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Immunotoxicity

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Effect on immunotoxicity: via oral route

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Endpoint:
immunotoxicity: acute oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not specified
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Study was not assessed for reliability; provided for information purposes only.
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this immunotoxicity study, atrophy of the thymus was found in rats given a single oral dose of the test material. Doses as low as 10 mg of test material per kg body wt decreased relative thymus weight.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
Male Wistar-derived rats of 4-5 weeks of age, obtained from the Central Institute for Breeding of Laboratory Animals, TNO, Zeist, The Netherlands, were used. Rats were housed in plastic cages and kept at room temperature of 23 ± 2 °C with 50-60 % relative humidity and a constant 12h light/dark cycle.
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Single oral dose.
Frequency of treatment:
Single oral dose.
Dose / conc.:
16.2 mg/kg bw/day (actual dose received)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
5 - 60 mg/kg
No. of animals per sex per dose:
4-5 rats per dose for dose-effect relationship method.
21 rats for time-effect relationship method.
Control animals:
yes
Details on study design:
Randomised groups of four to five rats, weighing 50-60 g, were given single oral doses of the test material (27 animals) ranging from 5 to 35 mg/kg. The test material was dissolved in absolute ethanol and intubated as a 5 % solution in corn oil (5 ml/kg body wt).
Sacrifice and pathology:
Body weights and weights of thymus, spleen, liver, kidneys and adrenals were recorded 4 days after intubation.
All excised organs were fixed in 10 % buffered formalin, dehydrated in graded series of ethanol and embedded in glycol methacrylate. Sections (1-2 µm) sere stained with May-Grünwald-Giemsa or bematoxylin and eosin.
Statistics:
The data were analysed for significance of differences by Student's t-test (two-tailed). A regression analysis was applied on the data from the dose-effect relationships.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Dose-effect relationships:
Dosages of 10 mg test material per kg rat or more caused is statistically significant weight reduction of the thymus. Reduction was dose-related and appeared maximal 4 days after intubation. Regression analysis of the dose-effect relationships revealed that the test material caused a decrease in relative thymus weight linear to the logarithm of the orally administered dose. The dose level calculated to give 50 % reduction of relative thymus weight was 29 mg/kg.
In rats given the test material, the relative spleen weights were decreased at dose levels of 35 and 60 mg/kg.

Time-effect relationships:
Thymus weight: During the relatively short test period of 9 days, the absolute thymus weights of control rats increased considerably, along with a rise in the number of cells that could be isolated from this gland. During the experimental period, the relative thymus weights remained at a constant level between 0.3 and 0.4 % of body wt. From the second day after a single oral dose of 50 µmol/kg rat, the relative thymus weights were significantly decreased. Concurrently, the total cell count was diminished significantly at day 4. These parameters, together indicative for thymus atrophy, were most severely reduced at day 4 after dosing of the test material. Recovery was complete within 7 days for the test material.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Dose-effect relationships:
On microscopic examination, a dose-related depletion of cortical lymphocytes was observed in thymus glands of the test material-exposed rats. At dose levels higher than 100 µmol/kg rat, the lymphocyte depletion was associated with a pronounced reduction in the width of the thymic cortex. As a result of the thymic involution, the mast cells lying in the connective tissue between the thymic lobes were seen more closely together when compared to the controls.
In rats given the test material, the relative spleen weights were decreased at dose levels of 35 and 60 mg/kg. Microscopic examination revealed a dose-dependent reduction in the periarteriolar lymphocyte sheaths and the red pulpa, resulting in a general reduction of the spleen size. The number of megakaryocytes per section was increased in the two highest dose levels.
In rats exposed to 10 mg/kg or more, reddening of mesenteric lymph nodes was frequently observed. Histologically, the appearance of erythrocytes was evident, often situated as rosettes around mononuclear cells in the sinuses of these lymph nodes.
Dose descriptor:
other: 50 % reduction in thymus weight
Effect level:
27 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios

Relative cell counts:

In thymocyte suspensions of control animals, the frequency of small cells (>130 µm^3) increased gradually within the 9 days of the test period, while the intermediate (130-225 µm^3) and large thymocytes (>225 µm^3) progressively decreased in number.

In TBTC, the most pronounced reduction in the frequency of large cells was found at 48 h (to 50 % of control; P<0.001). The lowest frequency of intermediate cells (70 % of control; P<0.01) was also reached on the second day. A significant increase in intermediate and large cells was observed at day 4 (both P<0.01), coinciding with a reduction in the frequency of small cells. One week after dosing, cell size distribution was normal again.

 

Incorporation of macromolecule precursors:

In association with the frequency of intermediate and large cells, the incorporation of Urd and TdR in thymocytes of control animals showed a tendency to decline during the 9 day period. Intubation of the test material markedly reduced the incorporation of the RNA precursor during the first 2 days. The tune-effect relationships for the incorporation of Urd were strikingly similar to the kinetics of the intermediate and large cells. The effects of the test material on the incorporation of TdR, however, were more severe and more persistant. Recovery occurred 1 day later (day 4) and a rebound may have taken place between day 4 and 7. Although the incorporation of Leu was less affected by the test material, the time-effect curves for this amino acid resembled the ones for the incorporation of Urd. Incorporation rates were normalised after 7-9 days after organotin intubation. In rats given the test material, the incorporation of TdR, Urd, and Leu was already reduced significantly after 2 days.

 

Absolute cell counts:

Using the frequency and the total cell count data, the absolute numbers of small, intermediate and large cells were determined. These absolute cell counts also indicate that the test material caused a selective reduction of the large cells in the first 2 days after dosing. At day 4, the small, intermediate and total cell counts were drastically diminished. In thymocyte suspensions prepared 4 days after organotin exposure, the frequency of the large cells was found to be markedly increased. However, the absolute number of large cells did not exceed the respective control value. As with the relative cell counts, the absolute numbers were normal again at day 9.

Conclusions:
50 % reduction in thymus weight was observed at 29 mg/kg bw of test material exposure.
Executive summary:

In this immunotoxicity study, atrophy of the thymus was found in rats given a single oral dose of the test material. Doses as low as 10 mg/kg body wt decreased relative thymus weight. 50 % reduction in thymus weight at 29 mg/kg bw of test material exposure.

Thymus atrophy caused by the test material is due to a selective effect on the population of large-sized, rapidly proliferating lymphoblasts. These cells, which are present in a limited number generate the large population of small, non-dividing cells that populate the thytmic cortex. As a result of this selective action on the lymphoblasts, a marked depletion of cortical lymphocytes develops several clays after exposure to the test material.

Endpoint:
immunotoxicity: short-term oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Information on animal numbers is not clear, however methods are well documented. Only two doses used. No information on test material or GLP status.
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this immunotoxicity study, atrophy of the thymus was found in rats given a single oral dose of the test material. Doses as low as 10 mg/kg body wt decreased relative thymus weight, to investugate the selective action on the thymus.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
Male Wistar albino rats (190-200 g) were obtained from the University of Surrey Animals Breeding Unit. Rats were housed in stainless-steel wire cages (2 animals per cage) at 22 ± 2 °C with 55-65 % relative humidity and a constant 12-h light/dark cycle.
Route of administration:
oral: feed
Vehicle:
peanut oil
Details on exposure:
DIET PREPARATION
The test material was dissolved in 5 mL of 100 % pure peanut oil (Petty, Wood and Co. Ltd., Andover, UK) and homogenised with 1 kg of feed (CRM rodent diet) to give dietary concentrations of 5 ppm and 25 ppm (mg kg^-1 diet). An equivalent amount of oil was added to the feed of the control group. Diet was prepared twice weekly and was stored in closed glass vessels at 5 °C in the dark until used.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days
Frequency of treatment:
Ad libitum
Dose / conc.:
5 ppm (nominal)
Dose / conc.:
25 ppm (nominal)
No. of animals per sex per dose:
8 animals recieved control diet
8 animals recieved the test material at 25 ppm
4 animals recieved the test material at 5 ppm
Control animals:
yes, plain diet
Sacrifice and pathology:
Half of the rats in the control group and each group receiving diets containing 25 ppm of the tin compounds were killed after 1 week. The other half of these groups and the group receiving diets containing 5 ppm of the tin compounds were killed after 4 weeks of feeding. In all cases, rats were killed by an overdose of pentobarbitone (Sagatal; May and Baker, UK).
At autopsy, major abdominal and thoracic organs were examined, and liver, spleen, kidneys, brain and thymus were weighed. In addition, mesenteric lymph nodes were sampled for histological examination.
Tissues for light microscopic examination were pre-pared by immersion fixation in 10 % neutral buffered formalin and embedded in paraffin wax. Sections, 4 µm thick, were cut and stained with haematoxylin and eosin.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Two of four rats receiving diets containing 25 ppm of pure test material showed abnormal 'burrowing' behaviour, immediately after injection with the anaesthetic prior to autopsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The differences in food consumption were reflected by reduced body weight gain in rats on diets containing 25 ppm of pure test material (Fig. 1B).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption of rats receiving diet containing 25 ppm of pure test material was consistently and significantly less than the controls (Fig. 1A). No such difference was observed in rats receiving diets containing 5 ppm of pure test material. The differences in food consumption were reflected by reduced body weight gain in rats on diets containing 25 ppm of pure test material (Fig. 1B). Actual intakes, expressed as mg tin per day, are presented in Table 1.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Animals killed after 28 days of exposure to 25 ppm of pure test material showed a marked decrease in weight gain.
Thymus weights showed both an absolute and a relative decrease (Table 3) in rats given pure test material. Lymph nodes in all rats exposed to the test material were markedly haemorrhagic. Histological examination showed partial atrophy of the nodes, with blood pools in the interstitial space. A few lymphoid follicles remained but these showed almost total depletion of the T-dependent areas. No other gross pathological changes were observed.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Animals killed after 28 days of exposure to 25 ppm of pure test material showed a marked decrease in weight gain.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathological examination of the thymus showed an almost complete restoration of normal structure. No histopathological abnormalities were detected in the spleen, liver or kidneys. The distribution of tin showed that concentrations in kidneys and livers were at least five times higher than in other organs.
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Conclusions:
At all times, the mean body weight gain and the food consumption was significantly less in rats treated with 25 ppm pure test material as compared to control rats.
Executive summary:

In a 1-month feeding trial, the test material was fed to rats at concentrations of 5 ppm and 25 ppm. At all times, the mean body weight gain and the food consumption was significantly less in rats treated with 25 ppm pure test material as compared to control rats.

Animals killed after 28 days of exposure to 25 ppm of pure test material showed a marked decrease in weight gain. It was also noted that two of four rats receiving diets containing 25 ppm of pure test material showed abnormal 'burrowing' behaviour, immediately after injection with the anaesthetic prior to autopsy. Thymus weights showed both an absolute and a relative decrease (Table 3) in rats given pure test material. Lymph nodes in all rats exposed to the test material were markedly haemorrhagic. Histological examination showed partial atrophy of the nodes, with blood pools in the interstitial space. A few lymphoid follicles remained but these showed almost total depletion of the T-dependent areas. No other gross pathological changes were observed. Histopathological examination of the thymus showed an almost complete restoration of normal structure. No histopathological abnormalities were detected in the spleen, liver or kidneys. The distribution of tin showed that concentrations in kidneys and livers were at least five times higher than in other organs.

Endpoint:
immunotoxicity: short-term oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No information on GLP and limited information on test material, however methods well reported.
Qualifier:
no guideline followed
Principles of method if other than guideline:
In 2-week feeding studies, the test material was fed to male weanling rats at dietary concentrations of 15, 50 and 150 ppm to evaluate their toxic effects, especially on the brains and the lymphoid organs, thymus and spleen.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
Specific pathogen-free Wistar rats were purchased from the Central Institute for Breeding of Laboratory Animals, TNO, Zeist, The Netherlands. The animals were housed in plastic cages at room temperature of 23 ± 2 °C with 50 to 60 % relative humidity, and a constant 12-hr light/dark cycle.
Route of administration:
oral: feed
Vehicle:
acetone
Details on exposure:
DIET PREPARATION
The diets were prepared 1 day before the experiment by dissolving the organotin compounds in 5 mL of acetone and mixing it thoroughly with a commercial feed (Trouw & Co., Putten, The Netherlands). Homogeneous distribution of tin was confirmed by atomic absorption, using a Perkin-Elmer 400 atomic absorption spectrometer with an acetylene-air flame. Diets and tap water were constantly available. Provided the organotin compounds are stored dry and dark, they are considered to be stable. In a diet containing 150 ppm test material even after 6 months no degradation products were found upon chromatographing hexane extracts of the diet (97 to 108 % recovery of tin).
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
2-week study and a 4-week study were conducted.
Frequency of treatment:
Ad libitum in diet.
Dose / conc.:
15 ppm (nominal)
Remarks:
2-week study
Dose / conc.:
50 ppm (nominal)
Remarks:
2-week study
Dose / conc.:
150 ppm (nominal)
Remarks:
2-week study
Dose / conc.:
100 ppm (nominal)
Remarks:
4-week study
No. of animals per sex per dose:
2-week study used 10 rats per dose.

4-week study used 6 rats per dose
Control animals:
yes, plain diet
Details on study design:
Two-week study:
Randomised groups of 10 male weanling rats, weighing 35 to 50 g, were used. The organotin compounds were fed at dietary concentrations of 0, 15, 50, and 150 ppm.

Four-week study:
The test material was fed to groups of six male weanling rats for a 4-week period at dietary concentrations of 0 and 100 ppm (rats weighing 35 to 40 g).
Observations and clinical examinations performed and frequency:
Two-week study:
Body weight and feed consumption were recorded twice a week.
Sacrifice and pathology:
All organs weighed were fixed in 10 % buffered formalin and embedded in paraplast, and 5-µm sections were cut according to routine procedures. Sections were stained with hematoxylin and eosin. In addition, thymus glands, spleens, and mesenteric lymph nodes from rats exposed to the test material were embedded in glycol methacrylate, and 1-µm sections were stained with May-Granwald-Giemsa.

Two-week study:
Body weights and the weights of thymus; spleen, liver, kidneys, adrenals, brain, and testes were recorded. In addition, five spleens obtained from the the test material feeding studies were dried and, after digestion in nitric acid, iron content of these spleens was determined by atomic absorption. A standard curve was prepared by dissolving iron sulfate in the appropriate solvent. Occult blood was determined in faeces of rats fed the test material.

Statistics:
The data were analysed for significance of differences by Student's t-test.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
2 -week sudy:
The test material induced a dose related and severe reduction of thymus and spleen weight. At dietary concentrations of 50 and 150 ppm relative liver weight was increased. Associated with a reduced feed intake of about 25 %, body and brain weight of rats fed 150 ppm were markedly decreased, whereas relative adrenal weight was slightly increased. Thymus weight was only 39 % of control weight in these animals.

4 -week study:
100 ppm of the test material in the diet of rats are presented in Table 3. The test material caused reduction of thymus weigh to 43 % of the controls. Body weights were moderately decreased. In contrast to the 2-week studies, no effects were observed on the weights of spleen and liver.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
2 -week sudy:
Microscopically, thymus atrophy was evident as judged by a numerical reduction of cortical lymphocytes, associated with a relative increase of reticulo-epithelial cells. The number of mitotic figures was also decreased. However, no signs of increased lymphocyte destruction were observed.

4 -week study:
Histologic examination of thymus glands of rats fed the test material revealed a severe reduction in lymphocyte density in the cortex. This feature was even more pronounced than in the 2-week feeding studies. Histology of the spleen and liver was normal, except for slight atrophy of the periarteriolar lymphocyte sheaths (PALS) in a few spleens of rats fed the test material.
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified

2 -week sudy:

When cell suspensions from thymus glands were made and the nucleated cell counts were determined, the severe and dose-dependent lymphocyte depletion could be accurately demonstrated (Table 2). Feeding of 50 ppm reduced the cell count to 50 % of control values, while in the 150-ppm group only 16 % of the cells could be isolated. Despite the reduction in spleen weight, no distinct signs of lymphocyte depletion were observed. Extramedullary haematopoiesis was similar in control and treatment groups. Using atomic absorbance spectrometry, the amount of iron per spleen was decreased in a dose-related manner (Table 2). However, the iron concentration (expressed in micrograms Fe per gram dry spleen weight) was not altered (Table 2). No morphologic changes were observed in the livers. On gross examination, reddening in only a few of the mesenteric lymph nodes was noticed in each dosage group. Microscopically, a dose-related increase in the number of erythrocytes, situated as rosettes around mononuclear cells, were seen throughout the medullary sinuses. No occult blood could be detected in faeces of all test material groups.

Recovey Experiment:

Upon feeding rats 100 ppm test material for 4 weeks, body weights were significantly reduced up to 3 weeks after terminating the treatment.

Adrenalectomy:

Adrenalectomy itself caused some growth retardation, but the subsequent treatment with 100 ppm in the diet for 13 days did not affect body weight any further. Adrenalectomy also resulted in an increase in thymus weight compared to the nonoperated or sham-adrenalectomised rats. This enlargement is considered to be a consequence of decreased adrenal activity. On careful examination, no adrenal remnants were found in 10 of the 12 adrenalectomised rats. Adrenalectomy did not abolish the test material-induced thymus atrophy. The relative thymus weight of these rats was decreased to the same extent as the nonoperated or sham-adrenalectomized rats.

Conclusions:
Atrophy of the thymus was considered to be the predominant effect of the test material. From this study it is concluded that the test material is primarily immunotoxic.
Executive summary:

In 2-week feeding studies, the test material was fed to male weanling rats at dietary concentrations of 15, 50 and 150 ppm to evaluate their toxic effects, especially on the brains and the lymphoid organs, thymus and spleen. The test material caused a dose-related reduction of thymus weight. At a dietary concentration of 150 ppm decreases in thymus weight to 39 % of controls were found following treatment. Microscopically, thymus atrophy was associated with a lymphocyte depletion in the thymic cortex. Only 16 % of the total number of nucleated thymocytes could be isolated from rats fed 150 ppm. These effects were completely reversed within 2 weeks. A dose-related decrease in spleen weight was noticed after 2 weeks feeding of the test material. Liver weights were increased in rats fed the test material for 2 weeks. Nevertheless, no enlarged livers and normal spleen weights were found upon feeding 100 ppm test material for 4 weeks, whereas thymus weight was severely decreased. Therefore, atrophy of the thymus was considered to be the predominant effect of the test material. From this study it is concluded that the test material is primarily immunotoxic.

Endpoint:
immunotoxicity: oral
Remarks:
developmental
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Limited test material information and no information on GLP. Equivalent to guideline study.
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.7800
Deviations:
not specified
Principles of method if other than guideline:
Ten rats/dose group, were dosed orally by gavage with the test material in olive oil at 0, 0.025, 0.25 and 2.5 mg/kg bw/day from day 8 of pregnancy. The doses of test material were based on existing immunotoxicity and reproductive data. Gestation day 8 was chosen for two reasons. Firstly, it is just prior to the cascade of events leading to sexual differentiation, and secondly, it precludes pre-implantation embryonic loss as a factor in these studies. Dosing continued at the same level through lactation. At weaning, (21 days of age) male and female pups were randomly assigned to various experiments and continued to receive the same level of test material as their respective dam until the age of 30 days (males and females), 60 days (females) or 90 days (males). To facilitate unbiased daily recording of body weights and weekly recording of food consumption, animals were slightly sedated with isofluorane and an electronic microchip was implanted.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Specific-pathogen-free pregnant female Sprague-Dawley rats were obtained from Charles River Canada Inc., (St. Constant, Quebec, Canada). The weight of the rats at arrival was 200-220 g. Upon arrival rats were identified, housed singly in plastic cages (Health Guard System, Research Equipment Company, Inc., Bryan, TX, USA) and maintained on rat chow (Purina 5001 Agribands Purina Canada Inc., Strathroy, ON) and tap water in accordance to policies established by the Canadian Council for Animal Care and protocols approved by Health Canada's Animal Care Committee, Ottawa, Ontario, Canada. The cages were placed on the rack so that the individual dose groups were not all at one level, i.e., the cages were ordered from top to bottom. Animals were housed in designated facilities, where the temperature was maintained at 21 ± 23 °C and the relative humidity between 50 and 60 %. The light/ dark cycle was maintained on 12-h intervals. Rat cages were cleaned and sanitised once per week.
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
The olive oil vehicle for test material administration was purchased from was purchased from Aldrich Diagnostics USA (St. Louis, MO). The tests material was purchased from Sigma-Aldrich Canada (Oakville, ON) and was stable for at least 4 years (98.8 % on a Sn basis, the remainder being dibutyltin). To provide a dosing solution, the test material was diluted in olive oil. The required dose/100 g body weight was delivered in 0.5 mL volumes. The dosing solutions were prepared fresh weekly and varied with the current weight of the rats. The test material is stable in olive oil for at least 2 months. The doses administered were as follows: Control: 0.00; Low: 0.025; Medium: 0.25 and High: 2.5 mg/kg bw/day. The test material was administered orally by gavage using the infant feeding tube method. Vehicle control rats were dosed with olive oil. The doses of test material selected for this study were based on the calculated TDI of 0.25 µg/kg bw/day and included the TDI dose of 0.25 mg/kg bw/day, one dose lower than the TDI and one higher than the TDI.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
The test material in olive oil at 0, 0.025, 0.25 and 2.5 mg/kg bw/day from day 8 of pregnancy. Dosing continued at the same level through lactation. At weaning, (21 days of age) male and female pups were randomly assigned to various experiments and continued to receive the same level of the test material as their respective dam until the age of 30 days (males and females), 60 days (females) or 90 days (males).
Dose / conc.:
0.025 mg/kg bw/day (actual dose received)
Dose / conc.:
0.25 mg/kg bw/day (actual dose received)
Dose / conc.:
2.5 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 rats per dose group.
Control animals:
yes, concurrent vehicle
Details on study design:
Ten rats/dose group, were dosed orally by gavage with the test material in olive oil at 0, 0.025, 0.25 and 2.5 mg/kg bw/day from day 8 of pregnancy. The doses of the test material were based on existing immunotoxicity and reproductive data. Gestation day 8 was chosen for two reasons. Firstly, it is just prior to the cascade of events leading to sexual differentiation, and secondly, it precludes pre-implantation embryonic loss as a factor in these studies. Dosing continued at the same level through lactation. At weaning, (21 days of age) male and female pups were randomly assigned to various experiments and continued to receive the same level of the test material as their respective dam until the age of 30 days (males and females), 60 days (females) or 90 days (males). To facilitate unbiased daily recording of body weights and weekly recording of food consumption, animals were slightly sedated with isofluorane and an electronic microchip (Bio Medic Data Systems, Inc., Seaford, DE) was implanted. Immunologic testing was as follows:
Sacrifice and pathology:
Thirty-day old male and female F1 pups:
When pups were 30 days old, four groups of male and four groups of female pups, 10 pups/group were sacrificed by exsanguination under isoflurane anaesthesia, spleens were collected and flow cytometric analysis was performed on spleen single-cell suspensions. Serum was collected also for total serum immunoglobulin (Ig) and cytokine determination.
Other examinations:
Sixty-day old female F1:
When pups were 60 days old, they were tested for effects on the immune system. The following parameters were examined: flow cytometric analysis, total serum Ig and cytokine levels, natural killer cell activity (NK) and lymphocyte transformation (LT) using spleen cells, 40 females (10 rats/group); delayed type hypersensitivity (DTH) response to oxazolone, 40 females (10 rats/group); L. monocytogenes infectivity assay, 48 females (12 rats/dose) and, determination of sheep red blood cell-specific antibody levels in the serum of immunized rats, (40 females, 10 rats/group).

Ninety-day old male F1:
When pups were 90 days old, they were tested for effects on the immune system. Group numbers and types of assays were identical to those for 60 days of age.
Statistics:
The ELISA data for total serum immunoglobulins and IgG subsets, anti-SRBC-IgM titres and serum cytokines were statistically evaluated using SigmaStat (Jandel Scientific, San Rafael, CA, USA). Data were analysed using both trend analyses and pairwise comparisons. The Pearson Product Moment correlation coefficient was used to measure the strength of association between dose and Ig concentration. Trends were considered significant when P ≤ 0.05. Data from control and treated rats were also compared using one-way analysis of variance (ANOVA) for multiple comparisons, followed by Dunnett's test for pairwise comparisons when necessary. The data on flow cytometic enumeration of leukocyte subsets were analysed for treatment effects using the Armitage's test for trend, i.e., an ANOVA model with linear and nonlinear effects of dose was used to test for dose related trends within each sub-experiment. Pairwise (t-test) comparisons to control were performed where significant results were found in the test for trend or for the nonlinear effect of dose. The lymphocyte transformation data were log trans-formed in order to stabilize the variance and normalize the data. Following this, data were analysed for treatment-related effects using a two-way ANOVA model with day and treatment as the main effects and the interaction term was fit separately for each mitogen. Nonsignificant interactions (P ≤ 0.05)
the response variables vs dose was also carried out. In all cases, data were tested for normality and screened for outliers. The DTH response data were analysed for treatment-related effects using the Kruskal-Wallis test. Histomorphoinetry data were analysed using both trend analyses and pairwise comparisons. Trends were considered significant when P ≤ 0.05. Data from control and treated rats were also compared using one-way ANOVA for multiple comparisons, followed by Dunnett's test for pairwise comparisons if necessary.
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no statistically significant effects on the T-cell population in any of the tissues examined compared to controls when slides were stained with the CD3 marker (P > 0.05). The histomorphometric evaluation of the thymus showed a significant reduction in the combined cortex + medulla area of the 30-day high dose male rats (P = 0.048) and in the medulla area of the 60-day high dose female rats (P = 0.042). In the 90-day high dose male rats there was a significant reduction in the area of the cortex, the medulla and the cortex + medulla (P = 0.0066, P = 0.0381 and P = 0.0063, respectively) (Table 15).
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross examination of lymphoid tissues did not reveal any significant effects.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Thymus: Cortical atrophy, characterised by decreased numbers of cortical lymphocytes, was observed in 4 out of 6 males (1 mild, 3 moderate) in the 30-day high dose group. Mild cortical atrophy occurred in 1 out of 5 in the 30-day medium dose males and in 1 out of 6 in the 30-day medium dose females.
Mesenteric Lymph Nodes: Mild generalized lymphoid atrophy, characterised by decreased numbers of lym-phocytes in the follicles and paracortex was observed in 1 out of 4 in the 30-day low dose males. Moderate generalised lymphoid atrophy occurred in 2 out of 6 in the 90-day medium dose males. Mild paracortical atrophy was observed in 1 out of 6 of the 30-day high dose males and in 1 out of 6 of the 90-day low dose males.
Popliteal Lymph Node: Generalized lymphoid atrophy was present in three rats from different dose groups, including 1 out of 5 in the 90-day low dose males (mild), 1 out of 5 in the 30-day low dose males (severe), and in 1 out of 6 in the 90-day medium dose males (severe). Mild atrophy in the B-lymphocyte dependent area, with decreased follicular and medullary cord area and reduced cord cell density, was found in 1 out of 5 in the 90-day low dose males. Two rats had moderate medullary cord hypertrophy, with large cords infiltrated by plasma cells, including 1 out of 5 90-day low dose male and 1 out of 6 90-day medium dose male. Mild paracortical atrophy occurred in 1 of the 90-day medium dose males.
Spleen: Six rats had mild atrophy of the marginal zones including 1 out of 5 in the 30-day medium dose males, 1 out of 6 in the 30-day high dose males, 2 out of 5 in the 60-day low dose females, and 2 out of 6 in the 60-day medium dose females.
Ileum: Five rats had slight involution of the T-dependent (interfollicular) regions including: 1 out of 2 males and 1 out of 5 females from the 30-day low dose groups, 1 out of 4 males from the 30-day medium dose group, 1 out of 6 females from the 60-day medium dose group, and 1 out of 4 males from the 90-day low dose group.
Bone Marrow: One male in the 90-day medium dose had an increased proportion of granulocytes relative to controls while one male in the 90-day high dose had moderate diffuse atrophy with fatty replacement.
Dose descriptor:
NOAEL
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Total serum Ig levels:

Thirty-day old female and male rats: there was a significant positive correlation (Pearson Product Moment Correlations) between serum total IgM and increasing test material doses in the 30-day old female rats (pg Ig/ml serumx 104 Mean ± SE Control: 51.6 ± 8.8, Low: 41.2 ± 6.8, Medium: 39.6 ± 5.8 and High: 66.5 ± 9.9 (P = 0.0243). However, there were no significant differences in IgM levels among dose groups using ANOVA. Also there were no treatment-related effects on Ig levels in the 30-day old male rats. Sixty-day old female rats: serum IgM levels (pg Ig/ml serumx 104 Mean ± SE) in the 60-day female rats were significantly higher than control levels in the low and high dose groups. Control: 34.0 ± 3.2, Low: 63.8 ± 5.8, Medium: 68.1 ± 16.4, High: 73.0 ± 15.0 (P 0.05, ANOVA and pairwise comparisons). Although levels looked higher in the meditun dose group as well, the differences were not statistically significant. There were no other significant Ig changes in female rats.

Ninety-day old male rats: there was a significant decrease in the IgA levels at. the middle dose group compared with the control. A significant increase was noted for serum IgM in the high dose group, for serum IgG in the medium and high doses and for IgG2a in the high dose (Table 1).

Flow cytometric analysis of splenocytes:

Thirty-day old female and male rats: In the 30-day female rats there was a statistically significant trend towards increased mean percentage and absolute NK cell numbers (P = 0.0016 and P = 0.0026, respectively) (Tables 2 and 3). Pairwise comparisons indicated that the treated to control differences were statistically significant for the high dose (P = 0.0227 and P = 0.0227 for the percentage and absolute numbers, respectively) (Tables 2 and 3). All other cell types were not affected significantly by treatment (P>0.05).

In the 30-day male rats there was a statistically significant treatment-related trend towards increased mean percent and absolute NK cell numbers (P = 0.00052 and P = 0.0027, respectively) (Tables 4 and 5). Pairwise comparisons indicated that the treated to control differences were statistically significant for the high dose (P = 0.009 and P = 0.0352 for the percentage and absolute numbers, respectively) (Tables 4 and 5). A statistically significant nonlinear trend towards decreased mean percentage CD8+ cell numbers was noted (P = 0.0247) which was due to a decrease in the medium dose in comparison to the control (P = 0.0217) This resulted in a nonlinear dose-related increase in the CD4+:CD8+ cell ratio (P = 0.0232) with pairwise comparisons being significant for the medium dose (P = 0.0107). All other cell types were not affected significantly by treatment (P >0.05).

Sixty-days old female rats: there was a dose-related trend towards a decrease in the mean percentage CD5+ (T lymphocytes) (P = 0.0269) but the differences between treated and control were not significant (P>0.05). In addition, there was an increase in the mean% double positive CD4+8+ (T lymphocytes) (trend P = 0.0139) with the middle and high doses being significantly different from the control (P = 0.164 and P = 0.054, respectively) (Table 6). The absolute CD4+8+ cell numbers were not affected significantly by treatment (Table 7). Also there was a dose-related trend towards an increase in the % NK cell numbers (P = 0.064) but the differences between treated and control were not significantly different (P> 0.05).

Ninety-day old male rats: statistically significant effects included the following: The mean % NK cell numbers were linearly increased with dose (trend P < 0.0001) with the high dose group being significantly different from the control (P = 0.0103) (Table 8).

There was also a significant increase in the low dose over the control in the mean % lymphocytes (CD5+ L Gate), the mean %Th/i; lymphocytes (CD5+4+ Lymphocyte Gate), the mean % Ts/c lymphocytes (CD5+8+ Lymphocyte Gate) (P = 0.0237, P = 0.0538 and P = 0.0018, respectively) (Table 8). The mean absolute B-cell numbers (CD45RA+ Lymphocyte Gate) in the low dose group were reduced compared with the control (P = 0.0321) (Table 9). All other cell types were not affected significantly by treatment (P = 0.05).

Response to SRBC antigens (IgM) The anti-SRBC IgM response in the 60-day old female rats and the 90-day old male rats determined either by the ELISA plaque forming cell assay or the haemolytic complement assay (titres) were not affected significantly by treatment.

Lymphocyte transformation:

There were no statistically significant treatment-related effects on the lymphoproliferative activity of splenocytes in response to mitogen stimulation determined in the 60-day old female rats and the 90-day old male rats.

Delayed-type hypersensitivity response:

At the termination of treatment, the 60-day old female rats and the 90-day old male rats were sensitized with oxazolone and challenged 5 days later. The DTH response was expressed as change (mm) in ear thickness over the acetone control. For the 60-day old female rats there were no treatment-related effects on DTH responses (P>0.05) (Table 10). For the 90-day old male rats there was a statistically significant trend towards a decrease in DTH response with increasing dose (linear trend P = 0.046 at 24 h and P = 0.047 at 48 h).

L. Monocytogenes infectivity assay:

The L. monocytogenes infectivity assay was carried out in the 60-day old female rats and the 90-day old male rats. In the 60-day old female rats the mean colony forming bacteria was non-linearly increased at 48 h post-infection (P = 0.044) and was statistically significant in pairwise comparisons only for the control vs. the medium dose group (48 h: P = 0.0207 and 72 h: P = 0.0334) (Table 11). In the 90-day old male rats there was a non-linear dose-response trend at 72 h post-infection (P = 0.045) but in pairwise comparisons there were no statistically significant treatment-related effects (Table 12).

Natural killer cell activity:

NK cell activity was measured in the 60-day old female rats and 90-day old male rats using the 4 h 51Cr release assay using three effector (E):target (T) cell ratios. For the 60-day old female rats there was a dose response statistically significant increase in NK cell activity at the 50:1 and 100:1 E:T cell ratios (P = 0.030 and P = 0.052, respectively) (Table 13). However, the pairwise comparisons (treated vs. control) were statistically significantly increased only in the low dose group (P = 0.007 and P = 0.015 for the E:T cell ratio of 50:1 and 100:1, respectively (Table 13). For the 90-day old male rats there was a statistically significant dose response trend towards increased levels of NK cell activity at the 25:1 and 50:1 cell ratios (P = 0.028 and P = 0.009 for the E:T cell ratios of 25:1 and 50:1, respectively). The pairwise comparisons (treated vs. control) were significant at all dose groups (for the E:T cell ratio 25:1 P = 0.025, P = 0.011 and P = 0.004 for the low, medium and high dose, respectively, and for the E:T cell ratio of 50:1 P = 0.003, P = 0.005 and P = 0.009 for the low, medium and high dose, respectively) (Table 14).

Cytokine levels in serum:

The ELISA basal serum levels for IL-2, TNF-α, IFN-γ and IL-1β in the treated rats were not significantly different from the control.

Conclusions:
Data resulting from the present studies indicated that the test material at levels which include the currently calculated TDI dose of 0.25 µg/kg bw/day, affected a number of specific and non-specific immune parameters
Executive summary:

The immunotoxic effects of the test material were examined in the offspring of Sprague-Dawley rats exposed in utero from day 8 of gestation, through lactation and post-weaning until pups reached the age of 30 days (male and female), 60 days (female) and 90 days (male). Daily oral (gavage) doses of 0.025, 0.25 and 2.5 mg/kg body weight/day were administered in olive oil 7 days/week. Immunologic endpoints were investigated at the termination of each study. Statistically significant results (P < 0.05) included the following: At 30 days, the mean percent and absolute natural killer (NK) cell numbers were increased in male and female rats treated with the high test material dose. At 60 days, female rats had increased mean serum IgM levels at the low and high doses, increased mean percentage CD4+8+ (immature) T lymphocytes at the middle and high doses, a non-linear dose-response increase in NK cell activity at the 50:1 and 100:1 effector:target cell ratios (pairwise comparisons significant at the low dose compared with control), and increased mean numbers of L. monocytogenes colony-forming bacteria on Day 2 post-infection (significant for trend) and Day 3 post infection (pairwise comparisons significant only in the middle dose). The 90-day male rats had decreased mean serum IgA levels at the middle dose group; increased IgM levels at the high dose group, increased IgG levels at the middle and high doses; decreased IgG2a in the high dose compared to the control; a dose-related increase in the mean percentage NK cell numbers (pairwise comparisons significant at the high dose compared with the control) and increased mean NK cell activity (pairwise comparisons significant at all dose groups compared with the control). The delayed-type hypersensitivity response to oxazolone was increased in the low and middle doses and decreased in the high dose. Thymus atrophy was observed in the high test material dose across all ages. Thus, in utero and post-natal treatment of F1 rats with low levels of the test material affected some aspects of humoral and cell mediated immunity as well as the number and function of cells which are involved in the host's immunosurveillance mechanisms against tumours and viral infections.

Effect on immunotoxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Effect on immunotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No key values were determined for immunotoxicity, the data were specific investigations for immunotoxic effects and were provided as further toxicological information of

the test material.

The results of the various supporting studies would indicate that the test material does have effects on immunotoxicity.

- In the Snoeij et al (1988) paper, atrophy of the thymus was found in rats given a single oral dose of the test material. Doses as low as 10 mg of test material per kg body wt decreased relative thymus weight. 50 % reduction in thymus weight at 29 mg/kg bw of test material exposure. Thymus atrophy caused by the test material is due to a selective effect on the population of large-sized, rapidly proliferating lymphoblasts. These cells, which are present in a limited number generate the large population of small, non-dividing cells that populate the thytmic cortex. As a result of this selective action on the lymphoblasts, a marked depletion of cortical lymphocytes develops several clays after exposure to the test material.

A reliability rating of 4 was assigned to this study, according to the criteria of Klimisch, 1997 as the method does not follow recognised guidelines and there is no GLP or purity information presented.

- In the Bressa et al (1991) paper, a 1-month feeding trialwas conducted in which the test material was fed to rats at concentrations of 5 ppm and 25 ppm. Throughout the study, the mean body weight gain and the food consumption was significantly less in rats treated with 25 ppm pure test material as compared to control rats. Animals killed after 28 days of exposure to 25 ppm of pure test material showed a marked decrease in weight gain. It was also noted that two of four rats receiving diets containing 25 ppm of pure test material showed abnormal 'burrowing' behaviour, immediately after injection with the anaesthetic prior to autopsy. Thymus weights showed both an absolute and a relative decrease in rats given pure test material

. Lymph nodes in all rats exposed to the test material were markedly haemorrhagic. Histological examination showed partial atrophy of the nodes, with blood pools in the interstitial space. A few lymphoid follicles remained but these showed almost total depletion of the T-dependent areas. No other gross pathological changes were observed. Histopathological examination of the thymus showed an almost complete restoration of normal structure. No histopathological abnormalities were detected in the spleen, liver or kidneys. The distribution of tin showed that concentrations in kidneys and livers were at least five times higher than in other organs.

A reliability rating of 4 was assigned to this study, according to the criteria of Klimisch, 1997 as the method does not follow recognised guidelines and there is no GLP or purity information presented.

 

-In the Snoeij et al (1985) paper, a 2-week feeding study was conducted in which the test material was fed to male weanling rats at dietary concentrations of 15, 50 and 150 ppm to evaluate their toxic effects, especially on the brains and the lymphoid organs, thymus and spleen. The test material caused a dose-related reduction of thymus weight. At a dietary concentration of 150 ppm decreases in thymus weight to 39 % of controls were found following treatment with the test material. Microscopically, thymus atrophy was associated with lymphocyte depletion in the thymic cortex. Only 16 % of the total number of nucleated thymocytes could be isolated from rats fed 150 ppm test material. These effects were completely reversed within 2 weeks. A dose-related decrease in spleen weight was noticed after 2 weeks feeding of the test material. Liver weights were increased in rats fed the test material for 2 weeks. Nevertheless, no enlarged livers and normal spleen weights were found upon feeding 100 ppm test material

for 4 weeks, whereas thymus weight was severely decreased. Therefore, atrophy of the thymus was considered to be the predominant effect of the test material. From this study it is concluded that the test material is primarily immunotoxic.

A reliability rating of 4 was assigned to this study, according to the criteria of Klimisch, 1997 as the method does not follow recognised guidelines and there is no GLP information presented.

 

- In the Tryphonas et al (2004) paper, the offspring of Sprague-Dawley rats exposed in utero from day 8 of gestation, through lactation and post-weaning until pups reached the age of 30 days (male and female), 60 days (female) and 90 days (male) were examined for immunotoxic effects. Daily oral (gavage) doses of 0.025, 0.25 and 2.5 mg/kg body weight/day were administered in olive oil 7 days/week. At 30 days, the mean percent and absolute natural killer (NK) cell numbers were increased in male and female rats treated with the the high test material dose. At 60 days, female rats had increased mean serum IgM levels at the low and high test material doses, increased mean percentage CD4+8+(immature) T lymphocytes at the middle and high doses, a non-linear dose-response increase in NK cell activity at the 50:1 and 100:1 effector:target cell ratios ), and increased mean numbers of L. monocytogenes colony-forming bacteria on Day 2 post-infection and Day 3 post infection. The 90-day male rats had decreased mean serum IgA levels at the middle dose group; increased IgM levels at the high dose group, increased IgG levels at the middle and high doses; decreased IgG2ain the high dose compared to the control; a dose-related increase in the mean percentage NK cell numbers (pairwise comparisons significant at the high dose compared with the control) and increased mean NK cell activity (pairwise comparisons significant at all dose groups compared with the control). The delayed-type hypersensitivity response to oxazolone was increased in the low and middle doses and decreased in the high dose. Thymus atrophy was observed in the high test material dose across all ages. Thus, in utero and post-natal treatment of F1 rats with low levels of the test material

affected some aspects of hutnoral and cell mediated immunity as well as the number and function of cells involved in the host's immunosurveillance mechanisms against tumours and viral infections.

A reliability rating of 2 was assigned to this study, according to the criteria of Klimisch, 1997 as the method is comparable to recognised guidelines but there in no GLP infomation presented..

Justification for classification or non-classification