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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
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 (109/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 (109/L)

8.82 ± 2.16

6.63 ± 1.66*

7.89 ± 2.28

7.60 ± 1.42

Absolute T-helper count (109/L)

5.31 ± 1.30

4.52 ± 1.19

5.41 ± 1.82

4.77 ± 1.12

Absolute T-suppressor count (109/L)

3.51 ± 1.11

2.11 ± 0.53*

2.48 ± 0.67*

2.83 ± 0.40

Absolute B-cell count (109/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 (109/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 (109/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 (109/L)

4.89 ± 1.33

4.07 ± 1.28

4.37 ± 1.35

4.98 ± 0.88

Absolute T-helper count (109/L)

3.32 ± 1.21

2.96 ± 1.10

3.07 ± 1.05

3.81 ± 0.87

Absolute T-suppressor count (109/L)

1.57 ± 0.31

1.11 ± 0.37*

1.30 ± 0.47*

1.18 ± 0.22**

Absolute B-cell count (109/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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
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
Details on test material:
Source: Atochem
Batch: 82551
Purity: 99.88%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
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 for males, 80-100 g for females 
- Weight at the start of the treatment: 185-256 g for males, 121-169 g  for females 
- Number of animals:  100 rats : 20 males + 20 females / dose group (4  dose groups + 1 control group)
- 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

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: 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.
Details on inhalation 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.
- Group            Target    Nominal  Analytical concentrations:      
1                 0     
2                10      20         10.17 ppm     
3               50    81         50.25 ppm   
4                 150      223        150.62 ppm     
5               250      373      246.59 ppm
Duration of treatment / exposure:
90 days
Frequency of treatment:
6 h/day; 5 d/week
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 50, 150, 250 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
Four groups of 20 male and 20 female Sprague-Dawley were exposed 6 hours/day, 5 days/week to 0, 10, 50, 150, or 250 ppm DMDS. The exposure of the 150 ppm group was terminated after 6 weeks and its treatment-free subgroup necropsied 2 weeks later. The remaining groups received a 13 week exposure period followed by four weeks for the treatment-free subgroups.
Positive control:
Not appropriate

Examinations

Observations and examinations performed and frequency:
- Clinical observations
* Morbidity and mortality All animals were examined twice daily to detect any which were dead or  moribund.
* Clinical signs All animals were examined once daily for signs of ill health or overt  toxicity. In addition each animal was given a detailed clinical  examination at weekly intervals. An individual record was maintained of  the clinical condition of each animal.
* Functional observation tests Observations were carried out on all animals prior to beginning treatment  and again during weeks 1, 4, 13 and the treatment-free animals in week  17. The observations were made prior to and following any exposure that day. Observations were recorded for the following parameters: ease of removal  from home cage, ease of handling, appearance of eyelids, lacrimation,  colour of tears, salivation, respiration, appearance of fur,  piloerection, writhing, vocalisation.

- Body weight Individual body weights were recorded before exposure on the first day of  the study, at weekly intervals thereafter and at necropsy.

- Food consumption The amount of food consumed by each cage of animals was determined weekly.

- Ophthalmoscopy The eyes of all animals were examined pre-dose and all control and high  dose animals in week 12.

- Laboratory investigations
Blood samples were obtained from groups 1, 4 and 5 for haematology and  clinical chemistry in week 6 and groups 1 and 5 for haematology and  clinical chemistry in week 12. The samples were collected from the main  study animals.
* Haematology: Haemoglobin, mean cell volume, red blood cell count and indices: mean  cell haemoglobin, mean cell haemoglobin concentration packed cell volume,  total and differential white blood cell count platelet count.
* Clinical chemistry: aspartate aminotransferase, alanine aminotransferase,  alkaline phosphatase, sodium, potassium, chloride, calcium inorganic phosphorus, glucose, urea, total bilirubin, creatinine, total  protein, albumin, albumin/globulin ratio total cholesterol.
Sacrifice and pathology:
- Pathology
* Necropsy
Full internal and external examination at sacrifice
* Organ weights
adrenals, brain, kidneys, liver, lungs, ovaries, pituitary, spleen, testes.
* Histology
adrenals, femur (including articular aorta surface)#, brain (including brain stem), heart, caecum, ileum colon, jejunum duodenum, kidneys, epididymides#, lachrymal gland#, eyes (with optic nerves), larynx, liver, sciatic nerve, lungs (with mainstem bronchi), skeletal muscle (quadriceps)#, lymph nodes (bronchial with tracheal bifurcation and mesenteric), skin and mammary gland#,  spinal cord (lumbar, cervical, thoracic)#, nasal passages, spleen, nasopharyngeal duct, sternum (and bone marrow), oesophagus, stomach, ovaries, testes, pancreas, thymus, pituitary, thyroids (with parathyroids), prostate#, tracheal bifurcation, rectum, urinary bladder, salivary glands (submaxillary, sublingual), uterus and all gross lesions. Samples of all tissues (except those annotated # above) from all main  study animals in the control and high dose group, the lungs and all gross  lesions of all main study animals and the nasal cavities of groups 2 and  3 terminal kill and groups 1, 2, 3 and 5 treatment-free animals were  evaluated by the study pathologist.
Statistics:
* ANOVA, T-test
Body weight: week 0
* ANOVA, Regression and Dunnett's
Body weight gains: weeks 0 to 6, 0 to 13, 13 to 17 weeks 6 to 8 (group 1  v group 4 only) 
Total food consumption: weeks 1 to 5, 7 to 12
Necropsy body weight: terminal kill and treatment-free 
Clinical  chemistry: weeks 6, 12 AST, ALT, ALK PROS, Na, K, Cl, Ca, P, GLUCOSE, UREA, T BILI, CREAT, T  PROT, ALBUMIN, AG RATIO, TOT CHOL Clinical chemistry: week 13 ALT, ALK PHOS (males), T BILI Clinical chemistry: week 17 ALT, ALK PHOS, T BILI 
Haematology: weeks 6, 12 Hb, RBC, PCV, MCV, MCH, MCHC, PLAT, WBC, Neutrophils and Lymphocytes 
-  absolute and percentages
Haematology: week 13 Hb, RBC, PCV, MCV, MCH, MCHC 
* ANCOVA, Dunnett's Organ weights (adjusted for necropsy body weight) 
- terminal kill and  treatment-free: adrenals, kidneys, spleen, liver, ovaries, gonads, lung, brain, pituitary
* Kruskal-Wallis, Terpstra-Jonckheere, Wilcoxon  Clinical chemistry
week 13 females ALK PHOS

No statistical analysis was considered necessary to interpret the results  of the functional observation tests.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only clinical signs attributable to treatment were salivation, lacrimation or reduced activity during exposure 1 and 2 of the 150 and 250 ppm groups and a low incidence of dyspnoea or wheezing in the early part of the study, particularly in the 250 ppm animals at week 1.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was no treatment-related mortality.
Two female animals, 145 (50 ppm) and 163 (150 ppm), showed ulcerative sores on the neck in week 1 of the study attributed to the ear tattooing procedure and were therefore removed from study and replaced with two spare animals designated 145Rl and 163Rl. Male animal 59 (50 ppm), treatment-free subgroup) died during anaesthesia for blood sampling in week 17. This death was considered to be incidental.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a dosage-related decrease in body weight gain (Tables 1 and 2) over the treatment period in treated groups compared with controls. Differences were statistically significant except for the 10 ppm group which was only significant for males over weeks 0 to 6. Improvements in the rate of body weight gain were observed in the test groups in the treatment-free period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Differences in food consumption (Table 3) paralleled those of body weight gain except the numerical differences did not achieve statistical significance in the 50 ppm males or the 10 ppm groups.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The eyes of the animals were unremarkable.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No effect was observed on the haematological profiles of the males on week 6 (0, 150 and 250 ppm groups). In females, a slight statistically significant decrease of MCV (58.7 vs 60.4 fl) and MCH (20.1 vs. 21.1 pg) was observed at 150 ppm and of MCHC (34.1 vs. 34.8 g/dl) at 250 ppm when compared to the control group.
Haematological profiles on week 12 (0 and 250 ppm groups, Table 4)) suggested a possible small reduction in Hb, RBC and PCV in the 250 ppm female group only. Haematological profiles on week 13 (0, 10 and 50 ppm groups, Table 5) were unremarkable (data not shown)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Blood chemistry examinations performed on week 6 (0, 150 and 250 ppm groups) showed in males a statistically significant increases of ALT (62 vs. 45 Iu/l) at 150 ppm and Alk. Phos. (450 vs. 338 Iu/l) and total bilirubin (5.2 vs. 3.9 µmol/l) at 250 ppm when compared to the control group. In females, significant increases of total bilirubin (4.1 vs. 3.1 µmol/l) was observed at 150 ppm and of alk. phos. (329 vs. 242 Iu/l) at 250 ppm. A decrease of the urea level was also observed at 150 ppm (6.6 vs. 7.7 mmol/l).
Blood chemistry examinations performed on week 12 (0 and 250 ppm groups, Table 6) and 13 (0, 10 and 50 ppm groups, Table 7) showed treatment-related changes in ALT, alkaline phosphatase and bilirubin. The changes did not include the 10 ppm group except for elevated ALT in occasional animals at week 13 and after the treatment-free period. Any changes in alkaline phosphatase and bilirubin in the 50 ppm group were equivocal.
Blood chemistry examinations performed on week 17 (0, 10, 50 and 250 ppm groups) did not show any treatment related effects on ALT, alk. phos. and total bilirubin.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Functional observation tests indicated treatment-related changes in response to handling and respiration, and in incidence of salivation, soiling of the fur and piloerection but no evidence of neurotoxicity.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no changes in organ weights that were considered to be treatment-related. Statistically significant elevations in lung weights in the 250 ppm males and females were of doubtful toxicological importance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic abnormalities at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the 10, 50 and 250 ppm animals examined microscopically there was a dose-related effect on nasal mucosa characterised by squamous metaplasia of the respiratory epithelium accompanied in 50 ppm and 250 ppm groups by atrophy and microcavitation in the anterior olfactory epithelium. In the 10 ppm group the effects were limited to a local, minor degree of squamous metaplasia of the anterior nasal cavity. The changes were still present in the 50 and 250 ppm groups after the treatment-free period but the 10 ppm group was generally unremarkable.
Histopathological findings: neoplastic:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEC
Remarks:
Systemic toxicity
Effect level:
10 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: no adverse effect
Dose descriptor:
LOAEC
Remarks:
Systemic toxicity
Effect level:
50 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
Key result
Dose descriptor:
LOAEC
Remarks:
Nasal irritation
Effect level:
10 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
10 ppm (analytical)
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
yes

Any other information on results incl. tables

Table 1: Group mean body weight gains (g), males

Week of study

Concentration (ppm)

0

10

50

150

250

Start

Mean

227.3

224.5

228.6

225.2

227.1

S.D.

15.69

13.15

13.36

14.36

14.82

0 to 6

Mean

160.2

139.0*

128.7**

115.2***

106.3***

S.D.

31.86

24.03

28.84

24.99

21.50

6 to 8

Mean

32.5-

-

-

47.5***

-

S.D.

10.67

-

-

8.68

-

0 to 13

Mean

233.0

225.6

181.8***

-

148.3***

S.D.

45.31

34.38

42.47

-

37.83

13 to 17

Mean

10.8

25.9

29.5

-

41.8**

S.D.

18.17

23.94

17.62

-

21.01

* p<0.05** p<0.01*** p<0.001

Table 2: Group mean body weight gains (g), females

Week of study

Concentration (ppm)

0

10

50

150

250

Start

Mean

149.6

146.0

144.9

145.6

144.2

S.D.

7.53

8.62

5.47

10.88

10.9

0 to 6

Mean

88.0

77.0

73.0**

68.1***

74.2**

S.D.

14.97

15.00

10.72

15.86

13.51

6 to 8

Mean

14.8

-

-

20.3*

-

S.D.

5.22

-

-

5.30

-

0 to 13

Mean

117.8

119.8

97.5**

-

93.2***

S.D.

22.69

22.63

17.13

-

17.99

13 to 17

Mean

3.0

5.5

9.5

-

9.6

S.D.

8.86

12.91

11.08

-

11.67

* p<0.05** p<0.01*** p<0.001

Table 3:mean food consumption (g/animal) over specified periods

Week of study

Concentration (ppm)

0

10

50

150

250

Males

1 to 5

Mean

810.2

779.4

752.3

772.4*

700.4**

S.D.

33.85

46.25

36.88

40.86

18.17

7 to 12

Mean

991.7

996.1

944.0

866.8***

S.D.

12.46

42.34

39.49

24.38

Females

1 to 5

Mean

585.5

558.5

527.5**

516.1**

535.6*

S.D.

17.77

19.63

14.23

31.86

19.34

7 to 12

Mean

741.1

728.4

674.2***

691.1**

S.D.

3.54

27.29

9.63

21.2

* p<0.05** p<0.01*** p<0.001

Table 4: Group mean haematology, Occasion: Week 12

Group

Hb

RBC

PCV

MCV

MCH

MCHC

PLAT

Sex

g/dl

mil/cmm

%

fl

pg

g/dl

1000/cmm

Males

0 ppm

Mean

15.0

8.16

45.9

56.4

18.4

32.6

926

S.D.

0.7

0.56

1.9

2.0

0.7

0.4

147

250 ppm

Mean

14.4

7.76

44.0

56.7

18.5

32.7

936

S.D.

0.8

0.44

2.5

1.3

0.5

0.3

128

Females

0 ppm

Mean

14.4

7.44

43.4

58.4

19.4

33.2

967

S.D.

0.7

0.34

2.1

1.5

0.6

0.3

140

250 ppm

Mean

13.5*

6.88**

41.0*

59.6

19.7

33.0

947

S.D.

0.8

0.42

2.3

1.7

0.6

0.4

73

* p<0.05 ** p<0.01 *** p<0.001

Table 5: Group mean haematology, Occasion: Week 12

Group

WBC 1000 / cmm (%)

Sex

TOTAL

N

L

M

E

B

Males

0 ppm

Mean

13.2

1.71(13)

11.23(85)

0.12(1)

0.12(1)

0.00(0)

S.D.

4.5

0.71(-)

3.91(-)

0.13(-)

0.14(-)

0.00(-)

250 ppm

Mean

11.7

1.31(11)

10.33(88)

0.01(0)

0.04(0)

0.00(0)

S.D.

3.5

0.50(-)

3.23(-)

0.04(-)

0.06(-)

0.00(-)

Females

0 ppm

Mean

11.0

1.25(12)

9.22(84)

0.28(3)

0.20(2)

0.00(0)

S.D.

2.7

0.82(-)

2.51(-)

0.15(-)

0.23(-)

0.00(-)

250 ppm

Mean

10.8

1.30(12)

9.13(85)

0.28(3)

0.10(1)

0.00(0)

S.D.

1.7

0.43(-)

1.45(-)

0.18(-)

0.08(-)

0.00(-)

Table 6: Group mean clinical chemistry, Occasion: Week 12

Group

AST
(GOT)

ALT
(GPT)

ALK
PHOS

Na

K

Cl

Ca

P

Sex

Iu/l

Iu/l

Iu/l

mmol/l

mmol/l

mmol/l

mmol/l

mmol/l

Males

0 ppm

Mean

96

51

222

143

3.9

108

2.41

1.8

S.D.

13

11

50

1

0.2

2

0.10

0.1

250 ppm

Mean

78**

47

302*

143

3.8

106

2.44

1.8

S.D.

9

7

82

1

0.3

2

0.09

0.1

Females

0 ppm

Mean

86

39

168

141

3.4

107

2.46

1.7

S.D.

9

6

37

1

0.2

1

0.09

0.1

250 ppm

Mean

84

51*

264*

139*

3.4

106

2.38*

1.7

S.D.

9

14

108

2

0.3

3

0.06

0.1

* p<0.05 ** p<0.01*** p<0.001

Group

GLUC

UREA

T BILI

CREAT

T PROT

ALBUMIN

AG RATIO

TOT CHOL

Sex

mmol/l

mmol/l

µmol/l

µmol/l

g/l

g/l

mol/l

Males

0 ppm

Mean

5.9

6.0

3.7

62

62

34

1.3

1.7

S.D.

0.7

1.0

0.6

3

4

1

0.1

0.3

250 ppm

Mean

6.0

6.1

4.8*

59*

62

36*

1.4

1.5

S.D.

0.5

0.9

1.2

2

3

1

0.1

0.3

Females

0 ppm

Mean

6.2

7.0

3.5

65

68

40

1.4

2.2

S.D.

0.3

0.9

0.6

5

3

2

0.1

0.4

250 ppm

Mean

6.1

6.7

5.7***

65

68

39

1.4

2.1

S.D.

0.6

1.1

1.1

2

5

1

0.2

0.4

* p<0.05 ** p<0.01*** p<0.001

Table 7: Group mean clinical chemistry, Occasion: Week 13

Group

ALT(GPT)

ALK PHOS

T BILI

Sex

Iu/l

Iu/l

µmol/l

Males

0 ppm

Mean

48

282

2.0

S.D.

5

45

1.0

10 ppm

Mean

61

345

2.5

S.D.

16

112

0.6

50 ppm

Mean

72*

375

2.2

S.D.

35

101

0.6

Females

0 ppm

Mean

46

218

1.2

S.D.

8

59

0.5

10 ppm

Mean

75

264

1.3

S.D.

42

47

0.5

50 ppm

Mean

57*

320

1.4

S.D.

10

128

0.9

* p<0.05 ** p<0.01*** p<0.001

Applicant's summary and conclusion

Conclusions:
Clear treatment-related effects were seen at 50 and 250 ppm and were present to a marginal degree at 10 ppm. It was concluded that the effect level was 50 ppm. The no-effect level was in the region of, but less than, 10 ppm due to the reversible changes in the nasal mucosa
Executive summary:

In an OECD 413 study, groups of 10 rats/sex were exposed by inhalation to DMDS 6 h/day, 5 d/week for 90 days to concentrations of 0, 10, 50, 150, 250 ppm (Collins, 1992). The exposure of the 150 ppm group was terminated after 6 weeks and its treatment-free subgroup necropsied 2 weeks later. The remaining groups received a 13 week exposure period followed by 4 weeks for the treatment-free subgroups. The only clinical signs attributable to treatment were salivation, lacrimation or reduced activity during exposures 1 and 2 of the 150 and 250 ppm groups and a low incidence of dyspnea or wheezing in the early part of the study, particularly in the 250 ppm animals at week 1. Functional observation tests indicated no evidence of neurotoxicity. Body weight gains and food consumption were decreased in all treatment groups; this effect was reversible during the recovery period. Hematological profiles suggested a possible small reduction in Hb, RBC and PCV in the 250 ppm female group only. Blood chemistry examinations showed treatment-related changes in ALT, alkaline phosphatase and bilirubin. The changes did not include the 10 ppm group except for elevated ALT in occasional animals at week 13 and after the treatment-free period. There were no changes in organ weights that were considered to be treatment-related and no treatment-related macroscopic abnormalities. Microscopic evaluations indicated a dose-related effect on nasal mucosa characterised by squamous metaplasia of the respiratory epithelium accompanied by atrophy and microcavitation in the anterior olfactory epithelium. In the 10 ppm group the effects were limited to a local, minor degree of squamous metaplasia of the anterior nasal cavity. The changes were still present in the 50 and 250 ppm groups after the treatment-free period but the 10 ppm group was generally unremarkable. Clear treatment-related effects were seen at 50 and 250 ppm and were present to a marginal degree at 10 ppm.