(butylamine)[[2,2'-thiobis[4-(1,1,3,3-tetramethylbutyl)phenolato]](2-)-O,O',S]nickel

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 August 1984 to 2 November 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Read-across

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

6,6'-di-tert-butyl-4,4'-thiodi-m-cresol

EC Number:

202-525-2

EC Name:

6,6'-di-tert-butyl-4,4'-thiodi-m-cresol

Cas Number:

96-69-5

Molecular formula:

C22H30O2S

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Monsanto Industrial Chemical Company (Akron, OH, USA); Lot 12
- Purity: 99%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
The dose formulations were prepared weekly by mixing 4,4'-thiobis(6-t-butyl-m-cresol) with feed (Table 11). Homogeneity and stability studies of the 250 and 25,000 ppm dose formulations were performed by the analytical chemistry laboratory. For the homogeneity and stability studies, dose formulations were analyzed by high performance liquid chromatography. Homogeneity was confirmed at the 100 and10,000ppm concentrations, and stability was established at these concentrations for at least 3 weeks at -20" C when stored in the dark and for 3 days when exposed to air and light.

Test animals

Species:

rat

Strain:

other: F344/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Frederick Cancer Research Center (Frederick, MD, USA)
- Age at study initiation: 43 days
- Weight at study initiation: 106-142 g
- Housing: 5 per cage; Polycarbonate cages, (Suburban Surgical Co., Inc., Wheeling, IL), changed twice weekly with SaniChip hardwood chips (PJ. Murphy Forest Products Corp., Rochelle Park, NJ), changed twice weekly
- Diet: NIH-07 open formula meal diet (Zeigler Brothers, Inc., Gardners, PA), available ad libinun, changed daily
- Water: Tap water (Woburn municipal supply) via automatic watering system (Hardco, Cincinnati, OH),available ad libitum


DETAILS OF FOOD AND WATER QUALITY:Refer to Appendix K

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.7°C
- Humidity (%):41% to 60%
- Air changes (per hr): 12 changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

- DIET PREPARATION
A premix of feed and 4,4'-thiobis(6-t-butyl-m-cresol) was prepared, then layered into the remaining feed and blended in a Patterson-Kelley twin-shell blender with the intensifier bar on for 5 minutes and off for 10 minutes. The dose formulations were prepared weekly by mixing 4,4'-thiobis(6-t-butyl-m-cresol) and feed to give the required concentrations (Appendix I - Table I1). Formulations were discarded 2 weeks after the date of preparation. The dose formulations were stored in sealed double plastic bags in plastic bins at at -18°C or less for the 13-week studies.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Periodic-analyses of the dose formulations of TBBC were conducted at the study laboratory and analytical chemistry laboratory (Midwest Research Institute (Kansas City, MO, USA) using high-performance liquid chromatography. During the 13-week study, the dose formulations were analyzed every 6 to 10 weeks (Appendix I - Table I3). Results of the periodic referee analyses performed by the analytical chemistry laboratory were in good agreement with the results obtained by the study laboratory (Appendix I - Table I5).

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 ppm

Remarks:

15 mg/kg bw/day in males/females

Doses / concentrations 2

Dose / conc.:

500 ppm

Remarks:

30/35 mg/kg bw/day in males/females

Doses / concentrations 3

Dose / conc.:

1 000 ppm

Remarks:

60/70 mg/kg bw/day in males/females

Doses / concentrations 4

Dose / conc.:

2 500 ppm

Remarks:

165/170 mg/kg bw/day in males/females

Doses / concentrations 5

Dose / conc.:

5 000 ppm

Remarks:

315/325 mg/kg bw/day in males/females

No. of animals per sex per dose:

10 males
10 females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
Groups of 10 male and 10 female F344/N rats were fed diets containing 0, 1,000, 2,500, 5,000, 10,000 or 25,000 ppm TBBC for 15 days. Rats given to 1,000, 2,500, 5,000, or 10,000 ppm received approximate doses of 95,235,335, or 365 mg TBBC per kilogram bodyweight per day (males) or 85, 220,325, or 270 mag per day (females). Approximate doses for rats receiving 25,000 ppmcould not be calculated due to early deaths. All 25,000ppm rats and three male and four female 10,000 ppm rats died. Surviving rats in the 10,000ppm groups had a significant weight loss and the final mean body weights of 5,000 and 10,000 ppm male and female rats were significantly lower than those of the controls. Male and female rats exposed to 5,OOO, 10,000, or 25,000 ppm TBBC consumed markedly less feed than the controls.

Diarrhea occurred in 5,000, 10,000, and 25,000 ppm males and females. The principal lesions attributed to the administration of TBBC were renal papillary and tubule necroses which occurred in 10,000 ppm rats. Focal necrosis or erosions of the glandular stomach also occurred in some 10,000ppm rats. Changes observed in the thymus and spleen were attributed to debilitation or stress; bone marrow depletion was attributed to nutrient deficiency accompanying weight loss (Tables 2,3, F1, G1).

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: Observed twice daily; clinical observations were recorded weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed initially, weekly, and at the end of the studies;

FOOD CONSUMPTION AND COMPOUND INTAKE
Feed consumption was recorded daily by cage

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified


HAEMATOLOGY: Yes
Blood was collected from all animals from the orbital sinus; hematocrit,hemoglobin, erythrocytes, mean erythrocyte volume, reticulocytes, leukocyte differentials, and nucleated erythrocytes


CLINICAL CHEMISTRY: Yes
Blood was collected by cardiac puncture; urea nitrogen, creatinine, alkaline phosphatase, alanine aminotransferase,and y-glutamyltranspeptidase


NEUROBEHAVIOURAL EXAMINATION: Yes
Male and female 0, l,OOO, and 2,500 ppm rats were tested for forelimb and hindlimb grip strength, tail flick, startle response, and foot splay.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Necropsy performed on all animals. Organs weighed were brain, heart, right kidney, liver, lung, spleen, right testis, and thymus.

HISTOPATHOLOGY: Yes
Tissues for microscopic examination were fmed and preserved in 10% neutral buffered formalin, processed andtrimmed,embedded in paraffin, sectioned to a thickness of 5 to 6 pm, and stained with hematoxylin and eosin.Complete histopathology was performed on 0, 1,000, 2,500, and 5,000 ppm rats. In addition to gross lesions and tissue masses, the tissues examined included: adrenal gland, brain, clitoral gland (rats), esophagus, gallbladder (mice), heart, kidney, large intestine (cecum. colon, rectum), liver, lung, mammary gland, mandibular or mesenteric lymph node, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland (rats), prostate gland, salivary gland, skin, small intestine (duodenum, jejunum, ileum), spleen, sternum and vertebra (including marrow), stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thyroid gland, thymus, trachea, urinary bladder, and uterus. Only the following tissues were examined from the 1.OOO and 2,500 ppm rats: liver and mandibular or mesenteric lymph node. The kidney from the 2,500 ppm rats was also examined.

Statistics:

Two approaches were employed to assess the significance of pair wise comparisons between exposed and control groupsin the analysis of continuous variables. Organ and body weight data, which have approximately normal distributions, were analyzed using the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Clinical chemistry and hematology data, which have typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964).

Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related trend (Dunnett’s or Dunn’s test). Average severity values were analyzed for significance using the Mann-Whitney U test (Hollander and Wolfe, 1973).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

All animals survived to the end of the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The final mean body weights of 5,000 ppm males and females were markedly lower than those of the controls; the mean bodyweight of males receiving 2,500 ppm was slightly but consistently lower than that of the controls throughout the study. Feed consumption by 5,OOO ppm rats was markedly lower than that by controls throughout the study. Feed consumption by 2,500 ppm males was somewhat reduced initially, but was similar to or greater than that by the controls after week 4. Rats exposed to 250, 500, 1,OOO. 2,500, or 5,OOO ppm received approximate doses of 15, 30, 60, 165, or 315 mg TBBC per kilogram body weight per day (males) or 15, 35, 70, 170, or 325 mgkg per day (females). Since reduction in feed consumption was apparent from the beginning of the study, the reduction would seem more likely to have been caused by decreased feed palatability than by anorexia resulting from toxicity. This conclusion is supported by the fact that diarrhea, the major clinical finding in 5,OOO ppm rats, did not appear in the males until day 64 (with the exception of one male in which diarrhea was observed on day 29) or in the females until day 57.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The final mean body weights of 5,000 ppm males and females were markedly lower than those of the controls; the mean bodyweight of males receiving 2,500 ppm was slightly but consistently lower than that of the controls throughout the study. Feed consumption by 5,OOO ppm rats was markedly lower than that by controls throughout the study. Feed consumption by 2,500 ppm males was somewhat reduced initially, but was similar to or greater than that by the controls after week 4. Rats exposed to 250, 500, 1,OOO. 2,500, or 5,OOO ppm received approximate doses of 15, 30, 60, 165, or 315 mg TBBC per kilogram body weight per day (males) or 15, 35, 70, 170, or 325 mgkg per day (females). Since reduction in feed consumption was apparent from the beginning of the study, the reduction would seem more likely to have been caused by decreased feed palatability than by anorexia resulting from toxicity. This conclusion is supported by the fact that diarrhea, the major clinical finding in 5,OOO ppm rats, did not appear in the males until day 64 (with the exception of one male in which diarrhea was observed on day 29) or in the females until day 57.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematocrit and hemoglobin concentrations in male rats exposed to l,OOO, 2,500, and 5,OOO ppm were significantly lower than those of the controls; these results suggest a mild anemia (Table G2). However, considering the diarrhea and unthriftiness that occurred in these animals, possible dehydration could be masking larger decreases, including decreases in erythrocyte counts, or could account for the absence of changes in hematocrit or hemoglobin values in females. Since reticulocyte counts in male rats were not higher than those of the controls, the anemia in the male rats was considered nonresponsive. Mean erythrocyte volume was significantly lower in males that received 1,OOO or 2,500 ppm TBBC and in males and females that received 5,OOO ppm; this effect is usually associated with a disturbance in hemoglobin production and has commonly been observed with anemias of chronic inflammation or iron deficiency.

Total leukocyte counts were significantly higher in 5,OOO ppm females and slightly increased in 5,OOO ppm males. Male and female rats that received 5,OOO ppm also exhibited significantly higher segrnented neutrophil counts. Band neutrophil counts were significantly higher in all exposed female groups than in controls; the largest increase occurred in 5,OOO ppm rats. These changes in leukocyte para- meters are consistent with an inflammatory response.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Serum alkaline phosphatase levels were significantly higher in 2,500 and 5,OOO ppm males and were slightly higher in the females exposed to 5,OOO ppm (Table G2). Males and females exposed to 2,500 or 5,OOO ppm TBBC had significantly higher serum alanine levels. Increased aminotransferase activity of y-glutamyl transpeptidase in rats exposed to 5,OOO ppm was not considered to be biologically significant.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

A significant increase in absolute and relative liver weights occurred in females that received 5,OOO ppm TBBC (Table F2).The relative, but not absolute, liver weight of 2,500 ppm males was significantly increased. As in the 15-day study, other significant differences in absolute or relative organ weights were considered due to much lower final mean body weights and not to organ-specific toxicity.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The principal lesions associated with the administration of TBBC for 13 weeks occurred in the liver and kidney, primarily in 2,500 and 5,OOO ppm males and females (Table 5).

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

Results of three neurotoxicity trials in 0, l,OOO, and 2,500 ppm rats demonstrated a significant dose related increase in forelimb and hindlimb grip strength (Table Hl). Foot splay, tail flick, and startle response reflexes were unaffected by exposure to TBBC.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The lesions in the liver consisted of scattered individual cell necrosis, individual or aggregates of enlarged Kupffer cells with abundant yellow-tan pigmented cytoplasm (Kupffer cell hypertrophy), focal accumulations of similar macrophages in or adjacent to the portal areas, and a slight increase in small bile ductules in the portal areas (Plate 3). By electron microscopy, the pigmented material in the cytoplasm of Kupffer cells was amorphous to finely granular and light to moderately electron dense with a scattering of irregular, highly electron-dense bodies. While the more abundant amorphous substance was not membrane bound, many of the smaller electron-dense bodies were partially surrounded by a plasma membrane. The cytoplasm of the Kupffer cells stained strongly positive with PAS, weakly to strongly by the Ziehl-Neelsen method for acid-fast material, and inconsistently weakly positive by Perl’s iron method. While not observed by the study pathologist, enlargement of centrilobular hepatocytes, relative to the periportal hepatocytes, in the 5,OOO ppm group was also observed by the Pathology Working Group. This finding is consistent with hepatocellular hypertrophy and with the higher activities of serum enzymes in the 2,500 and 5,OOO ppm groups.

The kidney lesions consisted of focal, segmental degeneration and necrosis of the proximal tubule epithelium, primarily in the outer stripe of the outer medulla, and extensive pigmentation of the proximal convoluted tubule epithelium (Plate 4). The degeneration and necrosis were characterized by faintly stained, pale cells with little cytoplasmic or nuclear detail, suggestive of cytolysis and karyolysis. The pigmentation was characterized by pale, yellow-red discoloration of the epithelial cytoplasm.

Both the size and number of macrophages were increased in the mesenteric lymph nodes of male and female rats exposed to 2,500 or 5,OOO ppmTBBC (Table 5)

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Effect level:

35 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

60 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

In a 90 day repeated dose toxicity study in F344/n rats treated with 250, 500, 1,000, 2,500, or 5,000 ppm TBBC, the NOAEL was 30/35 mg/kg bw/day for males/females.

Executive summary:

In a subchronic toxicity study (95-3166), TBBC (99%) was administered to groups of F344/N rats (10 animals/sex/group) in the diet at dose levels of 0, 250, 500, 1,000, 2,500, or 5,000 ppm (15, 30/35, 60/70, 165/170 and 315/325 mg/kg bw/day for males/females) for 7 days per week for 13 weeks.

All animals survived to the end of the study. The final mean body weights of 5,000 ppm males and females were markedly lower than those of the controls; the mean bodyweight of males receiving 2,500 ppm was slightly but consistently lower than that of the controls throughout the study. Feed consumption by 5,000 ppm rats was markedly lower than that by controls throughout the study. Feed consumption by 2,500 ppm males was somewhat reduced initially, but was similar to or greater than that by the controls after week 4. Since reduction in feed consumption was apparent from the beginning of the study, the reduction would seem more likely to have been caused by decreased feed palatability than by anorexia resulting from toxicity. This conclusion is supported by the fact that diarrhea, the major clinical finding in 5,OOO ppm rats, did not appear in the males until day 64 (with the exception of one male in which diarrhea was observed on day 29) or in the females until day 57.

A significant increase in absolute and relative liver weights occurred in females that received 5,000 ppm TBBC. The relative, but not absolute, liver weight of 2,500 ppm males was significantly increased. As in the 15-day study, other significant differences in absolute or relative organ weights were considered due to much lower final mean body weights and not to organ-specific toxicity.

Serum alkaline phosphatase levels were significantly higher in 2,500 and 5,000 ppm males and were slightly higher in the females exposed to 5,000 ppm. Males and females exposed to 2,500 or 5,000 ppm TBBC had significantly higher serum alanine levels. Increased aminotransferase activity of y-glutamyl transpeptidase in rats exposed to 5,000 ppm was not considered to be biologically significant. Hematocrit and hemoglobin concentrations in male rats exposed to 1,000, 2,500, and 5,000 ppm were significantly lower than those of the controls; these results suggest a mild anemia). However, considering the diarrhea and unthriftiness that occurred in these animals, possible dehydration could be masking larger decreases, including decreases in erythrocyte counts, or could account for the absence of changes in hematocrit or hemoglobin values in females. Since reticulocyte counts in male rats were not higher than those of the controls, the anemia in the male rats was considered nonresponsive. Mean erythrocyte volume was significantly lower in males that received 1,000 or 2,500 ppm TBBC and in males and females that received 5,OOO ppm; this effect is usually associated with a disturbance in hemoglobin production and has commonly been observed with anemias of chronic inflammation or iron deficiency. Total leukocyte counts were significantly higher in 5,000 ppm females and slightly increased in 5,000 ppm males. Male and female rats that received 5,000 ppm also exhibited significantly higher segrnented neutrophil counts. Band neutrophil counts were significantly higher in all exposed female groups than in controls; the largest increase occurred in 5,000 ppm rats. These changes in leukocyte parameters are consistent with an inflammatory response. Results of three neurotoxicity trials in 0, 1,000, and 2,500 ppm rats demonstrated a significant dose related increase in forelimb and hindlimb grip strength. Foot splay, tail flick, and startle response reflexes were unaffected by exposure to TBBC.

The principal lesions associated with the administration of TBBC for 13 weeks occurred in the liver and kidney, primarily in 2,500 and 5,000 ppm males and females. The lesions in the liver consisted of scattered individual cell necrosis, individual or aggregates of enlarged Kupffer cells with abundant yellow-tan pigmented cytoplasm (Kupffer cell hypertrophy), focal accumulations of similar macrophages in or adjacent to the portal areas, and a slight increase in small bile ductules in the portal areas. By electron microscopy, the pigmented material in the cytoplasm of Kupffer cells was amorphous to finely granular and light to moderately electron dense with a scattering of irregular, highly electron-dense bodies. While the more abundant amorphous substance was not membrane bound, many of the smaller electron-dense bodies were partially surrounded by a plasma membrane. The cytoplasm of the Kupffer cells stained strongly positive with PAS, weakly to strongly by the Ziehl-Neelsen method for acid-fast material, and inconsistently weakly positive by Perl’s iron method. While not observed by the study pathologist, enlargement of centrilobular hepatocytes, relative to the periportal hepatocytes, in the 5,000 ppm group was also observed by the Pathology Working Group. This finding is consistent with hepatocellular hypertrophy and with the higher activities of serum enzymes in the 2,500 and 5,000 ppm groups. The kidney lesions consisted of focal, segmental degeneration and necrosis of the proximal tubule epithelium, primarily in the outer stripe of the outer medulla, and extensive pigmentation of the proximal convoluted tubule epithelium. The degeneration and necrosis were characterized by faintly stained, pale cells with little cytoplasmic or nuclear detail, suggestive of cytolysis and karyolysis. The pigmentation was characterized by pale, yellow-red discoloration of the epithelial cytoplasm. Both the size and number of macrophages were increased in the mesenteric lymph nodes of male and female rats exposed to 2,500 or 5,000 ppm TBBC. The NOAEL for males was 30 mg/kg bw/day and for females was 35 mg/kg bw/day, based on the liver lesions.

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study (OECD 408) in rats.