Dimethyl disulphide

Endpoint summary

Description of key information

DMDS has been tested in three 90-day repeated dose toxicity studies by inhalation (Collins, 1992, Kim et al, 2006 and Nemec, 2006). The critical effect identified in 2 of these studies was local contact toxicity observed as epithelial changes in the nasal cavity, indicative of local irritation. This was a transient finding, generally reversible after four weeks and was observed from 10 ppm (Collins, 1992). A NOAEC of 5 ppm was identified (Nemec, 2006). In addition to local contact toxicity lower total motor activity counts were observed in week 12 at the concentration of 80 ppm, with a no-observed adverse effect concentration (NOAEC) of 20 ppm (Nemec, 2006). Reduced body weights and food consumption were observed from 25 ppm (Kim et al., 2006) with a NOAEC of 20 ppm (Nemec, 2006). Slight effects on red blood cells and some clinical chemistry parameters were observed at the top concentration of 250 ppm (Collins, 1992) with a NOAEC of 125 ppm (Kim et al., 2006).  Overall, the NOAECs for systemic toxicity and nasal irritation were 20 ppm (77 mg/m3) and 5 ppm (19.25 mg/m3), respectively.

DMDS has been tested in one 28-day repeated dose toxicity by dermal route in rabbits (Prinsen 1990). The no-adverse-effect level (NOAEL) of DMDS for systemic toxicity is 0.1 ml/kg/day (106.3 mg/kg bw/day) and the NOAEL for local irritation is less than 0.01 ml/kg/day (8.5 mg/cm²).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

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

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

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

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.

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

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

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

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

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.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2005 - February 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 424 (Neurotoxicity Study in Rodents)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours per day, 7 days per week

Remarks:

Doses / Concentrations:
5, 20 and 80 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

12

Control animals:

yes, sham-exposed

Key result

Dose descriptor:

NOAEC

Remarks:

systemic toxicity and neurotoxicity

Effect level:

20 ppm (analytical)

Sex:

male

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEC

Remarks:

systemic toxicity and neurotoxicity

Effect level:

>= 80 ppm (analytical)

Sex:

female

Basis for effect level:

other: No adverse effect at 80 ppm.

Key result

Dose descriptor:

NOAEC

Remarks:

local contact (nasal) irritation

Effect level:

5 ppm (analytical)

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

20 ppm (analytical)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Systemic toxicity of DMDS administered by whole-body inhalation exposure to Crl:CD(SD) rats for 13 weeks was observed at an exposure level of 80 ppm in males as evidenced by lower body weight gains and lower food consumption through the first 8 weeks of the exposure period. Lower total session motor activity counts were noted in the 80 ppm group males during study week 12. Local contact (nasal) irritation was observed at exposure levels of 20 and 80 ppm in males and females as a dose-dependent mild to moderate degeneration of the nasal olfactory epithelium.

Executive summary:

A subchronic neurotoxicity study via the inhalation route was conducted with dimethyl disulphide (DMDS) following the OECD guideline # 424. Four groups of 12 male and 12 female Crl:CD(SD)BR were exposed to either clean filtered or DMDS vapor atmospheres or 5, 20 or 80 ppm for 6 hours daily in whole-body inhalation chambers for 13 consecutive weeks. All animals were observed twice daily for mortality and moribundity and clinical examinations were perfortned daily. Body weights and food consumption were recorded weekly. Functional observational battery and locomotor activity assessments were evaluated prior to initiation of exposure and at the time of peak effect (one hour post exposure) during study weeks 3, 7 and 12. Brain weights and dimensions were determined and neuropathologic evaluations were performed. In addition, a microscopic examination of nasal tissues was performed.

DMDS-related, dose-dependent findings in the 20 and 80 ppm group males and females included lower food consumption and corresponding lower body weight gains during study week 0 to 1. Thereafter, body weight gain and food consumption for the 20 and 80 ppm group females and 20 ppm group males returned to levels comparable to the control group beginning during study week 1 to 2, but lower food consumption and body weight gains were sustained in the 80 ppm group males for the duration of the study. As a result, body weights of the males were reduced compared to the control group and cumulative body weight gains were reduced for the duration of the study for both sexes.

Lower total motor activity counts were observed in the 80 ppm group males during all sub-intervals of the study week 12 evaluation. Habituation patterns for these animals were unremarkable. Neuropathologic parameters affected by test article exposure included lower brain length in the males. Minimal to moderate degeneration of the olfactory epithelium on nasal Level II was observed in all 80 ppm males and females. In general, the olfactory epithelium of the females was more severely affected. Minimal to moderate degeneration of the olfactory epithelium was noted on nasal Level II in 6/6 males and 4/6 females in the 20 ppm group. Olfactory epithelial degeneration, characterized by a loss of the adluminal cytoplasmic layer of the sustentacular cells, was also found on Levels III and IV in the 80 ppm group. Minimal olfactory epithelial degeneration on Levels III and IV was noted in 1/6 males in the 20 ppm group. The only test article-related finding in the 5 ppm group consisted of minimal degeneration of the olfactory nasal epithelium on nasal Level III in 1/6 males. On Level II, degeneration of the olfactory epithelium was most noticeable on the dorsal arches, while on Levels III and IV, the alteration typically affected the medial aspects of turbinates in the dorsal meatus.

The no-observed-adverse-effect concentration (NOAEC) for systemic toxicity and neurotoxicity of DMDS via whole-body inhalation exposure to Crl:CD(SD) rats for 13 consecutive weeks was 20 ppm for males and 80 ppm for females. The NOAEL for local contact (nasal) irritation of DMDS via whole-body inhalation exposure for 13 consecutive weeks was 5 ppm for both males and females.

Reference 3

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Principles of method if other than guideline:

This study was carried out according to the test guidelines from the Korea National Toxicology Program (KNTP) and Organisation for Economic Cooperation and Development (OECD) guidelines for the testing of chemicals under modern Good Laboratory Practice Regulations.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Inc. (Kanagawa, Japan)
- Age at study initiation: 6-wk-old
- Weight at study initiation: no data
- Fasting period before study: not appropriate
- Housing: individually in wire-bottomed stainless steel wire mesh cages
- Diet : commercial rodent chow (LabDiet 5002, PMI Nutrition, USA), ad libitum.
- Water: sterilized tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 3°C
- Humidity (%): 50 +/- 10%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12 hrs

IN-LIFE DATES: no data

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainlesssteel chamber (1000 L).
- Method of holding animals in test chamber: no data
- Source and rate of air: no data
- Method of conditioning air: no data
- System of generating particulates/aerosols: not appropriate
- Temperature and humidity in air chamber: 22.1-25.7°C, 45.2-59.6%
- Air flow rate: no data
- Air change rate: no data
- Method of particle size determination: no data
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used:
Gas chromatography (Shimadzu Co., Japan) : detector, flame ionization detector; column, silicon DC-200 15% chromosorb with mesh of 80/100 and a 0.5 m length; detector temperature, 200°C; oven temperature, 100°C; injector temperature, 200°C; and injection volume, 1 ml of gas sample.
- Samples taken from breathing zone: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

DMDS vapor concentrations in the chambers during exposure were measured every 15 min and were controlled to be within ±6% of the target concentration using a personal computer. The mean concentration measured every 15 min for 6 h was taken as the value on a given day. This was then averaged over the 13-wk exposure in order to obtain the mean and standard deviations, and the daily gas concentrations in the 3 chambers were measured at 5.3 ± 0.16, 25.3 ± 0.43, and 125.0 ± 1.34 ppm, respectively.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 h/day, 5 days/wk

Dose / conc.:

5 ppm

Dose / conc.:

25 ppm

Dose / conc.:

125 ppm

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale:
The experimental concentrations were selected based on the results of a preliminary dose-range-finding study. Groups of 5 rats of each gender were exposed to DMDS via whole-body inhalation at concentrations of 33, 100, and 300 ppm for 2 wk. Males at 300 ppm and females at above 33 ppm showed reduced body weight gain and/or decreased food intake.
- Post-exposure recovery period in satellite groups: none

Positive control:

Not appropriate

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
All animals were observed twice daily (before and after exposure) throughout the study period for any clinical signs of toxicity, moribundity, and mortality.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
Body weights of each rat were measured at the beginning of exposure and once a week during the exposure period.

FOOD CONSUMPTION: Yes
Food consumption was measured at the beginning of exposure and once a week during the exposure period. The amounts of food were calculated before they were supplied to each cage, and their remnants were measured on the next day in order to calculate the difference, which was regarded as daily food consumption (g/rat/day).

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
External eye examination on all males and females was carried out shortly before the beginning of the experiments and in the last week of the exposure period. The ocular fundus was examined in the last week of the exposure period using an indirect binocular ophthalmoscope (IO-H, Neitz Instruments Co., Japan). The conjunctiva, sclera, cornea, lens, and iris of each eye were also examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before necropsy
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: Yes, over night
- How many animals: all
- Parameters checked: red blood cell (RBC, erythrocyte) count, hemoglobin concentration, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), RBC distribution width (RDW), platelet count, mean platelet volume (MPV), white blood cell (WBC, leukocyte) count, and differential WBC count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters checked: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine phosphokinase (CPK), glucose, total protein (TP), albumin, albumin/globulin ratio (A/G ratio), blood urea nitrogen (BUN), creatinine, triglyceride, phospholipid, total cholesterol, total bilirubin, calcium, and inorganic phosphorus.

URINALYSIS: Yes
During the last week of exposure, urinalysis of five males and five females per group was carried out with fresh urine to determine the specific gravity, pH, protein, glucose, ketone body, occult blood, bilirubin, urobilinogen, and nitrite by using a CliniTek-100 urine chemistry analyzer (Ames Division, Miles Laboratory, USA).

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the end of experiments, all surviving animals were anesthetized by an intraperitoneal administration of sodium pentobarbital (50 mg/kg body weight) for blood sample collection. The rats were then sacrificed by exsanguination from the abdominal aorta. Complete gross postmortem examinations were performed on all terminated animals.

ORGAN WEIGHTS
The absolute and relative (organ-to-body weight ratios) weights of the following organs were measured: brain, pituitary gland, adrenal glands, liver, spleen, kidneys, heart, lung, thymus, testes, epididymides, and ovaries.

HISTOPATHOLOGY: Yes
The following tissues were obtained from all animals: abnormal lesions, skin, mammary gland, spleen, pancreas, jejunum, stomach, duodenum, ileum, cecum, colon, mesenteric lymph node, salivary gland, submandibular lymph node, ovaries, uterus, vagina, urinary bladder, epididymides, prostates, seminal vesicles, rectum, kidneys, adrenal glands, liver, sternum, thymus, heart, lung, trachea, esophagus, thyroids (including parathyroids), tongue, aorta, sciatic nerve, skeletal muscle, femur, thoracic spinal cord, Harderian glands, brain, pituitary gland, eyes, testes, nasal cavity, nasal turbinates, and Zymbal glands. Eyes and testes were preserved in Davidson’s fixative and Bouin’s fixative, respectively. The other tissues were fixed with a 10% neutral buffered formalin solution. The tissues were routinely processed, embedded in paraffin, and sectioned at 3–5 µm. The sections were stained with hematoxylin–eosin stain for microscopic examination. The nasal passages and nasal turbinates were decalcified prior to being embedded and sectioned.
The nasal cavity was sectioned at the levels of posterior to the upper incisors, the incisive papilla, the second palatine ridge, and the first molar teeth (Young, 1981). All organs and tissues taken from all animals in the vehicle control and the high dose groups were examined microscopically. All gross lesions, as defined by the study pathologist, were also included in the examination.

Statistics:

Statistical analyses were performed by comparing the treatment groups with the vehicle control group using SAS software (SAS Institute, Inc., 1997). Data were presented as means±SD. Variance of numerical datawas checked by Bartlett’s test (1937). If the variancewas homogeneous, the data were subjected to oneway analysis of variance (ANOVA); if not, they were analyzed by the Kruskal–Wallis nonparametric ANOVA (1952). If either of the tests showed a significant difference among the groups, the data were analyzed by the multiple-comparison procedure of Dunnett’s post hoc test (1964). Results of urinalysis obtained with reagent strips were analyzed by the Kruskal–Wallis test followed by multiple comparisons using Dunnett’s test. Clinical signs, necropsy findings, and histopathological findings were represented as frequencies and were subjected to Fisher’s exact probability test (1970) when necessary. The significant probability values p < .05 or p < .01 were noted.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain of male rats was statistically significantly suppressed in the 25-ppm group on days 14, 28, 48, 56, 63, 70, 77, and 86 when compared with the control group. The body weight gain of the male 125-ppm group was also statistically significantly suppressed from day 14 on test to termination in comparison with controls. That of female rats was also significantly lower in the 125-ppm group on days 14 to 48 than that in the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, food consumption was significantly less on days 0, 7, and 63 in the 25-ppm group and on days 0, 7, and 86 in the 125-ppm group than in the vehicle control group, respectively. In females, food consumption of the 125-ppm group was significantly decreased on days 0, 7, and 28 when compared with the vehicle control group.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

RBC and hematocrit were statistically significantly increased in the male 5-ppm group when compared with the control group. RBC, WBC, and lymphocytes were also statistically significantly increased in the male 25-ppm group, and MCVand MCH were significantly decreased in comparison with those of the control group. The male 125-ppm group showed a statistically significant increase in the RBC, and a statistically significant decrease in the MCV and MCH compared with the controls. In females (data not shown), a statistically significant decrease in the hemoglobin concentration was found in the 125-ppm group when compared with the control group.
However, all these variations are not considered to be treatment-related because there are of low intensity and/or not dose-related and in the range of the historical control data.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males, a statistically significant decrease in AST, ALT, and BUN was observed in the 25-ppm group in comparison with the control group. The male 125-ppm group showed a statistically significant decrease in AST, ALT, BUN, CPK, and triglyceride, and a statistically significant increase in serum glucose when compared with the control group.
In females, a statistically significant decrease in AST and BUN was observed in the 25-ppm group when compared with the control group. The 125-ppm group exhibited a statistically significant decrease in AST, BUN, and CPK when compared with the control group.
However, all these variations are not considered to be treatment-related because there are of low intensity and/or not dose-related and in the range of the historical control data.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

A significant decrease in the specific gravity and white blood cell at 5 ppm and the white blood cell at 125 ppm was observed in males without any dose-response relationship (data not shown). There were no significant differences between the treatment groups and controls regarding any of the urinary parameters examined in females.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

In males, absolute liver weight was significantly decreased in the 125-ppm group and relative weights of kidneys and adrenal glands were significantly increased in the same dose group in comparison with the control group. In females, absolute thymus weight in the 125-ppm group was significantly lower than that in the control group, while there were no significant differences in the relative organ weights between the control and treatment groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At the scheduled necropsy, a dark red spot on the thymus was observed in a single male of the control group, but there were no treatment-related gross findings in any of the treated animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In males, bile duct proliferation, macrophage infiltration in the bronchioles, atrophy of the salivary glands, pseudogland formation in the nasal cavity, atrophy of the prostate, and chromodacryorrhea of the Harderian gland were observed in 1 case each in the 125-ppm group. Two cases of a goblet cell proliferation in the nasal cavity, 3 cases of glomerular atrophy of the kidney, 5 cases of protein cast in the kidney, 6 cases of epithelial destruction of the small intestinal villi, 8 cases of functional hyperplasia of the prostate, and 10 cases of bile pigment infiltration in the spleen were also observed in the 125-ppm group. In females, centriacinar telangiectasis, bile-duct proliferation, protein cast in the kidney, ultimobranchial cyst in the thyroid gland, ectopic thymus, and lymphoid cell infiltration in the Harderian gland were observed in 1 case each in the 125-ppm group. Two cases of chromodacryorrhea of the Harderian gland, 3 cases each of epithelial destruction of the small intestinal villi and goblet-cell proliferation in the nasal cavity, and 10 cases of bile pigment infiltration in the spleen were also observed in the 125-ppm group. The incidence and severity of the histopathological alterations observed in the high dose group of both genders were similar to controls.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEC

Remarks:

Systemic toxicity

Effect level:

5 ppm (analytical)

Sex:

male

Basis for effect level:

other: no adverse effect

Dose descriptor:

LOAEC

Remarks:

Systemic toxicity

Effect level:

25 ppm (analytical)

Sex:

male

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOAEC

Remarks:

Systemic toxicity

Effect level:

25 ppm (analytical)

Sex:

female

Basis for effect level:

other: no adverse effect

Dose descriptor:

LOAEC

Remarks:

Systemic toxicity

Effect level:

125 ppm (analytical)

Sex:

female

Basis for effect level:

body weight and weight gain

Critical effects observed:

no

TABLE 1 : Haematological findings in male rats exposed to dimethyl disulfide for 13 wk

	Dimethyl disulfide (ppm)
Parameters	0	5	25	125
RBC (×1012/L)	8.47 ± 0.575	9.34 ± 0.586** #	9.30 ± 0.455** #	9.25 ± 0.484* #
HB (g/dl)	15.65 ± 0.433	16.19 ± 0.943	16.01 ± 0.500	15.45 ± 0.493
HCT (%)	39.73 ± 2.653	42.97 ± 2.573* #	42.34 ± 1.819	41.72 ± 2.270
MCV (fl)	46.91 ± 0.631	46.03 ± 0.996	45.54 ± 0.648** #	45.12 ± 0.658** #
MCH (pg)	18.56 ± 1.301	17.37 ± 0.929	17.26 ± 1.121* #	16.74 ± 0.847** #
MCHC (g/dl)	39.53 ± 2.673	37.74 ± 2.196	37.89 ± 2.175	37.12 ± 1.909
RDW (%)	15.17 ± 0.254	15.21 ± 0.376	15.13 ± 0.250	15.42 ± 0.336
PLT (×109/L)	522.5 ± 21.83	559.3 ± 58.13	582.0 ± 156.9	584.9 ± 29.30
WBC (×109/L)	4.17 ± 0.578	4.78 ± 0.890	5.41 ± 0.597** #	4.60 ± 0.534
NEU (×109/L)	1.03 ± 0.217	1.40 ± 0.319	1.20 ± 0.246	1.07 ± 0.200
LYM (×109/L)	2.95 ± 0.436	3.19 ± 0.786	4.05 ± 0.473**	3.36 ± 0.465
MON (×109/L)	0.18 ± 0.051	0.19 ± 0.076	0.15 ± 0.040	0.17 ± 0.047
EOS (×109/L)	0.002 ± 0.004	0.005 ± 0.007	0.009 ± 0.009	0.001 ± 0.003
BAS (×109/L)	0.00 ± 0.000	0.001 ± 0.003	0.004 ± 0.007	0.000 ± 0.000

Note. Values are presented as mean ± SD. RBC, red blood cells; HB, hemoglobin, HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW, red cell distribution width; PLT, platelet; NEU, neutrophil; LYM, lymphocyte; MON, monocyte; EOS, eosinophil; and BAS, basophil.

* Significant difference at p .05 level when compared with the control group.

** Significant difference at p .01 level when compared with the control group.

# values in the range of the laboratory's historical data (n=84) (Kang BH, Son HY, Ha CS et al. (1995) Reference values of hematology and serum chemistry in Ktc: Sprague-Dawley rats. Korean J Lab Anim Sci; 11: 141-5): RBC: 7.41 - 9.96, HCT: 38.17 - 53.28, MCV: 49.30 - 56.39, MCH: 16.35 - 19.83, WBC: 4.68 - 17.85.     

 

TABLE 2 : Serum biochemical findings in male rats exposed to dimethyl disulfide for 13 wk

	Dimethyl disulfide (ppm)
Parameters	0	5	25	125
AST (IU/L)	169.2 ± 21.61	143.8 ± 28.77	122.6 ± 26.35** #	110.7 ± 10.58** #
ALT (IU/dl)	68.6 ± 17.91	59.9 ± 11.84	45.8 ± 7.75** #	47.9 ± 5.17** #
ALP (mg/dl)	510 ± 49.2	508 ± 54.6	513 ± 59.5	480 ± 56.7
BUN (mg/dl)	18.6 ± 1.50	18.2 ± 1.16	16.9 ± 1.28* #	15.9 ± 1.61** #
CRTN (mg/dl)	0.59 ± 0.102	0.52 ± 0.096	0.48 ± 0.083* #	0.54 ± 0.050
GLU (mg/dl)	128.5 ± 17.38	163.5 ± 54.59	154.1 ± 35.60	182.5 ± 23.14** #
T-CHO (mg/dl)	68.8 ± 5.96	74.1 ± 5.88	65.3 ± 6.20	66.0 ± 6.13
T-BIL (mg/dl)	0.101 ± 0.008	0.095 ± 0.016	0.101 ± 0.010	0.108 ± 0.008
TP (g/dl)	6.59 ± 0.199	6.77 ± 0.391	6.38 ± 0.246	6.50 ± 0.164
ALB (g/dl)	4.42 ± 0.075	4.53 ± 0.145	4.43 ± 0.115	4.39 ± 0.152
CPK (IU/L)	1630 ± 626.0 ##	1268 ± 442.1 ##	1187 ± 361.7 ##	1038 ± 398.3* ##
TG (mg/dl)	75.5 ± 34.19	64.8 ± 24.30	75.1 ± 27.29	37.9 ± 19.70** #
Ca (mg/dl)	9.30 ± 0.121	9.69 ± 0.838	9.22 ± 0.239	9.16 ± 0.186
IP (mg/dl)	5.96 ± 1.283	7.66 ± 2.889	6.05 ± 0.586	5.93 ± 0.551
PL (mg/dl)	120.3 ± 8.14	122.3 ± 9.59	114.7 ± 10.29	117.1 ± 6.01
A/G (ratio)	2.04 ± 0.135	2.04 ± 0.162	2.28 ± 0.169	2.10 ± 0.258

Note.Values are presented as means±SD. AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; BUN, blood urea nitrogen; CRTN, creatinine; GLU, glucose; T-CHO, total cholesterol; T-BIL, total bilirubin; TP, total protein; ALB, albumin; CPK, creatine phosphokinase; TG, triglyceride; Ca, calcium; IP, inorganic phosphate; PL, phospholipid; and A/G, albumin/globulin.

* Significant difference atp.05 level when compared with the control group.

** Significant difference atp.01 level when compared with the control group.

# values in the range of the laboratory's historical data (n=84) (Kang BH, Son HY, Ha CS et al. (1995) Reference values of hematology and serum chemistry in Ktc: Sprague-Dawley rats. Korean J Lab Anim Sci; 11: 141-5): AST : 50.46 - 171.97, ALT: 25.29 - 84.24, BUN: 11.64 - 20.20, CRTN: 0.19 - 0.85, GLU: 104.25 - 217.56, TG : 12.20 - 242.35

## value higher than the historical control data in all groups (CPK: 67.80 - 620.20)

 

TABLE 3 : Serum biochemical findings in female rats exposed to dimethyl disulfide for 13 wk

	Dimethyl disulfide (ppm)
Parameters	0	5	25	125
AST (IU/L)	122.1 ± 20.40	111.2 ± 12.20	98.0 ± 16.07** #	96.2 ± 8.27** #
ALT (IU/dl)	43.7 ± 8.45	45.9 ± 8.22	41.7 ± 8.91	39.6 ± 5.38
ALP (mg/dl)	488 ± 86.8	472 ± 38.2	457 ± 57.8	444 ± 58.1
BUN (mg/dl)	18.6 ± 2.31	17.9 ± 1.97	16.0 ± 1.70* #	15.0 ± 1.39** #
CRTN (mg/dl)	0.45 ± 0.084	0.44 ± 0.050	0.47 ± 0.056	0.46 ± 0.062
GLU (mg/dl)	97.6 ± 22.74	117.3 ± 24.82	116.4 ± 20.38	121.8 ± 27.29
T-CHO (mg/dl)	82.8 ± 12.26	84.3 ± 11.58	78.5 ± 7.95	81.0 ± 8.52
T-BIL (mg/dl)	0.105 ± 0.008	0.104 ± 0.008	0.101 ± 0.013	0.121 ± 0.008
TP (g/dl)	6.21 ± 0.378	6.09 ± 0.316	6.27 ± 0.304	6.30 ± 0.158
ALB (g/dl)	4.27 ± 0.133	4.29 ± 0.108	4.32 ± 0.100	4.29 ± 0.061
CPK (IU/L)	970 ± 298.3 ##	725 ± 162.8 ##	820 ± 521.3 ##	617 ± 208.7* ##
TG (mg/dl)	11.8 ± 3.34	11.7 ± 6.82	10.7 ± 1.49	10.8 ± 1.96
Ca (mg/dl)	7.08 ± 1.660	6.08 ± 0.639	6.62 ± 1.441	7.18 ± 1.314
IP (mg/dl)	5.96 ± 1.283	7.66 ± 2.889	6.05 ± 0.586	5.93 ± 0.551
PL (mg/dl)	136.6 ± 20.05	142.5 ± 19.23	133.8 ± 11.90	141.5 ± 14.52
A/G (ratio)	2.24 ± 0.342	2.42 ± 0.360	2.25 ± 0.251	2.15 ± 0.155

Note. Values are presented as means ± SD. AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; BUN, blood urea nitrogen; CRTN, creatinine; GLU, glucose; T-CHO, total cholesterol; T-BIL, total bilirubin; TP, total protein; ALB, albumin; CPK, creatine phosphokinase; TG, triglyceride; Ca, calcium; IP, inorganic phosphate; PL, phospholipid; and A/G, albumin/globulin.

* Significant difference at p .05 level when compared with the control group.

** Significant difference at p .01 level when compared with the control group.

# values in the range of the laboratory's historical data (n=84) (Kang BH, Son HY, Ha CS et al. (1995) Reference values of hematology and serum chemistry in Ktc: Sprague-Dawley rats. Korean J Lab Anim Sci; 11: 141-5): AST : 58.97 - 173.75, BUN : 11.58 - 25.60

## value higher than the historical control data in all groups (CPK: 68.70 - 476.56)

Conclusions:

The 13-wk repeated exposure of rats to DMDS caused decreases in the body weight gain and food intake in males at 25 ppm and decreases in the body weight gain, food intake and thymus weight and increases adrenal glands weight at 125 ppm of both sexes. The target organ could not be determined in rats under these experimental conditions. The no-observed-adverse-effect concentration (NOAEC) was considered to be 5 ppm, 6 h/day for male rats and 25 ppm, 6 day for female rats, respectively.

Executive summary:

In a study was carried out following the OECD guideline # 413, groups of 10 F344 rats of each sex were exposed to dimethyl disulphide (DMDS) vapor by whole-body exposure at concentrations of 0, 5, 25, or 125 ppm for 6 h/day, 5 days/wk for 13 wk. All the rats were sacrificed at the end of treatment period. During the test period, clinical signs, mortality, body weights, food consumption, ophthalmoscopy, urinalysis, hematology, serum biochemistry, gross findings, organ weights, and histopathology were examined. At 25 ppm, a decrease in the body weight gain and food intake was observed in the males, but not in the females. However, at 125 ppm, a decrease in the body weight gain, food intake, and thymus weight and an increase in the weights of adrenal glands were observed in both genders. In contrast, no treatment-related effects were observed in the 5 ppm group. In these experimental conditions, the target organ was not determined in rats. The no-observed-adverse-effect concentration (NOAEC) was found to be 5 ppm, 6 h/day for male rats and 25 ppm, 6 h/day for female rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

77 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP guideline studies

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2005 - February 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 424 (Neurotoxicity Study in Rodents)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours per day, 7 days per week

Remarks:

Doses / Concentrations:
5, 20 and 80 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

12

Control animals:

yes, sham-exposed

Key result

Dose descriptor:

NOAEC

Remarks:

systemic toxicity and neurotoxicity

Effect level:

20 ppm (analytical)

Sex:

male

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

food consumption and compound intake

Key result

Dose descriptor:

NOAEC

Remarks:

systemic toxicity and neurotoxicity

Effect level:

>= 80 ppm (analytical)

Sex:

female

Basis for effect level:

other: No adverse effect at 80 ppm.

Key result

Dose descriptor:

NOAEC

Remarks:

local contact (nasal) irritation

Effect level:

5 ppm (analytical)

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

20 ppm (analytical)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Systemic toxicity of DMDS administered by whole-body inhalation exposure to Crl:CD(SD) rats for 13 weeks was observed at an exposure level of 80 ppm in males as evidenced by lower body weight gains and lower food consumption through the first 8 weeks of the exposure period. Lower total session motor activity counts were noted in the 80 ppm group males during study week 12. Local contact (nasal) irritation was observed at exposure levels of 20 and 80 ppm in males and females as a dose-dependent mild to moderate degeneration of the nasal olfactory epithelium.

Executive summary:

A subchronic neurotoxicity study via the inhalation route was conducted with dimethyl disulphide (DMDS) following the OECD guideline # 424. Four groups of 12 male and 12 female Crl:CD(SD)BR were exposed to either clean filtered or DMDS vapor atmospheres or 5, 20 or 80 ppm for 6 hours daily in whole-body inhalation chambers for 13 consecutive weeks. All animals were observed twice daily for mortality and moribundity and clinical examinations were perfortned daily. Body weights and food consumption were recorded weekly. Functional observational battery and locomotor activity assessments were evaluated prior to initiation of exposure and at the time of peak effect (one hour post exposure) during study weeks 3, 7 and 12. Brain weights and dimensions were determined and neuropathologic evaluations were performed. In addition, a microscopic examination of nasal tissues was performed.

DMDS-related, dose-dependent findings in the 20 and 80 ppm group males and females included lower food consumption and corresponding lower body weight gains during study week 0 to 1. Thereafter, body weight gain and food consumption for the 20 and 80 ppm group females and 20 ppm group males returned to levels comparable to the control group beginning during study week 1 to 2, but lower food consumption and body weight gains were sustained in the 80 ppm group males for the duration of the study. As a result, body weights of the males were reduced compared to the control group and cumulative body weight gains were reduced for the duration of the study for both sexes.

Lower total motor activity counts were observed in the 80 ppm group males during all sub-intervals of the study week 12 evaluation. Habituation patterns for these animals were unremarkable. Neuropathologic parameters affected by test article exposure included lower brain length in the males. Minimal to moderate degeneration of the olfactory epithelium on nasal Level II was observed in all 80 ppm males and females. In general, the olfactory epithelium of the females was more severely affected. Minimal to moderate degeneration of the olfactory epithelium was noted on nasal Level II in 6/6 males and 4/6 females in the 20 ppm group. Olfactory epithelial degeneration, characterized by a loss of the adluminal cytoplasmic layer of the sustentacular cells, was also found on Levels III and IV in the 80 ppm group. Minimal olfactory epithelial degeneration on Levels III and IV was noted in 1/6 males in the 20 ppm group. The only test article-related finding in the 5 ppm group consisted of minimal degeneration of the olfactory nasal epithelium on nasal Level III in 1/6 males. On Level II, degeneration of the olfactory epithelium was most noticeable on the dorsal arches, while on Levels III and IV, the alteration typically affected the medial aspects of turbinates in the dorsal meatus.

The no-observed-adverse-effect concentration (NOAEC) for systemic toxicity and neurotoxicity of DMDS via whole-body inhalation exposure to Crl:CD(SD) rats for 13 consecutive weeks was 20 ppm for males and 80 ppm for females. The NOAEL for local contact (nasal) irritation of DMDS via whole-body inhalation exposure for 13 consecutive weeks was 5 ppm for both males and females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

19 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP guideline study

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-04-24 to 1989-05-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

yes

Remarks:

see below: Any other information ...

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS:
- Source: ENKI-Konijnenfarm, someren, the Netherlands
- Age at reception: 12 weeks old - Weight at the start of the treatment: no data
- Number of animals:  The control and top-dose group comprised 10 males  and 10 females, whereas the low- and mid-dose group comprised 5 males and  5 females.
- Aclimatation period: 13 days

HOUSING
The animals were housed individually in suspended, galvanized cages,  fitted with a wire-mesh floor and front.

FOOD and WATER
- Food: standard laboratory rabbit diet ad libitum
- Water: tap water, ad libitum 

ENVIRONMENTAL CONDITIONS
- Temperature : 18 ± 3°C
- Relative humidity : at least 40%
- Light/dark cycle : 12h/12h
- Ventilation : at least 10 changes/hour

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

Doses were applied by volume, viz. 1.0, 0.1, and 0.01 ml/kg body weight  for the top-dose, the mid-dose, and the low-dose, respectively. The  respective amounts of the test substance were applied topically to the  intact, shaven skin area by means of a syringe fitted with a blunt  needle. Upon contact with the shaven skin, the test substance immediately  spread spontaneously, so covering application sites of circa 2 x 2 cm, 6  x 6 cm, and 15 x 15 cm in the low-, mid-, and top-dose group,  respectively. Immediately after application, the test site of each rabbit  was covered with porous gauze dressing fixed onto a non-irritating tape.  The entire trunk of each rabbit was wrapped with this tape to maintain  the gauze dressing in position and to retard evaporation of volatile  substances. The animals of the control group were sham-treated with the patches only.  The dermal exposure period was approximately 6 hours per day and 5 days  per week for a period of 4 weeks for the control, low-dose, and mid-dose  group. Because of the high mortality that occurred in the top-dose group  in week 3 of the study, the animals of this group were treated only  during the first 2 1/2 week of the study, i.e. 13 exposures to DMDS. 

Details on analytical verification of doses or concentrations:

Not appropriate

Duration of treatment / exposure:

0.01 and 0.1 ml/kg/d groups were treated 5 days/week during a four-week period, whereas the 1 ml/kg/d group was treated with for 2 1/2 weeks (i.e. 13 days of treatment).

Frequency of treatment:

DMDS was administered daily, by dermal occlusive application (6 hours daily) to four groups of albino rabbits.

Remarks:

Doses / Concentrations:
0.01, 0.1, 1 ml/kg/day (10.63, 106.3 and 1063 mg/kg bw/d)
Basis:
nominal per unit body weight

No. of animals per sex per dose:

The control and 1.0 ml/kg/d group consisting of 10 males and 10 females, and the 0.01 and 0.1 ml/kg/d group consisting of 5 males and 5 females.

Control animals:

other: sham treated with the occlusive dressing

Details on study design:

After nominal day 16 of the study, the surviving animals  of the top-dose group  were kept without further treatment during the remainder of the study. 

Positive control:

Not appropriate

Observations and examinations performed and frequency:

- Clinical signs: twice a day on exposure days and once a day on  non-exposure days.
- Mortality: twice a day.
- Dermal reactions: At the start of the study and prior to each daily administration,  individual skin reactions were evaluated in all (four) groups by the  method of Draize et al. (J. Pharmacol. Exp. Ther. 82 (1944) 377-390). In  addition, on day 28 and day 29 of the study just prior to autopsy, again  skin effects were recorded.
- Body weight: at the start of the study, twice a week thereafter, and on  the day of autopsy, i.e. on day 0, 3, 7, 10, 14, 17, 21, 24, 28, and day  29 of the study.
- Food consumption: on day 0-3, day 3-7, day 7-10, day 10-14, day 14-17,  day 17-21, day 21-24, and day 24-28.
- Ophthalmoscopic examination: no
- Blood examinatrions: On nominal day 23 for males and on nominal day 24 for females (Le. 7 and  8 days after the last treatment to DMDS for males and females of the  top-dose group, respectively), haematology and clinical chemistry  determinations were conducted in blood or plasma of the animals, which  were deprived of food for approximately 24 hours prior to the time of  blood sampling * Haematology: Hemoglobin, hematocrit, red blood cell count, white blood cell count,  differential leukocyte count, platelet count, mean cell volume, mean cell  haemoglobin concentration, mean cell haemoglobin * Biochemistry:  . Electrolytes: calcium, chloride, phosphorous, potassium, sodium,  . Enzymes: alkaline phosphatase, alanine-aminotransferase,  aspartate-aminotransferase, gamma-glutamyl-transferase  . Other: albumin, blood creatinine, blood urea nitrogen,  albumin/globulin, glucose, total bilirubin, total cholesterol, total  serum protein, bile acids
- Urinanalysis: no

Sacrifice and pathology:

On day 28 and 29 of the study (i.e. 12 and 13 days after the last  treatment to DMDS for males and females of the top-dose group), animals  were sedated by intravenous injection with Nembutaltm and subsequently  killed by opening the abdominal aorta. Next, the animals were examined  grossly for pathological changes.
- Weighted organs: adrenals, brain, heart, kidneys, liver, lungs,  ovaries, spleen, testes, thyroid and thymus.
- Microscopic examinations:  Histopathological examination was done on the skin (treated and  untreated), heart, brain, lungs, trachea, liver, kidneys, adrenals,  spleen, testes, ovaries and thymus of the animals of the control group,  the mid-dose and the high-dose groups. Examination of the bone-marrow  (sternum), popliteal, submandibular and mesenteric lymph nodes, spinal  cord and sciatic nerve were carried out in the rabbits of the control-  and the top-dose group. The skin, the thymus and the heart were also examined in the low- dose group because of possible  treatment-related effects. In addition, histopathological examination was  also done on the brain of the animals of the control group, the mid-dose,  and the top-dose group.

Statistics:

Body-weight: Covar + Dunnetts tests (two-sided)
Food intake: Anova + Dunnetts tests (two-sided)
Haematology: Anova + Dunnetts tests (two-sided) and Kruskal-Wallis Anova + Mann Whitney u-test
Blood chemistry: Anova + Dunnetts tests (two-sided)
Organ weight: Anova + Dunnetts tests (two-sided)
Histophatology: pairwise Fisher's test

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

After each daily exposure to DMDS, approximately 10-15 minutes after administration, all animals of the mid dose group were slightly lethargic whilst animals in the high dose group had distinct or severe lethargy or were even unconscious (4/10 animals on day 11). Symptoms disappeared during week-end days (d5-6 and d12-13) when no treatment occurred.
In addition, a number of high dose males and females repeatedly showed clear signs of pain (screaming) after application of DMDS. At the end of the second week, one male and three females of the high dose group showed spasms after treatment and two of the females also showed spasms during the subsequent weekend when there was no treatment. On the last exposure day for the high dose animals (study day 16) all remaining males and females showed spasms after treatment.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

DMDS was absorbed by the skin or had disappeared otherwise within one minute after application. After removal of the patches, no remains of DMDS were present on the skin or on the gauze dressing of the covering patches.
During the four-week test period none of the controls showed any signs of irritation on the skin area treated with the control patches. Repeated dermal administration of DMDS caused severe, dose-dependent skin irritation in all dose groups.
During week 1 the following dermal effects were observed in DMDS treated animals:
- low-dose group: very slight, well-defined or moderate erythema, very slight or slight oedema, and ischemic necrosis
- mid dose group: well-defined, moderate or severe erythema, slight, moderate, or severe oedema, ischemic necrosis, and slight encrustation
- high dose group: well-defined, moderate, or severe erythema, moderate or severe oedema, ischemic necrosis, haemorrhages, and very slight, slight, or moderate encrustation.

During the second week of exposure, the treated skin of all test animals showed incrustation, which almost completely covered the treated skin area. The degree of erythema and oedema could only be scored on the small areas of the application site that did not show incrustation, or at the periphery. At the end of the second week, upon regeneration of the skin underneath, the crusts loosened. In addition, due to the inflexibility, the formed crusts cracked, especially in the animals of high dose group. Administration of DMDS to the newly formed skin underneath these cracks caused small wounds. During the third and fourth week of exposure, the severity of the encrustation in most animals of the three dose groups was such that scoring of erythema and oedema was no longer possible. After study day 16 (i.e. exposure day 13) it was decided to stop treatment of the animals of the high dose group, because of the high mortality that had occurred. Furthermore, it was decided not to record skin effects of this group on a daily basis. Instead, skin effects were recorded only on study day 21 (i.e exposure day 16) and on study day 28 or 29, just prior to autopsy in order to determine the reversibility or irreversibility of the lesions.

In the animals of the high dose group the severity of the skin effects had decreased during the recovery period. However, the 12 to 13-day recovery period was clearly not long enough to fully evaluate the reversibility or irreversibility of the dermal effects observed.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two males in the control group were prematurely sacrificed due to a traumatic injury. One male in the low dose group was prematurely sacrificed due to a poor general condition. One male in the top dose group was prematurely sacrificed due to a traumatic injury. These 4 deaths were not considered to be treatment-related.
Five females and 4 males of the top-dose group were found dead. These 9 deaths were considered to be treatment-related.
Control group.
On nominal day 17 of the study, one male (A6) of the control group was found with a broken hindleg, probably because the leg had been caught between the wire mesh floor and the side panel of the cage. The animal was killed; subsequent gross observation at autopsy did not reveal any macroscopical changes.
One nominal day 24 of the study, one male (A20) of the control group was found with paralyzed hindquarters. It was decided to kill this animals. At autopsy it appeared to have a broken backbone. The cause of this injury could not be found.
Low dose group
One male (B50) of the low-dose group showed very poor defecation from nominal day 10 of the study. On day 14 of the study, the general condition of this animal was very poor, therefore, it was decided to kill the animal.
Top dose group
One nominal day 14 of the study, one male (D84) of the top dose group was found with paralyzed hindquarters. On day 15, it was decided to kill this animals. At autopsy it appeared to have a broken backbone. The cause of this injury could not be found.
One nominal day 8 of the study, one female (D91) of the top dose group was found with the lower body part paralyzed and on day 9 it was decided to kill this animals. However, at autopsy indications of a broken backbone were not found.
Five females of the top dose group were found dead between 45 minutes and 6 hours after treatment on days 8 (D89), 10 (D81), 11 (D97), 15 (D87) and 16 (D83)). Four males (D86, D94, D98 and D100) of the top-dose group were found dead on nominal day 15 at 6 hours after treatment. These deaths were considered to be treatment-related. Therefore, it was decided to no longer treat the animals of the top-dose group after day 16 of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

High dose group males had reduced body weights in comparison to controls up to study day 17. Following termination of treatment after study day 16, males of this group regained weight and at necropsy, no significant differences were observed. Body weights of the low and mid dose groups were unaffected by treatment. In females, there were no clear differences in mean body weights between the groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, food intake in the high dose group animals was somewhat reduced during the first two weeks of the study. There were no indications for a treatment-related effect on food intake of females in any of the dose groups.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related haematological changes were confined to males in the high dose groups. Red blood cell count (RBC) and haemoglobin concentration (Hb) were decreased. This was accompanied by an increased mean corpuscular volume (MCV), a decreased mean corpuscular haemoglobin concentration (MCHC) and an increased number of reticulocytes (retics). Furthermore, Heinz bodies were seen in erythrocytes of most animals in this group. This group also showed a decreased number of white blood cells (WBC) due to a decrease in the number of lymphocytes (lymph).
The combination of decreased RBC and haemoglobin with an increased number of reticulocytes and the presence of Heinz bodies indicates a haemolytic anaemia, accompanied by increased erythropoiesis as compensation. The increased MCV also indicates the presence of a relatively large number of young red blood cells, which are larger than old erythrocytes. Haemolytic anaemia is also observed in ruminants fed with Brassica oleraceae (like kale, cabbage etcetera). These plants contain large amouts of S-methylcysteine sulphoxide which is converted in ruminants to DMDS (R.H. Smith, The veterinary Record, July 5, 1980, page 12-15).

The effects on the white blood cells, especially the lymphocytes in males of the high dose group may be related to the severe effects of treatment on the skin of the animals. The effects on plasma sodium and creatinine concentration in males of the high dose group may be due to their poor health.
The occurrence of these effects in males and their complete absence in females indicate either a sex-dependent difference in sensitivity or in metabolism of the test compound.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males of the high dose group showed decreased plasma creatinine and sodium concentrations when compared with the control group. There were no treatment related findings in the mid and low dose groups. None of the findings in females were considered to be of toxicological significance.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The absolute and organ weights relative to brain weight measured at necropsy did not show statistically significant differences; however group mean organ weight adjusted to mean necropsy body weight was not calculated in the study report.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopy of the animals that died or were killed when moribund during the experimental period did not reveal a distinct treatment-related cause of death. At the scheduled necropsy examination, with the exception of the application sites, there were no findings that were related to DMDS treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination identified treatment-related changes in the treated skin and the heart. The dermal lesions were seen in the treated skin of nearly all rabbits treated with topical DMDS. The changes mainly consisted of acanthosis, hyper- and/or parakeratosis, subcutaneous infiltrates of mononuclear cells and/or polymorphonuclear inflammatory cells, oedema and incidentally congestion. The severity of some of the lesions was slightly higher in the mid dose than in the low dose animals. In the high dose female rabbits that survived the experimental period, acanthosis and mononuclear cell infiltrate in the subcutis were somewhat more pronounced than in intercurrent deaths, whereas parakeratosis, subcutaneous oedema and polymorphonuclear inflammatory cell infiltrate were less pronounced. In the males of the high dose group the skin lesions of survivors and intercurrent deaths were comparable. The untreated skin of test and control animals did not reveal any abnormalities.

In the heart myocarditis and myocardial degeneration were observed in one male rabbit and in 8 female rabbits of the high dose group. This finding was most pronounced in the animals that died during the study; in severe cases it may have caused the death of the animal.

The clinically-observed neurological changes were not accompanied by histopathological changes in the brain, the spinal cord or the sciatic nerve.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Dose descriptor:

NOEL

Remarks:

systemic toxicity

Effect level:

10.63 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: no effect

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

106 mg/kg bw/day

Sex:

male/female

Basis for effect level:

clinical signs

Key result

Dose descriptor:

LOAEL

Remarks:

local irritation

Effect level:

8.5 mg/cm² per day (nominal)

Sex:

male/female

Basis for effect level:

dermal irritation

Critical effects observed:

no

Table 1 Mortality

 

Days Dose (mg/kg bw)	Number of death (killed because moribound or found dead)
	Males	Females
0	2/10	0/10
10.6	1/5	0/5
106.3	0/5	0/5
1063	5/10	6/10

Table 2 Summary of dermal reactions in rabbits, draize scores

 

Day of test	Dose level (mg/kg)
	Males				Females
	0	10.6	106.3	1063	0	10.6	106.3	1063
Erythema
2	0	1.8	2.8	3.9	0	2.4	3.2	3.3
7	0	2.2	2.6	2.3	0	2.6	3.2	3.4
14	0	?	?	-^1	0	?	?	-^1
Autopsy (21)	0	?	?	1^2	0	2.6	?	2.25^2
Oedema
2	0	1.4	2.6	3	0	2	3	3
7	0	3	4	4	0	2.6	3.8	4
14	0	?	?	-^1	0	?	?	-^1
Autopsy (21)	0	?	?	1^2	0	2.8	?	1.5^2
Incrustation
4	0	0	0.4	1.8	0	0	0.4	1
7	0	1.8	2.8	3.7	0	1.8	2.8	3.1
14	0	3	4	-^1	0	3.4	3.8	-^1
Autopsy (21)	0	2	3.8	3^2	0	2.8	4	1.75^2

? Because of the degree of incrustation present on the entire area of the application site erythema and/or oedema could not be observed

¹animals of the high dose group were no longer treated with DMDS; skin effects not recorded

²skin reaction were recorded in order to determine the reversibility or the irreversibility of the skin lesions (4/10 females and 5/10 males examined)

Table 3 Group mean haematology (selected parameters – males- Day 23)

 

Dose mg/kg bw/day		RBC 10^12/L	Hb mmol/L	PCV l/l	MCV fl	MCH fmol	MCHC mmol/L	Retics /1000	Heinz /1000	WBC 10^9/L	Lymph 10^9/L
0	Mean sem n	5.85 0.09 9	7.9 0.1 9	0.368 0.004 9	62.9 1.0 9	1.35 0.02 9	21.4 0.2 9	31.8 5.4 9	0.0 0.0 9	11.0 0.7 9	6.4 0.4 9
10.6	Mean sem n	6.05 0.17 4	8.1 0.3 4	0.384 0.013 4	63.6 1.2 4	1.34 0.03 4	21.1 0.1 4	28.5 5.0 4	0.0 0.0 4	12.3 1.9 4	5.5 0.7 4
106.3	Mean sem n	6.26 0.26 5	7.8 0.2 5	0.365 0.009 5	58.5 1.6 5	1.25* 0.03 5	21.3 0.4 5	23.2 4.1 5	0.0 0.0 5	11.5 0.8 5	5.3 0.6 5
1063	Mean sem n	4.84** 0.24 5	6.6** 0.4 5	0.349 0.018 5	72.2** 2.0 5	1.36 0.03 5	18.9** 0.5 5	102.8*** 18.3 5	9.6 5.7 5	7.7* 0.7 5	3.6** 0.7 5

Statistics: * p<0.05, **P<0.01, ***P<0.001

sem – standard error of the mean

Table 4       Group mean clinical chemistry (selected parameters) – day 23

 

Dose mg/kg bw/day		Creatinine µmol/L	Sodium mmol/L
0	Mean sem n	133.1 4.6 9	140.6 0.4 9
10.6	Mean sem n	129.8 9.4 4	141.6 1.0 4
106.3	Mean sem n	131.5 3.1 5	141.6 0.4 5
1063	Mean sem n	108.0* 6.5 5	138.4* 0.3 5

Table 5 Incidence of selected histopathological findings - terminal kill

 

Observation   Dose (ppm)	Nasal cavity findings (10 animals/group)
	Males				Females
	0	10.6	106.3	1063	0		10.6	106.3	1063
Heart myocarditis
Very slight	0	0	0	0	0		0	0	2
Slight	0	0	0	0	0		0	0	3
Moderate	0	0	0	0	0		0	0	2
severe	0	0	0	0	0		0	0	1
Total incidence	0	0	0	0	0		0	0	8***
Myocardial degeneration
slight	0	0	0	1	0		0	0	3
Moderate	0	0	0	0	0		0	0	1

***p < 0.001

 

Conclusions:

In this study, dermal application of DMDS resulted in treatment-related mortality in the males and females of the high dose group. In addition, dose-dependent clinical effects on the central nervous system (slight to severe lethargy) were induced by DMDS in the males and the females of the mid and high dose group. In addition, a number of high dose males and females repeatedly showed clear signs of pain (screaming) after application of DMDS. However, the observed neurological changes were of a temporary nature and were not accompanied by histopathological changes in the brain, spinal cord or sciatic nerve.

Severe dose-dependent dermal effects were observed in the males and females of all three dose groups, whereas adverse effects on body weight were only observed in males of the high dose group.
Dermal application of DMDS in this study resulted in clear effects on the red blood cell system in males of the high dose group. Microscopical examination revealed dose-dependent changes in the treated skin of the males and the females of all three dose-groups, confirming the macroscopic findings observed during the study and at necropsy. Treatment-related changes were also observed in the heart of one male and several females of the top-dose group, which died during the study or were killed at necropsy.

The no-adverse-effect level of DMDS for systemic toxicity is 106.3 mg DMDS/kg body weight/day, when applied dermally. The overall no-adverse-effect level of DMDS including local skin effects is lower than 10.63 mg DMDS/kg body weight/day.

Executive summary:

In a study performed according to the OECD guideline # 410, dimethyl disulphide was administered daily, by dermal occlusive application (6 hours daily) to four groups of albino rabbits. The dose levels applied were 0, 0.01, 0.1, and 1.0 ml/kg body weight/day, which is equivalent to 0, 10.63, 106.3, and 1063 mg/kg body weight/day, respectively. The control and 1.0 ml/kg/d group consisting of 10 males and 10 females, and the 0.01 and 0.1 ml/kg/d group consisting of 5 males and 5 females.. The animals of the 0.01 and 0.1 ml/kg/d group were treated five days a week during a four-week period, whereas animals of the 1 ml/kg/d group were treated with DMDS for 2 1/2 weeks (i.e. 13 days of treatment).

After each daily treatment with DMDS, temporary effects on the central nervous system (CNS) were observed in the animals of the 0.1 and 1 ml/kg/d group. The observed behavioural effects consisted of slight lethargy in the 0.1 ml/kg/d group and distinct to severe lethargy and unconscinousness in the 1 ml/kg/d group. At the end of each daily exposure these effects were no longer observed. During the four-week test period, treatment-related signs of abnormal behaviour were not observed in the animals of the 0.01 ml/kg/d group or in the controls. During the second and third week of the study treatment-related mortality occurred in males and females of the 1 ml/kg/d group. Therefore, it was decided to discontinue the treatment of the 1 ml/kg/d group on nominal day 16 of the study, i.e. after 13 days of treatment.

Repeated dermal administration of DMDS caused severe, dose-dependent skin irritation in all dose groups.

During the treatment period, decreased body weights were observed in males of the 1 ml/kg/d group. Food intake of males was somewhat decreased during the same period in the 1 ml/kg/d group. Mean body weight and food intake figures of the 0.01 and 0.1 ml/kg/d group were comparable with those of the controls.

Haematology and clinical chemistry examinations of the 1 ml/kg/d males, 7 days after the last exposure to DMDS, revealed treatment-related changes in several red blood cell variables and in the number of white blood cells, and treatment-related changes in plasma creatinine and sodium concentrations. In the females no effects on clinical chemistry variables were observed that could be ascribed to treatment with DMDS.

The absolute and relative organ weights measured at autopsy did not show statistically significant differences that could be ascribed to treatment. However, the mean thymus weight of both males and females tended to be lower in the two higher dose groups than in the controls.

Macroscopic examination at autopsy did not reveal any treatment-related changes other than the dermal lesions induced during the treatment with DMDS. Microscopic examination revealed treatment-related changes in the heart of males and females of the 1 ml/kg/d group and in the treated skin of all three dose-groups, confirming the macroscopic lesions observed during the study and at autopsy. No treatment-related changes were found in the brain, spinal cord, sciatic nerve, or thymus.

The dose of 1.0 ml/kg/day (1063 mg/kg bw/day) is a clear effect level and exceeds the maximal tolareted dose. The no-adverse-effect level (NOAEL) of DMDS for systemic toxicity is 0.1 ml/kg/day (106.3 mg/kg bw/day) and the NOAEL for local irritation is less than 0.01 ml/kg/day (10.63 mg/kg bw/day).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

106.3 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

GLP guideline study

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-04-24 to 1989-05-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

yes

Remarks:

see below: Any other information ...

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS:
- Source: ENKI-Konijnenfarm, someren, the Netherlands
- Age at reception: 12 weeks old - Weight at the start of the treatment: no data
- Number of animals:  The control and top-dose group comprised 10 males  and 10 females, whereas the low- and mid-dose group comprised 5 males and  5 females.
- Aclimatation period: 13 days

HOUSING
The animals were housed individually in suspended, galvanized cages,  fitted with a wire-mesh floor and front.

FOOD and WATER
- Food: standard laboratory rabbit diet ad libitum
- Water: tap water, ad libitum 

ENVIRONMENTAL CONDITIONS
- Temperature : 18 ± 3°C
- Relative humidity : at least 40%
- Light/dark cycle : 12h/12h
- Ventilation : at least 10 changes/hour

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

Doses were applied by volume, viz. 1.0, 0.1, and 0.01 ml/kg body weight  for the top-dose, the mid-dose, and the low-dose, respectively. The  respective amounts of the test substance were applied topically to the  intact, shaven skin area by means of a syringe fitted with a blunt  needle. Upon contact with the shaven skin, the test substance immediately  spread spontaneously, so covering application sites of circa 2 x 2 cm, 6  x 6 cm, and 15 x 15 cm in the low-, mid-, and top-dose group,  respectively. Immediately after application, the test site of each rabbit  was covered with porous gauze dressing fixed onto a non-irritating tape.  The entire trunk of each rabbit was wrapped with this tape to maintain  the gauze dressing in position and to retard evaporation of volatile  substances. The animals of the control group were sham-treated with the patches only.  The dermal exposure period was approximately 6 hours per day and 5 days  per week for a period of 4 weeks for the control, low-dose, and mid-dose  group. Because of the high mortality that occurred in the top-dose group  in week 3 of the study, the animals of this group were treated only  during the first 2 1/2 week of the study, i.e. 13 exposures to DMDS. 

Details on analytical verification of doses or concentrations:

Not appropriate

Duration of treatment / exposure:

0.01 and 0.1 ml/kg/d groups were treated 5 days/week during a four-week period, whereas the 1 ml/kg/d group was treated with for 2 1/2 weeks (i.e. 13 days of treatment).

Frequency of treatment:

DMDS was administered daily, by dermal occlusive application (6 hours daily) to four groups of albino rabbits.

Remarks:

Doses / Concentrations:
0.01, 0.1, 1 ml/kg/day (10.63, 106.3 and 1063 mg/kg bw/d)
Basis:
nominal per unit body weight

No. of animals per sex per dose:

The control and 1.0 ml/kg/d group consisting of 10 males and 10 females, and the 0.01 and 0.1 ml/kg/d group consisting of 5 males and 5 females.

Control animals:

other: sham treated with the occlusive dressing

Details on study design:

After nominal day 16 of the study, the surviving animals  of the top-dose group  were kept without further treatment during the remainder of the study. 

Positive control:

Not appropriate

Observations and examinations performed and frequency:

- Clinical signs: twice a day on exposure days and once a day on  non-exposure days.
- Mortality: twice a day.
- Dermal reactions: At the start of the study and prior to each daily administration,  individual skin reactions were evaluated in all (four) groups by the  method of Draize et al. (J. Pharmacol. Exp. Ther. 82 (1944) 377-390). In  addition, on day 28 and day 29 of the study just prior to autopsy, again  skin effects were recorded.
- Body weight: at the start of the study, twice a week thereafter, and on  the day of autopsy, i.e. on day 0, 3, 7, 10, 14, 17, 21, 24, 28, and day  29 of the study.
- Food consumption: on day 0-3, day 3-7, day 7-10, day 10-14, day 14-17,  day 17-21, day 21-24, and day 24-28.
- Ophthalmoscopic examination: no
- Blood examinatrions: On nominal day 23 for males and on nominal day 24 for females (Le. 7 and  8 days after the last treatment to DMDS for males and females of the  top-dose group, respectively), haematology and clinical chemistry  determinations were conducted in blood or plasma of the animals, which  were deprived of food for approximately 24 hours prior to the time of  blood sampling * Haematology: Hemoglobin, hematocrit, red blood cell count, white blood cell count,  differential leukocyte count, platelet count, mean cell volume, mean cell  haemoglobin concentration, mean cell haemoglobin * Biochemistry:  . Electrolytes: calcium, chloride, phosphorous, potassium, sodium,  . Enzymes: alkaline phosphatase, alanine-aminotransferase,  aspartate-aminotransferase, gamma-glutamyl-transferase  . Other: albumin, blood creatinine, blood urea nitrogen,  albumin/globulin, glucose, total bilirubin, total cholesterol, total  serum protein, bile acids
- Urinanalysis: no

Sacrifice and pathology:

On day 28 and 29 of the study (i.e. 12 and 13 days after the last  treatment to DMDS for males and females of the top-dose group), animals  were sedated by intravenous injection with Nembutaltm and subsequently  killed by opening the abdominal aorta. Next, the animals were examined  grossly for pathological changes.
- Weighted organs: adrenals, brain, heart, kidneys, liver, lungs,  ovaries, spleen, testes, thyroid and thymus.
- Microscopic examinations:  Histopathological examination was done on the skin (treated and  untreated), heart, brain, lungs, trachea, liver, kidneys, adrenals,  spleen, testes, ovaries and thymus of the animals of the control group,  the mid-dose and the high-dose groups. Examination of the bone-marrow  (sternum), popliteal, submandibular and mesenteric lymph nodes, spinal  cord and sciatic nerve were carried out in the rabbits of the control-  and the top-dose group. The skin, the thymus and the heart were also examined in the low- dose group because of possible  treatment-related effects. In addition, histopathological examination was  also done on the brain of the animals of the control group, the mid-dose,  and the top-dose group.

Statistics:

Body-weight: Covar + Dunnetts tests (two-sided)
Food intake: Anova + Dunnetts tests (two-sided)
Haematology: Anova + Dunnetts tests (two-sided) and Kruskal-Wallis Anova + Mann Whitney u-test
Blood chemistry: Anova + Dunnetts tests (two-sided)
Organ weight: Anova + Dunnetts tests (two-sided)
Histophatology: pairwise Fisher's test

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

After each daily exposure to DMDS, approximately 10-15 minutes after administration, all animals of the mid dose group were slightly lethargic whilst animals in the high dose group had distinct or severe lethargy or were even unconscious (4/10 animals on day 11). Symptoms disappeared during week-end days (d5-6 and d12-13) when no treatment occurred.
In addition, a number of high dose males and females repeatedly showed clear signs of pain (screaming) after application of DMDS. At the end of the second week, one male and three females of the high dose group showed spasms after treatment and two of the females also showed spasms during the subsequent weekend when there was no treatment. On the last exposure day for the high dose animals (study day 16) all remaining males and females showed spasms after treatment.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

DMDS was absorbed by the skin or had disappeared otherwise within one minute after application. After removal of the patches, no remains of DMDS were present on the skin or on the gauze dressing of the covering patches.
During the four-week test period none of the controls showed any signs of irritation on the skin area treated with the control patches. Repeated dermal administration of DMDS caused severe, dose-dependent skin irritation in all dose groups.
During week 1 the following dermal effects were observed in DMDS treated animals:
- low-dose group: very slight, well-defined or moderate erythema, very slight or slight oedema, and ischemic necrosis
- mid dose group: well-defined, moderate or severe erythema, slight, moderate, or severe oedema, ischemic necrosis, and slight encrustation
- high dose group: well-defined, moderate, or severe erythema, moderate or severe oedema, ischemic necrosis, haemorrhages, and very slight, slight, or moderate encrustation.

During the second week of exposure, the treated skin of all test animals showed incrustation, which almost completely covered the treated skin area. The degree of erythema and oedema could only be scored on the small areas of the application site that did not show incrustation, or at the periphery. At the end of the second week, upon regeneration of the skin underneath, the crusts loosened. In addition, due to the inflexibility, the formed crusts cracked, especially in the animals of high dose group. Administration of DMDS to the newly formed skin underneath these cracks caused small wounds. During the third and fourth week of exposure, the severity of the encrustation in most animals of the three dose groups was such that scoring of erythema and oedema was no longer possible. After study day 16 (i.e. exposure day 13) it was decided to stop treatment of the animals of the high dose group, because of the high mortality that had occurred. Furthermore, it was decided not to record skin effects of this group on a daily basis. Instead, skin effects were recorded only on study day 21 (i.e exposure day 16) and on study day 28 or 29, just prior to autopsy in order to determine the reversibility or irreversibility of the lesions.

In the animals of the high dose group the severity of the skin effects had decreased during the recovery period. However, the 12 to 13-day recovery period was clearly not long enough to fully evaluate the reversibility or irreversibility of the dermal effects observed.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two males in the control group were prematurely sacrificed due to a traumatic injury. One male in the low dose group was prematurely sacrificed due to a poor general condition. One male in the top dose group was prematurely sacrificed due to a traumatic injury. These 4 deaths were not considered to be treatment-related.
Five females and 4 males of the top-dose group were found dead. These 9 deaths were considered to be treatment-related.
Control group.
On nominal day 17 of the study, one male (A6) of the control group was found with a broken hindleg, probably because the leg had been caught between the wire mesh floor and the side panel of the cage. The animal was killed; subsequent gross observation at autopsy did not reveal any macroscopical changes.
One nominal day 24 of the study, one male (A20) of the control group was found with paralyzed hindquarters. It was decided to kill this animals. At autopsy it appeared to have a broken backbone. The cause of this injury could not be found.
Low dose group
One male (B50) of the low-dose group showed very poor defecation from nominal day 10 of the study. On day 14 of the study, the general condition of this animal was very poor, therefore, it was decided to kill the animal.
Top dose group
One nominal day 14 of the study, one male (D84) of the top dose group was found with paralyzed hindquarters. On day 15, it was decided to kill this animals. At autopsy it appeared to have a broken backbone. The cause of this injury could not be found.
One nominal day 8 of the study, one female (D91) of the top dose group was found with the lower body part paralyzed and on day 9 it was decided to kill this animals. However, at autopsy indications of a broken backbone were not found.
Five females of the top dose group were found dead between 45 minutes and 6 hours after treatment on days 8 (D89), 10 (D81), 11 (D97), 15 (D87) and 16 (D83)). Four males (D86, D94, D98 and D100) of the top-dose group were found dead on nominal day 15 at 6 hours after treatment. These deaths were considered to be treatment-related. Therefore, it was decided to no longer treat the animals of the top-dose group after day 16 of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

High dose group males had reduced body weights in comparison to controls up to study day 17. Following termination of treatment after study day 16, males of this group regained weight and at necropsy, no significant differences were observed. Body weights of the low and mid dose groups were unaffected by treatment. In females, there were no clear differences in mean body weights between the groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, food intake in the high dose group animals was somewhat reduced during the first two weeks of the study. There were no indications for a treatment-related effect on food intake of females in any of the dose groups.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related haematological changes were confined to males in the high dose groups. Red blood cell count (RBC) and haemoglobin concentration (Hb) were decreased. This was accompanied by an increased mean corpuscular volume (MCV), a decreased mean corpuscular haemoglobin concentration (MCHC) and an increased number of reticulocytes (retics). Furthermore, Heinz bodies were seen in erythrocytes of most animals in this group. This group also showed a decreased number of white blood cells (WBC) due to a decrease in the number of lymphocytes (lymph).
The combination of decreased RBC and haemoglobin with an increased number of reticulocytes and the presence of Heinz bodies indicates a haemolytic anaemia, accompanied by increased erythropoiesis as compensation. The increased MCV also indicates the presence of a relatively large number of young red blood cells, which are larger than old erythrocytes. Haemolytic anaemia is also observed in ruminants fed with Brassica oleraceae (like kale, cabbage etcetera). These plants contain large amouts of S-methylcysteine sulphoxide which is converted in ruminants to DMDS (R.H. Smith, The veterinary Record, July 5, 1980, page 12-15).

The effects on the white blood cells, especially the lymphocytes in males of the high dose group may be related to the severe effects of treatment on the skin of the animals. The effects on plasma sodium and creatinine concentration in males of the high dose group may be due to their poor health.
The occurrence of these effects in males and their complete absence in females indicate either a sex-dependent difference in sensitivity or in metabolism of the test compound.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males of the high dose group showed decreased plasma creatinine and sodium concentrations when compared with the control group. There were no treatment related findings in the mid and low dose groups. None of the findings in females were considered to be of toxicological significance.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The absolute and organ weights relative to brain weight measured at necropsy did not show statistically significant differences; however group mean organ weight adjusted to mean necropsy body weight was not calculated in the study report.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopy of the animals that died or were killed when moribund during the experimental period did not reveal a distinct treatment-related cause of death. At the scheduled necropsy examination, with the exception of the application sites, there were no findings that were related to DMDS treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination identified treatment-related changes in the treated skin and the heart. The dermal lesions were seen in the treated skin of nearly all rabbits treated with topical DMDS. The changes mainly consisted of acanthosis, hyper- and/or parakeratosis, subcutaneous infiltrates of mononuclear cells and/or polymorphonuclear inflammatory cells, oedema and incidentally congestion. The severity of some of the lesions was slightly higher in the mid dose than in the low dose animals. In the high dose female rabbits that survived the experimental period, acanthosis and mononuclear cell infiltrate in the subcutis were somewhat more pronounced than in intercurrent deaths, whereas parakeratosis, subcutaneous oedema and polymorphonuclear inflammatory cell infiltrate were less pronounced. In the males of the high dose group the skin lesions of survivors and intercurrent deaths were comparable. The untreated skin of test and control animals did not reveal any abnormalities.

In the heart myocarditis and myocardial degeneration were observed in one male rabbit and in 8 female rabbits of the high dose group. This finding was most pronounced in the animals that died during the study; in severe cases it may have caused the death of the animal.

The clinically-observed neurological changes were not accompanied by histopathological changes in the brain, the spinal cord or the sciatic nerve.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Dose descriptor:

NOEL

Remarks:

systemic toxicity

Effect level:

10.63 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: no effect

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

106 mg/kg bw/day

Sex:

male/female

Basis for effect level:

clinical signs

Key result

Dose descriptor:

LOAEL

Remarks:

local irritation

Effect level:

8.5 mg/cm² per day (nominal)

Sex:

male/female

Basis for effect level:

dermal irritation

Critical effects observed:

no

Table 1 Mortality

 

Days Dose (mg/kg bw)	Number of death (killed because moribound or found dead)
	Males	Females
0	2/10	0/10
10.6	1/5	0/5
106.3	0/5	0/5
1063	5/10	6/10

Table 2 Summary of dermal reactions in rabbits, draize scores

 

Day of test	Dose level (mg/kg)
	Males				Females
	0	10.6	106.3	1063	0	10.6	106.3	1063
Erythema
2	0	1.8	2.8	3.9	0	2.4	3.2	3.3
7	0	2.2	2.6	2.3	0	2.6	3.2	3.4
14	0	?	?	-^1	0	?	?	-^1
Autopsy (21)	0	?	?	1^2	0	2.6	?	2.25^2
Oedema
2	0	1.4	2.6	3	0	2	3	3
7	0	3	4	4	0	2.6	3.8	4
14	0	?	?	-^1	0	?	?	-^1
Autopsy (21)	0	?	?	1^2	0	2.8	?	1.5^2
Incrustation
4	0	0	0.4	1.8	0	0	0.4	1
7	0	1.8	2.8	3.7	0	1.8	2.8	3.1
14	0	3	4	-^1	0	3.4	3.8	-^1
Autopsy (21)	0	2	3.8	3^2	0	2.8	4	1.75^2

? Because of the degree of incrustation present on the entire area of the application site erythema and/or oedema could not be observed

¹animals of the high dose group were no longer treated with DMDS; skin effects not recorded

²skin reaction were recorded in order to determine the reversibility or the irreversibility of the skin lesions (4/10 females and 5/10 males examined)

Table 3 Group mean haematology (selected parameters – males- Day 23)

 

Dose mg/kg bw/day		RBC 10^12/L	Hb mmol/L	PCV l/l	MCV fl	MCH fmol	MCHC mmol/L	Retics /1000	Heinz /1000	WBC 10^9/L	Lymph 10^9/L
0	Mean sem n	5.85 0.09 9	7.9 0.1 9	0.368 0.004 9	62.9 1.0 9	1.35 0.02 9	21.4 0.2 9	31.8 5.4 9	0.0 0.0 9	11.0 0.7 9	6.4 0.4 9
10.6	Mean sem n	6.05 0.17 4	8.1 0.3 4	0.384 0.013 4	63.6 1.2 4	1.34 0.03 4	21.1 0.1 4	28.5 5.0 4	0.0 0.0 4	12.3 1.9 4	5.5 0.7 4
106.3	Mean sem n	6.26 0.26 5	7.8 0.2 5	0.365 0.009 5	58.5 1.6 5	1.25* 0.03 5	21.3 0.4 5	23.2 4.1 5	0.0 0.0 5	11.5 0.8 5	5.3 0.6 5
1063	Mean sem n	4.84** 0.24 5	6.6** 0.4 5	0.349 0.018 5	72.2** 2.0 5	1.36 0.03 5	18.9** 0.5 5	102.8*** 18.3 5	9.6 5.7 5	7.7* 0.7 5	3.6** 0.7 5

Statistics: * p<0.05, **P<0.01, ***P<0.001

sem – standard error of the mean

Table 4       Group mean clinical chemistry (selected parameters) – day 23

 

Dose mg/kg bw/day		Creatinine µmol/L	Sodium mmol/L
0	Mean sem n	133.1 4.6 9	140.6 0.4 9
10.6	Mean sem n	129.8 9.4 4	141.6 1.0 4
106.3	Mean sem n	131.5 3.1 5	141.6 0.4 5
1063	Mean sem n	108.0* 6.5 5	138.4* 0.3 5

Table 5 Incidence of selected histopathological findings - terminal kill

 

Observation   Dose (ppm)	Nasal cavity findings (10 animals/group)
	Males				Females
	0	10.6	106.3	1063	0		10.6	106.3	1063
Heart myocarditis
Very slight	0	0	0	0	0		0	0	2
Slight	0	0	0	0	0		0	0	3
Moderate	0	0	0	0	0		0	0	2
severe	0	0	0	0	0		0	0	1
Total incidence	0	0	0	0	0		0	0	8***
Myocardial degeneration
slight	0	0	0	1	0		0	0	3
Moderate	0	0	0	0	0		0	0	1

***p < 0.001

 

Conclusions:

In this study, dermal application of DMDS resulted in treatment-related mortality in the males and females of the high dose group. In addition, dose-dependent clinical effects on the central nervous system (slight to severe lethargy) were induced by DMDS in the males and the females of the mid and high dose group. In addition, a number of high dose males and females repeatedly showed clear signs of pain (screaming) after application of DMDS. However, the observed neurological changes were of a temporary nature and were not accompanied by histopathological changes in the brain, spinal cord or sciatic nerve.

Severe dose-dependent dermal effects were observed in the males and females of all three dose groups, whereas adverse effects on body weight were only observed in males of the high dose group.
Dermal application of DMDS in this study resulted in clear effects on the red blood cell system in males of the high dose group. Microscopical examination revealed dose-dependent changes in the treated skin of the males and the females of all three dose-groups, confirming the macroscopic findings observed during the study and at necropsy. Treatment-related changes were also observed in the heart of one male and several females of the top-dose group, which died during the study or were killed at necropsy.

The no-adverse-effect level of DMDS for systemic toxicity is 106.3 mg DMDS/kg body weight/day, when applied dermally. The overall no-adverse-effect level of DMDS including local skin effects is lower than 10.63 mg DMDS/kg body weight/day.

Executive summary:

In a study performed according to the OECD guideline # 410, dimethyl disulphide was administered daily, by dermal occlusive application (6 hours daily) to four groups of albino rabbits. The dose levels applied were 0, 0.01, 0.1, and 1.0 ml/kg body weight/day, which is equivalent to 0, 10.63, 106.3, and 1063 mg/kg body weight/day, respectively. The control and 1.0 ml/kg/d group consisting of 10 males and 10 females, and the 0.01 and 0.1 ml/kg/d group consisting of 5 males and 5 females.. The animals of the 0.01 and 0.1 ml/kg/d group were treated five days a week during a four-week period, whereas animals of the 1 ml/kg/d group were treated with DMDS for 2 1/2 weeks (i.e. 13 days of treatment).

After each daily treatment with DMDS, temporary effects on the central nervous system (CNS) were observed in the animals of the 0.1 and 1 ml/kg/d group. The observed behavioural effects consisted of slight lethargy in the 0.1 ml/kg/d group and distinct to severe lethargy and unconscinousness in the 1 ml/kg/d group. At the end of each daily exposure these effects were no longer observed. During the four-week test period, treatment-related signs of abnormal behaviour were not observed in the animals of the 0.01 ml/kg/d group or in the controls. During the second and third week of the study treatment-related mortality occurred in males and females of the 1 ml/kg/d group. Therefore, it was decided to discontinue the treatment of the 1 ml/kg/d group on nominal day 16 of the study, i.e. after 13 days of treatment.

Repeated dermal administration of DMDS caused severe, dose-dependent skin irritation in all dose groups.

During the treatment period, decreased body weights were observed in males of the 1 ml/kg/d group. Food intake of males was somewhat decreased during the same period in the 1 ml/kg/d group. Mean body weight and food intake figures of the 0.01 and 0.1 ml/kg/d group were comparable with those of the controls.

Haematology and clinical chemistry examinations of the 1 ml/kg/d males, 7 days after the last exposure to DMDS, revealed treatment-related changes in several red blood cell variables and in the number of white blood cells, and treatment-related changes in plasma creatinine and sodium concentrations. In the females no effects on clinical chemistry variables were observed that could be ascribed to treatment with DMDS.

The absolute and relative organ weights measured at autopsy did not show statistically significant differences that could be ascribed to treatment. However, the mean thymus weight of both males and females tended to be lower in the two higher dose groups than in the controls.

Macroscopic examination at autopsy did not reveal any treatment-related changes other than the dermal lesions induced during the treatment with DMDS. Microscopic examination revealed treatment-related changes in the heart of males and females of the 1 ml/kg/d group and in the treated skin of all three dose-groups, confirming the macroscopic lesions observed during the study and at autopsy. No treatment-related changes were found in the brain, spinal cord, sciatic nerve, or thymus.

The dose of 1.0 ml/kg/day (1063 mg/kg bw/day) is a clear effect level and exceeds the maximal tolareted dose. The no-adverse-effect level (NOAEL) of DMDS for systemic toxicity is 0.1 ml/kg/day (106.3 mg/kg bw/day) and the NOAEL for local irritation is less than 0.01 ml/kg/day (10.63 mg/kg bw/day).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

8.5 mg/cm²

Study duration:

subacute

Species:

rabbit

Quality of whole database:

GLP guideline study

Additional information

Inhalation exposure
DMDS has been tested in three 90-day repeated dose toxicity studies by inhalation (Collins CJ, 1992 and Kim et al, 2006 and Nemec MD, 2006).
 
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. 

In a study carried out following the OECD guideline # 413, groups of 10 F344 rats of each sex were exposed to dimethyl disulphide (DMDS) vapor by whole-body exposure at concentrations of 0, 5, 25, or 125 ppm for 6 h/day, 5 days/wk for 13 wk (Kim et al., 2006). All the rats were sacrificed at the end of treatment period. During the test period, clinical signs, mortality, body weights, food consumption, ophthalmoscopy, urinalysis, hematology, serum biochemistry, gross findings, organ weights, and histopathology were examined. At 25 ppm, a decrease in the body weight gain and food intake was observed in the males, but not in the females. However, at 125 ppm, a decrease in the body weight gain, food intake, and thymus weight and an increase in the weights of adrenal glands were observed in both genders. In contrast, no treatment-related effects were observed in the 5 ppm group. In these experimental conditions, the target organ was not determined in rats.

In the subchronic neurotoxicity study via the inhalation route conducted with dimethyl disulphide (DMDS) following the OECD guideline # 424 and reported in IUCLID section 7.9.1. (Nemec, 2006), four groups of 12 male and 12 female Crl:CD(SD)BR were exposed to either clean filtered or DMDS vapor atmospheres or 5, 20 or 80 ppm for 6 hours daily in whole-body inhalation chambers for 13 consecutive weeks. A sacrifice, a microscopic examination of nasal tissues was performed. Minimal to moderate degeneration of the olfactory epithelium on nasal Level II was observed in all 80 ppm males and females. In general, the olfactory epithelium of the females was more severely affected. Minimal to moderate degeneration of the olfactory epithelium was noted on nasal Level II in 6/6 males and 4/6 females in the 20 ppm group. Olfactory epithelial degeneration, characterized by a loss of the adluminal cytoplasmic layer of the sustentacular cells, was also found on Levels III and IV in the 80 ppm group. Minimal olfactory epithelial degeneration on Levels III and IV was noted in 1/6 males in the 20 ppm group. The only test article-related finding in the 5 ppm group consisted of minimal degeneration of the olfactory nasal epithelium on nasal Level III in 1/6 males. On Level II, degeneration of the olfactory epithelium was most noticeable on the dorsal arches, while on Levels III and IV, the alteration typically affected the medial aspects of turbinates in the dorsal meatus. 

Dermal exposure
In a study performed according to the OECD guideline # 410, dimethyl disulphide was administered daily, by dermal occlusive application (6 hours daily) to four groups of albino rabbits (Prinsen, 1990). The dose levels applied were 0, 0.01, 0.1, and 1.0 ml/kg body weight/day, which is equivalent to 0, 10.63, 106.3, and 1063 mg/kg body weight/day, respectively. The control and 1.0 ml/kg/d group consisting of 10 males and 10 females, and the 0.01 and 0.1 ml/kg/d group consisting of 5 males and 5 females.. The animals of the 0.01 and 0.1 ml/kg/d group were treated five days a week during a four-week period, whereas animals of the 1 ml/kg/d group were treated with DMDS for 2 1/2 weeks (i.e. 13 days of treatment).
After each daily treatment with DMDS, temporary effects on the central nervous system (CNS) were observed in the animals of the 0.1 and 1 ml/kg/d group. The observed behavioural effects consisted of slight lethargy in the 0.1 ml/kg/d group and distinct to severe lethargy and unconscinousness in the 1 ml/kg/d group. At the end of each daily exposure these effects were no longer observed. During the four-week test period, treatment-related signs of abnormal behaviour were not observed in the animals of the 0.01 ml/kg/d group or in the controls. During the second and third week of the study treatment-related mortality occurred in males and females of the 1 ml/kg/d group. Therefore, it was decided to discontinue the treatment of the 1 ml/kg/d group on nominal day 16 of the study, i.e. after 13 days of treatment.
Repeated dermal administration of DMDS caused severe, dose-dependent skin irritation in all dose groups.
During the treatment period, decreased body weights were observed in males of the 1 ml/kg/d group. Food intake of males was somewhat decreased during the same period in the 1 ml/kg/d group. Mean body weight and food intake figures of the 0.01 and 0.1 ml/kg/d group were comparable with those of the controls.
Haematology and clinical chemistry examinations of the 1 ml/kg/d males, 7 days after the last exposure to DMDS, revealed treatment-related changes in several red blood cell variables and in the number of white blood cells, and treatment-related changes in plasma creatinine and sodium concentrations. In the females no effects on clinical chemistry variables were observed that could be ascribed to treatment with DMDS.
The absolute and relative organ weights measured at autopsy did not show statistically significant differences that could be ascribed to treatment. However, the mean thymus weight of both males and females tended to be lower in the two higher dose groups than in the controls.Macroscopic examination at autopsy did not reveal any treatment-related changes other than the dermal lesions induced during the treatment with DMDS. Microscopic examination revealed treatment-related changes in the heart of males and females of the 1 ml/kg/d group and in the treated skin of all three dose-groups, confirming the macroscopic lesions observed during the study and at autopsy. No treatment-related changes were found in the brain, spinal cord, sciatic nerve, or thymus.
The dose of 1.0 ml/kg/day (1063 mg/kg bw/day) is a clear effect level and exceeds the maximal tolareted dose. The no-adverse-effect level (NOAEL) of DMDS for systemic toxicity is 0.1 ml/kg/day (106.3 mg/kg bw/day) and the NOAEL for local irritation is less than 0.01 ml/kg/day (10.63 mg/kg bw/day).

Justification for classification or non-classification

Based on the available data, no classification for repeated toxicity of DMDS is relevant according to the Regulation EC n°1272/2008.

Justification of the non-classification (RAC) : 

Via the inhalation route there were no adverse effects other than those in the nasal epithelium observed in rats in the repeated dose studies at concentration ranges of 38-962 mg/m3. Via dermal exposure in rabbits at 60 < C ≤ 600, there were no adverse effects in organs other than skin. There were no studies of repeated dose toxicity by oral route. Based on the existing evidence RAC considers that data are conclusive, but not sufficient for classification for (STOT RE).