 4.3
%Sig-kappa+	19.4 ± 2.9	24.2 ± 4.0**	23.1 ± 4.0*	19.9 ± 3.6
Absolute T-cell count (10^9/L)	8.82 ± 2.16	6.63 ± 1.66*	7.89 ± 2.28	7.60 ± 1.42
Absolute T-helper count (10^9/L)	5.31 ± 1.30	4.52 ± 1.19	5.41 ± 1.82	4.77 ± 1.12
Absolute T-suppressor count (10^9/L)	3.51 ± 1.11	2.11 ± 0.53*	2.48 ± 0.67*	2.83 ± 0.40
Absolute B-cell count (10^9/L)	2.25 ± 0.65	2.19 ± 0.66	2.52 ± 0.84	2.10 ± 0.58

* p< 0.05

** p < 0.01

Table 2 : Absolute white blood cell (WBC) counts, % of lymphocytes, absolute lymphocytes counts, percentage of CD4+ T-helper, CD8+ T-suppressor and SIg-kappa+ B-cells and absolute numbers of T-cells, CD4+ T-helper cells, CD8+ T-suppressor cells and SIg-kappa+ B-cells at week 17.

Mean ± sd	Control	10 ppm	50 ppm	250 ppm
n	10	10	10	10
Total WBC count (10^9/L)	9.6 ± 2.6	7.4 ± 1.6*	7.8 ± 2.4	9.2 ± 1.8
% lymphocytes	83.9 ± 6.9	83.1 ± 12.3	85.1 ± 2.4	81.3 ± 5.3
Absolute lymphocyte count (10^9/L)	7.7 ± 2.0	6.2 ± 1.7	6.6 ± 2.1	7.4 ± 1.4
% CD4+	42.6 ± 4.5	46.9 ± 6.2	46.3 ± 5.1*	51.1 ± 5.0***
% CD8+	21.3 ± 5.2	18.5 ± 6.1	20.4 ± 5.8	16.4 ± 4.2*
%Sig-kappa+	22.4 ± 3.3	25.9 ± 6.4	25.9 ± 4.0	24.1 ±3.8
Absolute T-cell count (10^9/L)	4.89 ± 1.33	4.07 ± 1.28	4.37 ± 1.35	4.98 ± 0.88
Absolute T-helper count (10^9/L)	3.32 ± 1.21	2.96 ± 1.10	3.07 ± 1.05	3.81 ± 0.87
Absolute T-suppressor count (10^9/L)	1.57 ± 0.31	1.11 ± 0.37*	1.30 ± 0.47*	1.18 ± 0.22**
Absolute B-cell count (10^9/L)	1.71 ± 0.43	1.59 ± 060	1.74 ± 0.69	1.78 ± 0.37

* p< 0.05

** p < 0.01

*** p < 0.001

Conclusions:

The immunologic phase of the study suggests that lower lymphocyte counts were noted for the DMDS exposed groups. However, the finding was not dose responsive and not treatment-related as the only statistically significant difference was noted for the low exposed group. The lower lymphocytes counts appear to be result of higher than historical control value for the control group and the method used for determining the total lymphocytes. Evaluations of the percentage of lymphocytes to leukocytes showed that all DMDS exposed groups were similar to control. Based on the evaluation above, it can be concluded that DMDS is not immunotoxic.

Executive summary:

The inhalation immunotoxicity of dimethyl disulfide (DMDS) was assessed in the male Sprague-Dawley rat following exposure, 6 hours/day, 5 days/week over a thirteen week period to vapor concentrations of 0, 10, 50 or 250 ppm. The animals used formed part of a subchronic toxicity study reported separately (see section 7.5.3, Collins, 1992). Blood samples were obtained at week 12 and corresponding recovery animals at week 17. The following parameters were evaluated for these samples: total white cells, total lymphocytes, pan B-tells, pan T-cells. T helper cells and T suppressor cells. In addition, IgG and IgM titres were determined in serum from these samples. Organ weights were determined for the popliteal lymph node, submandibular lymph node, mesenteric lymph node and thymus prior to fixation. Histologic evaluations were performed on the popliteal lymph node, suhmandibular lymph node, mesenteric lymph node, mid colon lymphoid tissue, spleen and thymus following staining with hematoxylin-eosin saffron, with slow Giemsa and with immunocytochemical staining.

 

B and T cell analysis did not show any evidence of treatment-related changes in T-helper or B-tells. Reductions in absolute lymphocyte counts at week 12 in treated groups were primarily attributable to a specific reduction in T-suppressor cells and were inversely proportional to dose level. The reduction in T-suppressor cells persisted to the end of the recovery period. There was no evidence of any treatment-related differences in serum concentrations of IgG or IgM in the samples taken at the week 14 necropsy. The only treatment-related change in the myelograms taken at necropsy was a trend towards decreased lymphocytes in the treated groups.

There were no organ weight differences except for minor changes attributable to non-specific stress or reduced body weights in the high dose group.

The immunohistopathology study of a range of lymphoid organs showed various inflammatory lesions in the three examined lymph nodes. For mandibular nodes, the inflammatory reaction was greater in the high dose group compared with the control group. This difference was considered to be attributable to treatment.

Atrophy of the white pulp of the spleen was observed in most animals, including two controls. Nevertheless histometry demonstrated a significant atrophy of the white pulp and marginal zone in the high dose group. The regression line of the atrophy was significantly negative indicating a dose-response.

 

The report concluded that the reduced lymphocyte counts even et 10 ppm were attributable to a reduction in T-suppressor cells, which were inversely related to dose level and persisted to the end of the recovery period. This finding was confirmed by the trend to reduced Lymphocytes in the myelograms. The immunohistopathology revealed an increase in Inflammation of the mandibular lymph nodes at the high dose level of 250 ppm and a dosage-related increase in splenic lymphoid atrophy that were attributed to treatment. Histometry confirmed significant atrophy of the spleen white pulp and marginal Zone in the high dose group. There was no evidence of sensitization. The immunologic phase of the study suggests that lower lymphocyte counts were noted for the DMDS exposed groups. However, the finding was not dose responsive and not treatment-related as the only statistically significant difference was noted for the low exposed group. The lower lymphocytes counts appear to be result of higher than historical control value for the control group and the method used for determining the total lymphocytes. Evaluations of the percentage of lymphocytes to leukocytes showed that all DMDS exposed groups were similar to control. Based on the evaluation above, it can be concluded that DMDS is not immunotoxic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

962 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP guideline study

Effect on immunotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

 

The inhalation immunotoxicity of dimethyl disulphide (DMDS) was assessed in the male Sprague-Dawley rat following exposure, 6 hours/day, 5 days/week over a thirteen week period to vapor concentrations of 0, 10, 50 or 250 ppm (Collins, 1994). The animals used formed part of a subchronic toxicity study reported separately (Collins, 1992). Blood samples were obtained at week 12 and corresponding recovery animals at week 17. The following parameters were evaluated for these samples: total white cells, total lymphocytes, pan B-tells, pan T-cells. T helper cells and T suppressor cells. In addition, IgG and IgM titres were determined in serum from these samples. Organ weights were determined for the popliteal lymph node, submandibular lymph node, mesenteric lymph node and thymus prior to fixation. Histologic evaluations were performed on the popliteal lymph node, suhmandibular lymph node, mesenteric lymph node, mid colon lymphoid tissue, spleen and thymus following staining with hematoxylin-eosin saffron, with slow Giemsa and with immunocytochemical staining. B and T cell analysis did not show any evidence of treatment-related changes in T-helper or B-cells. Reductions in absolute lymphocyte counts at week 12 in treated groups were primarily attributable to a specific reduction in T-suppressor cells and were inversely proportional to dose level. The reduction in T-suppressor cells persisted to the end of the recovery period. There was no evidence of any treatment-related differences in serum concentrations of IgG or IgM in the samples taken at the week 14 necropsy. The only treatment-related change in the myelograms taken at necropsy was a trend towards decreased lymphocytes in the treated groups. There were no organ weight differences except for minor changes attributable to non-specific stress or reduced body weights in the high dose group. The immunohistopathology study of a range of lymphoid organs showed various inflammatory lesions in the three examined lymph nodes. For mandibular nodes, the inflammatory reaction was greater in the high dose group compared with the control group. This difference was considered to be attributable to treatment. Atrophy of the white pulp of the spleen was observed in most animals, including two controls. Nevertheless histometry demonstrated a significant atrophy of the white pulp and marginal zone in the high dose group. The regression line of the atrophy was significantly negative indicating a dose-response. The report concluded that the reduced lymphocyte counts even et 10 ppm were attributable to a reduction in T-suppressor cells, which were inversely related to dose level and persisted to the end of the recovery period. This finding was confirmed by the trend to reduced Lymphocytes in the myelograms. The immunohistopathology revealed an increase in inflammation of the mandibular lymph nodes at the high dose level of 250 ppm and a dosage-related increase in splenic lymphoid atrophy that were attributed to treatment. Histometry confirmed significant atrophy of the spleen white pulp and marginal zone in the high dose group. There was no evidence of sensitization. The immunologic phase of the study suggests that lower lymphocyte counts were noted for the DMDS exposed groups. However, the finding was not dose responsive and not treatment-related as the only statistically significant difference was noted for the low exposed group. The lower lymphocytes counts appear to be result of higher than historical control value for the control group and the method used for determining the total lymphocytes. Evaluations of the percentage of lymphocytes to leukocytes showed that all DMDS exposed groups were similar to control. Based on the evaluation above, it can be concluded that DMDS is not immunotoxic.

Justification for classification or non-classification

From the results of the subchronic immunotoxicity study and in accordance with Regulation (EC) No 1272/2008, DMDS does not warrant classification with regard to Specific Target Organ Toxicity – Single or Repeated Exposure.