Dioctadecyl disulphide

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Start of the study: 1976-12-02 Termination of the study: 1979-05-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed and reported GLP-study with design equivalent to OECD 452/451.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dioctadeclydisulphide (DOS2)

IUPAC Name:

Dioctadeclydisulphide (DOS2)

Constituent 2

Reference substance name:

Antioxydant Hoechst TM SE 10

IUPAC Name:

Antioxydant Hoechst TM SE 10

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

white powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: obtained from a preceeding reproduction study (with the same test item and dosages; please refer to IUCLID Chapter 7.8.1) with 60 male and 120 female SPF rats (Cpb: WU, Wistar random); these parent rats were obtained from the Central Institute for the Breeding of Laboratory Animals TNO, Zeist, The Netherlands
- Age at study initiation: weaning age (parent animals were also treated with the test substance while reproduction phase)
- Weight at study initiation: males: 52 g; females: 51 g
- Housing: in groups of 5 in suspended stainless steel cages fitted with wire-mesh floors and fronts
- Diet (e.g. ad libitum): CIVO stock diet, ad libitum (before conducting blood glucose-, and urea nitrogen determination, all rats were fasted overnight, and before urine examinations all rats were deprived of food and water for 24 h)
- Water (e.g. ad libitum): water, ad libitum
- Acclimation period: n.a.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 1°C
- Humidity (%): 55 - 85%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES: From: 2. Dec. 1976 To: End of May 1979

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: CIVO stock diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): The test diets were normally prepared in batches of 40 kg once every 4 to 6 weeks, but occasionally batches of 20 kg were prepared.

VEHICLE
- Concentration in diet: 0.6, 1.2, 2.4%

The test substance was thoroughly mixed with CIVE stock diet by means of a mechanical belnder (Lögige type) at levels of 0 (control), 0.6, 1.2 or 2.4%. Levels of nutrients in the stock diet were periodically determined. The contaminants were also periodically determined in stock diets and drinking water. Samples from the feed containers were taken at intervals and sent to Hoechst AG, Frankfurt, for determination of Dioctadecaldisulphide.

Mean test substance concentration in diet:
control: < 0.03%
0.6% group: 0.58%
1.2% group: 1.18%
2.4% group: 2.35%

Since the time interval between sampling the diets and analysis was one to two and a half months, it appears that no appreciable loss of the test substance occurred upon storage.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Results of determination of Dioctadecylsulphide (DOS2) in test diets

Intended % DOS2
in the diet % DOS2 in feed samples in week
45 60 85

0 0.04 0.04 < 0.01
0.6 0.56 0.60 0.57
1.2 1.17 1.24 1.13
2.4 2.28 2.39 2.37

Duration of treatment / exposure:

130 weeks via diet (test animals were pretreated in utero via parent animals and lactation)

Frequency of treatment:

continuously

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 %
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.6 %
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1.2 %
Basis:
nominal in diet

Doses / concentrations 4

Remarks:

Doses / Concentrations:
2.4 %
Basis:
nominal in diet

No. of animals per sex per dose:

50

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: This long-term study was preceeded by a reproduction phase which was started at 13 Sept. 1976. This reproduction study with the same test substance and dose levels did not reveal any adevers effects on reproduction performance. Administration of DOS2 at dose levels of 0.1 and 1% to rats for 90 days revealed no treatment-related abnormalities either (Hoechst 1967)

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
The animals were observed at intervals for signs of illness and tumours. All signs if ill-health or toxicity with any changes in behaviour were recorded, together with the progression of such abnormalities. Animals which were ill were caged individually. They were returned to their cage if condition improved or killed if conditions deteriorated.

BODY WEIGHT: Yes
Individual body weights were recorded at the end of weeks 0, 1, 2, 3, 4, 6, 8, 10 and 12 and once every 4 weeks thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)/FOOD EFFICIENCY:
Food intake (g/rat/day) of 20 rats/sex/group was determined during the first four weeks and in weeks 11 and 12, 23 and 24, 35 and 36, 47, 59 and 60, 71 and 72, 83 and 84 and 95 and 96. From the body weight gain and the total amount of food consumed in the first four weeks, the food efficiency (g body weight gain per g food) was calculated.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 13, 26, 52, 78, 102 and 127
- Animals fasted: Yes (please refer to "Details on test animals and environmental conditions")
- How many animals: 10/sex/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 26, 52 and 102
- Animals fasted: Yes (please refer to "Details on test animals and environmental conditions")
- How many animals: 10/sex/group

URINALYSIS: Yes
- Time schedule for collection of urine: weeks 13, 26, 52, 78 and 102
- Animals fasted: Yes, urine was collectedd during the last 16 hours of a 24-hour period of deprivation from food and water

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Body weights, results of hematological-, blood chemical-, and urine analysis, and relative organ weights were analysed by the Student t-test. Mortality figures and incidence of neoplastic and non-neoplastic lesions were evaluated by Chi square test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

mortality in males slightliy higher at 2.4% diet

Mortality:

mortality observed, treatment-related

Description (incidence):

mortality in males slightliy higher at 2.4% diet

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

decreased

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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):

decreased red blood cell count, increase in total white cells

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increased relative weights of liver, spleen and ovaries

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

abnormalities in spleen and mesenteric lymph nodes

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

changes in liver, spleen, ovaries, adrenals, bone marrow (sternum) and mesenteric lymph nodes

Histopathological findings: neoplastic:

no effects observed

Details on results:

Clinical signs:
There were no overt signs of reaction to DOS2. After 18 months ageing symptoms developed in all groups.

Mortality:
Mortality in males receiving DOS2 was slightly higher than in controls during the last six months of the study. No such difference occurred in females.

Body weights/food effeciency:
Statistically significantly lower body weights were observed in males at 0.6% from week 40 onwards, at 1.2% from week 60 onwards and at 2.4% from week 80 onwards. In females lower body weights were observed at all dose levels from week 20 onwards. The differences with the controls were less pronounced in the top-dose group than in the lower dose groups. It should be noticed that at the initiation of the study the mean body weights of males of the 0.6% group and females of both the 0.6% and the 1.2% group were already lower than in controls. Moreover, body weights in all test groups were similar to the corresponding control values in the period of rapid growth.

No treatment-related differences in food intake or food efficienc were observed between the groups.

Hematology:
Decreased values for hemoglobin content, packed cell volume and red blood cell count were observed in the treated groups of both sexes. The decreases, which were generally inversely dose-related, did not aggravate in the curse of time. Only slight to marked increases in neutrophilic cells were observed in the test groups in both sexes. The differences with the controls were often most pronounced in the lowest dose group.

Blood chemistry:
Enzyme activities or levels of Glucoes, Urea and Protein of the blood did not show any treatment-related effects.

Kidney function/Urinalysis:
No indication of kidney damage was noticed in the volume, specific gravity or Glutamic-oxalacetic Transaminase activity of the urine. Urine composition was essentially normal.

Relative Organ weights:
There were increases in the relative weight of the liver, spleen and ovaries of all treatment groups. The increases showed a negative correlation with the feeding level of the test item.

Gross examination:
Macroscopic examination revealed some abnormalities in the spleen and mesenteric lymph nodes, such as enlargement and hemorrhages in treated animals. Microscopic examination of these organs indicated that these gross changes were treatment-related.

Microscopy:
Treatment-related histopathological changes were found in the liver, spleen, ovaries, adrenals, bone marrow (sternum) and mesenteric lymph nodes. The changes consisted of accumulations of a crystalline material, usually accompanied by multinucleated giant cells. The mesenteric lymph nodes were most heavily involved and are considered to be the major target organs. The abnormalities observed were more pronounced in the low- and mid-dose groups than in the high dose group.

Neoplasic lesion:
The incidence of hemangio-endotheliomas in mesenteric lymph nodes was increased in each of the test groups. The increases were considered to be related to treatment although a positive dose-response relationship was absent.

Effect levels

Dose descriptor:

NOAEL

Effect level:

0.6 other: % in diet

Sex:

male/female

Basis for effect level:

other: based on secondary effects generated by an overload of the animals´ metabolism/excretion capacity

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

After dosing rats (50 males and 50 females per dose group) with 0.6, 1.2 or 2.4% Dioctadecyldisulphide (Hostanox SE 10) in diet for 130 weeks increased neutrophil counts, relative weights of liver, spleen and ovaries combined with microscopic changes in these organs were observed. These abnormalities were generally more pronounced in the low and mid dose groups than in the high dose group in both sexes. All effects might be caused by the reported deposition and storage of a unknown compound resulting in inflammatory reactions, characterised by multinucleated giant cells and they are considered to be of secondary nature generated by an overload of the test animals´metabolism/excretion capacity. Therefore, the NOAEL is consiedered to be 0.6% (corrsponding to 300 mg/kg bw/d, considering a diet conversion factor (ppm to mg/kg bw/d) of 20 for older rats).

Executive summary:

Each 50 male and 50 female rats were fed with the test substance at dietary levels of 0, 0.6, 1.2 and 2.4 % for 130 weeks. During the treatment period animals were at least monthly observed for general health, mortality, food consumption and body weight development. Blood and urine samples were taken at treatment weeks 13, 26, 78, 102 and 127 for hematological and biochemical investigations. After 130 weeks of feeding the surviving animals were killed and were subjected to macroscopic and microscopic investigations for non-neoplastic as well as for neoplastic lesions.

From the treatment week 112 onwards the mortality in males of high dose was significantly increased compared to controls. No such difference occurred in other groups of males or in females.

From the treatment week 20 onward significantly decreased body weights of treated animals from all dose groups were observed compared to control values, with a more pronounced difference in animals of the low and mid dose groups.

No difference was observed for food consumption for treated and control animals.

Evident decreases for hemoglobin, packed cell volume and red blood count were found in females of all dose groups at the treatment week of 78 and 102. Additionally, increases in the neutrophil counts were observed for treated males and females almost all along the animal treatment period, partially accompanied with increased white blood cell counts.

Upon necropsy, increased organ weights were found for liver, spleen and gonads for all treated groups, whereas the increases for low or mid dose groups were more evident than for high dose groups. For spleen and mesenteric lymph nodes, enlargement and discoloration were found additionally, again the high dose group less severely affected than the mid- or low dose groups.

Non-neoplastic histopathological changes were found in the liver, spleen, ovaries, adrenals, bone marrow (sternum) and mesenteric lymph nodes. The changes consisted of accumulation of crystalline materials, usually accompanied by multinucleated giant cells. The abnormalities observed were more pronounced in the low and mid dose groups than in the high-dose group.

Treatment related neoplastic changes were found in animals of low and mid dose groups. Eight males in low dose group and three and five females in low and mid dose groups respectively exhibited hemangio-endotheliomas in the mesenteric lymph nodes, whereas no or only one animal in both, high and control group showed comparable changes. Since the incidence of hemangiomas in mesenteric lymph nodes of both male and female Wistar rats was well within the range of controls for Wistar-derived rat strains, the development of hemangiomas at this single site was considered to be unrelated to the administration of Hostanox SE 10. This was strenghtened by the absence of an increase in any other tumor type in treated rats following lifetime exposure to massive amounts of compounds, as well as a lack of genotoxic potential and reproductive toxicity of Hostanox SE 10, and the complete absence of any evidence of vascular endothelial cell proliferation in dogs with similar compound-associated foreign material in their tissues after two years (please refer to "WoE_2 year oral toxicity (diet)_dog_Hoechst_1980).

The result obtained from this chronic study in Wistar rats reflects the effect of phagocytic cells attempting to engulf amd dispose of the test substance, and constituted the expected response of dosing animals with large amounts of foreign material over a very long period of time. Therefore, the changes observed are considered to be secondary effects (inflammation, fibrosis etc.) generated by an overload of the animals´capacity to metabolize/excrete the substance. With regard to the apparent inverse dose-response relationship for many of the observed effects, it was noted that mortality was directly related to the dose, indicating that there were symptoms of greater toxicity at the high dose. It is supposed, that there may have been a higher degree of fibrosis, due to destruction of normal tissue, in the high dose group that could have led ultimately to the increased mortality and, in essence, masked the effects that were noted at lower doses.

Based on the above mentioned considerations (observation of secondary effects due to overload effects) the NOAEL of Hostanox SE 10 is considered to be 0.6% (corresponding to 600 mg/kg bw/d; assuming a mean food intake of 10 g/100 g bw/d).

Based on the findings mentioned above the NOAEL could not be established in this study.