2,3,5,6-tetrachloropyridine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Sensory activity was not measured but this was not a requirement at the time the study was conducted.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,3,5,6-tetrachloropyridine

EC Number:

219-283-9

EC Name:

2,3,5,6-tetrachloropyridine

Cas Number:

2402-79-1

Molecular formula:

C5HCl4N

IUPAC Name:

2,3,5,6-tetrachloropyridine

Details on test material:

Reference: AGR 289294
Purity = 98.6% (subsequently re-analysed and found to be of 99.1% purity)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Adult male and female Sprague-Dawley rats (Charles River Breeding Laboratories, Kingston, NY), approximately 10 weeks of age, were purchased for the study. Extra rats of both sexes were ordered to ensure that a sufficient number of animals of acceptable health and weight were available to conduct the study as designed. This strain of rat was selected because of its general acceptance and suitability for toxicity testing, reliability of the commercial supplier, and availability of historical background data. Upon arrival at the laboratory) the rats were examined for health status by the laboratory veterinarian and acclimated at least one week to laboratory conditions according to the Standard Operating Procedures of the Reproductive Toxicology Group. For the randomization procedure, the rats were weighed and ranked according to body weight, and those at the extremes of the distribution were identified and removed from the test population until the required number of animals remained. The remaining rats were assigned to the exposure groups by weight. This procedure was intended to increase the probability of uniform group mean body weights and standard deviations at the initiation of the study. All rats not placed on study were removed from the study room. Rats were identified by inserting a uniquely coded alphanumeric metal tag in the ear of each rat.

Rats were housed singly in wire-mesh, stainless steel cages provided with cageboard to minimize odor and maintain a clean environment, or in plastic cages provided with ground corn cob nesting material during gestation and lactation. The animal rooms of the facility were designed to maintain humidity at approximately 40-60%, temperature at approximately 20°C, photoperiod at 12 hours light : 12 hours dark, and air flow at 10 changes / hour. A feed crock and pressure-activated stainless steel water nipple were components of all cages. Feed (Certified Laboratory Rodent Chow No. 5002, Purina Mills, Inc., St. Louis, MO), the vehicle for the test material, and municipal tap water were available ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% METHOCEL A4M

Details on oral exposure:

The test material was administered as a suspension in an aqueous solution of 0.5% METHOCEL.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the dose suspensions indicated that each mixture were within 90 to 97% of the targeted concentration and that the stability of SYMTET in 0.05% aqueous METHOCEL was at least 43 days. Homogeneity analyses revealed that the test material was uniformly distributed in 0.05% aqueous METHOCEL.

Duration of treatment / exposure:

52 days in the main study, with a 14-day palatability study.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
5 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
25 mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
150 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels in the main study were selected based on the results of the 14 day oral gavage study.

The parental rats were maintained on control feed throughout the study. Body weights were recorded daily and dose volumes were adjusted daily based on individual body weights. After 14 days of dosing, the parental rats were mated (one male to one female of the respective treatment group) for a maximum of 14 days to produce the Fl litters. Dosing of both sexes continued throughout the gestation and lactation periods. All parental animals and litters were observed daily for changes in behavior or demeanor. Any animal which died, or appeared moribund was submitted for a complete necropsy. Feed consumption was measured at selected intervals. Pups were euthanatized on Day 4 of lactation and examined macroscopically. Approximately 24 hours after the dam's litter had been euthanatized ("lactation" Day 5), each dam was fasted overnight, and submitted for a complete necropsy by a veterinary pathologist. Adult males and females that did not deliver a litter were fasted overnight and necropsied. Blood samples were taken from the first 10 fasted adult male rats/dose and the first 10 fasted dams/dose at necropsy for hematologic and clinical chemistry determinations. Selected organs were weighed and examined microscopically.

Breeding Procedure.
After 14 days of treatment, each female was placed with a male (one female and one male of the respective treatment group) until mating had occurred or until 14 days had elapsed. Vaginal lavage samples were evaluated each morning for the presence of sperm as an indication of mating. The day on which sperm was detected or a vaginal plug was observed in situ was considered Day 0 of gestation. Sperm positive females were then separated and placed in nesting cages. Females which failed to mate were separated from the males and placed in nesting cages at the end of the I4-day mating
period.

Positive control:

Not applicable.

Examinations

Observations and examinations performed and frequency:

Physical Observations.
Each rat on study was observed at least once daily for changes in behavior or demeanor. All adult rats found dead or in a moribund condition were submitted for a gross pathologic examination. Any pup found dead during the lactation phase was examined grossly by personnel from the Reproductive Toxicology Group, to the extent possible, and discarded.

Litter Data.
All litters were examined as soon as possible after delivery. The following parameters were recorded on each litter: the date of parturition, litter size on the day of parturition (Day 0), the number of live and dead pups on Days 0, 1 and 4 postpartum, and the sex and the weight of each pup on Days 1 and 4 of lactation. Any visible physical abnormalities or demeanor changes in the neonates were recorded during the lactation period. Any pup that died during the test period was examined macroscopically to the extent possible and discarded. On lactation Day 4 following collection of litter data, all pups were euthanatized by personnel from the Reproductive Toxicology Group, examined macroscopically and discarded.

Body Weights and Feed Consumption.
All adult animals had feed consumption recorded weekly during the 14-day prebreeding treatment period, beginning on the week prior to the start of the study. Body weights for males and females were recorded daily throughout the study. Body weights for sperm-positive females on Days 0, 7, 14 and 21 of gestation were chosen for statistical analysis, as were body weights on lactation Days 1 and 4 for females that delivered litters. During breeding, feed consumption was not measured in males or females due to cohousing. Following completion of the breeding periods, weekly feed consumption was again measured in males and females that did not breed. During gestation, feed consumption was measured at weekly intervals in sperm-positive females. After parturition, the feed consumption of females with litters was measured during lactation Days 1 through 4.

Sacrifice and pathology:

Pathology.
A complete necropsy was conducted by a veterinary pathologist on all adults. Dams were necropsied approximately 24 hours after euthanasia of their litters ("lactation" Day 5). If "lactation" Day 5 fell on a weekend, dams were necropsied on the following workday. Adult males and adult females that did not deliver a litter were submitted for necropsy on test Day 53. The adults were fasted overnight, weighed, anesthetized with methoxyflurane and euthanatized. The eyes were examined in situ as described previously. Weights of the liver, kidneys, thymus, testes, and epididymides on the adult animals were recorded during necropsy. Tissues routinely collected were saved from these rats and preserved in neutral, phosphate-buffered 10% formalin, with the exception of the testes and epididymides which were preserved in Bouin's fixative. The lungs were infused with formalin to their approximate normal inspiratory volume. The nasal cavity was flushed with formalin via the pharyngeal duct to ensure rapid fixation of the tissue. Rats that died or were euthanatized due to their moribund condition also were given a complete necropsy, however, organ weights were not obtained. Histologic examination of the liver, kidneys, adrenals, brain, heart, spleen, ovaries, testes, epididymides and potential target organs were conducted on the control and high-dose groups. Examination of tissues from the low and middle groups was limited to those tissues which demonstrated treatment¬related histologic changes in the high-dose group. Examination of the uteri for the number of implantation sites and the ovaries for corpora lutea was conducted by personnel from the Reproductive Toxicology Group. The uteri of apparently non-pregnant animals were stained with a 10% aqueous solution of sodium sulfide (Kopf et al., 1964) and examined for evidence of early resorptions.

Other examinations:

Clinical Chemistry.
Blood samples from the first 10 fasted adult male rats/dose and the first 10 fasted dams/dose were obtained at necropsy. Blood was drawn from the orbital sinus of the rats under light methoxyflurane anesthesia. Clinical biochemical determinations of alkaline phosphatase (AP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine phosphokinase (CPK) activities as well as urea nitrogen
concentration (UN), creatinine (CREAT), total protein (TP), albumin (ALB), globulin (GLOB calculated), glucose (GLUC), cholesterol (CHOL), triglycerides (TG), and total bilirubin (TBILI) were made on all serum samples. Electrolyte levels (Na, K, P, Cl, Ca) were also determined. All analyses were conducted on a Monarch 2000 Chemistry System (Instrument Laboratory Inc., Lexington, MA.).

Hematology.
The same blood samples as above were used to obtain hematology measurements. Hematocrit (HCT), hemoglobin (HGB) concentration, erythrocyte count (RBC), total leukocyte count (WBC) and platelet count (PLAT) were evaluated for each animal sampled, using an ELT-8 (Ortho Instruments, Westwood, MA). Blood smears were prepared and stained with Wright's stain. Complete blood smear examinations included
differential leukocyte counts and an assessment of erythrocyte, leukocyte and platelet morphology.

Statistics:

Descriptive statistics (means and standard deviations) were reported for feed consumption for the 14-day dietary phase and the repeat dose and reproductive/developmental toxicity screen. For the 14-day oral gavage phase, descriptive statistics (means and standard deviations) were reported for all parameters measured. Statistical outliers were identified using a sequential method, but only values for feed consumption were routinely excluded unless justified by sound scientific reasons unrelated to treatment.

Body weights, body weight gains, absolute and relative organ weights, clinical chemistry data and appropriate hematology data first were evaluated by Bartlett's test for equality of variances. Based upon the outcome of Bartlett's test, either a parametric or nonparametric analysis of variance (ANOVA) was conducted. If the ANOVA was significant, a Dunnett's test or the Wilcoxon Rank-Sum test with Bonferroni's correction was performed. Gestation length and average time to mating were analyzed using a nonparametric ANOVA. If the ANOVA was significant, the Wilcoxon Rank-Sum test with Bonferroni's correction was performed. The fertility indices were analyzed by the Fisher exact probability test. Evaluation of the neonatal sex ratio was performed by the binomial distribution test. Survival indices and other incidence data among neonates were analyzed using the litter as the experimental unit by the Censored Wilcoxon test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Clinical Observations.
Three female rats either died or were euthanatized moribund prior to the scheduled necropsy. One female given 5 mg/kg/day was found dead on test Day 32. Although no significant clinical observations were made on this female prior to her death, gross pathologic examination of this animal indicated multifocal gastric erosions and/or ulcers, hemorrhagic and edematous lungs, icterus, pale anemic skin and enlarged spleen. The liver was also enlarged and the left lateral lobe was ruptured. The animal was pregnant with normal fetuses. Based on the gross pathologic examine, the cause of death was attributed to leukemia and liver rupture. A second female given 5 mg/kg/day was euthanatized on test Day 16 due to its moribund condition. Clinical observations of this rat included excessive chromodacryorrhea, facial soiling, hemorrhagic/bloody muzzle and nose, salivation and bone fragments protruding from the soft palate. Gross pathologic findings confirmed these clinical observations and diagnosed a fractured skull. The moribund condition of this animal was attributed to the fracture which appeared to be caused by the male which she was cohoused with during breeding. One female rat given 150 mg/kg/day was euthanatized on test Day 35. Clinical observations on this rat included facial soiling, excessive chromodacryorrhea and chromorhinorrhea, decreased activity, perineal soiling and vaginal bleeding. Gross pathologic examination, revealed focal gastric erosions and/or ulcers, hemolyzed blood in the digestive tract, perineal soiling constisting of blood, fetal resorptions and mottled kidneys. The moribund condition of this animal was subsequently shown to be treatment-related kidney toxicity.

Feed Consumption.
No significant treatment-related effects on feed consumption were observed at any dose level in males or females throughout the entire study.

Body Weights.
No significant treatment-related effects on body weights were observed at any dose level in males or females throughout the entire study.

Body Weight Gains.
No treatment-related effects on body weight gains were observed at any dose level in males or in females throughout the entire study.

Hematology.
Statistically identified differences in hematologic data occurred in male and female rats given 150 mg/kg/day and in male rats given 25 mg/kg/day. Inthe male rats, the differences consisted of a slightly lower hemoglobin concentration and hematocrit at 150 mg/kg/day and a slightly lower hemoglobin concentration at 25 mg/kg/day. In females given 150 mg/kg/day, the red blood cell count, hemoglobin concentration and hematocrit were also slightly lower than the controls. These decreased red blood cell parameters were not accompanied by treatment-related histopathologic changes in the bone marrow or spleen of either male or female rats given 150 mg/kg/day, and were within or in close proximity to the normal range for Sprague-Dawley rats (Charles River, 1984). Therefore these statistical differences were not attributed to toxicity to either the developing or mature red cells and were not interpreted to be biologically significant. The differential WBC count and cell morphology from all rats were considered to be within normal limits.

Clinical Chemistry.
There were no statistically identified differences in clinical chemistries of treated animals when compared with control. A number of clinical chemistry parameters of female rats given 150 mg/kg/day were altered and statistically identified. Increased urea nitrogen and creatinine levels were consistent with the inflammatory and degenerative kidney effects noted in female rats given 150 mg/kg/day and were secondary to kidney toxicity. A minor decrease in albumin and increase in cholesterol values in rats given 150 mg/kg/day may be related to alterations in liver function, as a result of hepatocellular hypertrophy. Decreased ALT activity occurred in female rats given 150 mg/kg/day and was not interpreted to be toxicologically significant. Increased ALT activity can be expected with liver, skeletal muscle and cardiac muscle diseases. The lower ALT activity in female rats given 150 mg/kg/day may reflect normal variation in activity of this enzyme or may indicate a lower background level of cellular degeneration in either the liver or muscle.

Organ Weights.
Absolute and relative liver weights of male and female rats given 150 mg/kg/day were increased and statistically identified. These weight differences were attributed to the centrilobular hepatocellular hypertrophy noted histologically in these rats. The relative liver weight of male rats given 25 mg/kg/day also was increased and statistically identified, however, this was not associated with a histopathologic liver effect and was not considered to be toxicologically significant. Liver weight and histologic differences were interpreted to be an adaptive change, secondary to the metabolism of SYMTET. This effect was not seen in conjunction with hepatocellular degeneration and necrosis. In addition although the males given 25 and 150 mg/kg/day were more effected than females, there was no indication of any adverse liver effects on serum chemistry parameters (AST,AP, TP, ALB). Absolute and relative kidney weights of male and female rats given 150 mg/kg/day also were increased and statistically identified. In male rats, this was associated with protein droplet nephropathy and epithelial cell pigmentation; whereas in female rats, the weight differences were associated with treatment-related inflammatory and degenerative kidney lesions. Absolute and relative epididymal weights in male rats given 150 mg/kg/day were decreased and statistically identified, however, there were no histopathologic lesions in either the epididymides or testes that indicated these weight differences were biologically or toxicologically significant. In addition, testes weights were not affected by treatment at any dose level tested.

Gross Pathologic Observations.
Alterations in the appearance of tissues occurred in male and/or female rats given 150 mg/kg/day SYMTET which consisted of pale foci in the renal cortex, and mottled or pale kidneys. Kidney alterations were interpreted to be treatment related. Other gross pathologic observations were considered to represent spontaneous changes that can occur in rats of this age, strain and sex.

Histopathologic Observations.
Treatment-related effects occurred in the liver and kidneys of male and female rats. Liver effects consisted of a slight centrilobular hepatocellular hypertrophy and occurred in male and female rats given 150 mg/kg/day. Similar alterations were not observed in male and female rats given 0, 5, or 25 mg/kg/day. Kidney effects in male and female rats were histologically dissimilar. An increased incidence of protein droplet nephropathy (PDN) and pigmentation of renal tubular epithelial cells occurred in male rats given 25 or 150 mg/kg/day, as compared to male rats given 0 or 5 mg/kg/day. PDN was subjectively characterized by the presence of increased numbers of apparent protein droplets with the cytoplasm of cortical tubular epithelial cells. These droplets appeared as eosinophilic to semi-translucent granules that were quite variable in size (approximately four to 60 µm in diameter). Occasional pyknotic renal tubular epithelial cells, dilated tubules and granular casts also were observed. Pigment occurred within the cytoplasm of renal tubular epithelial cells and appeared brown-yellow, spherical and approximately two to four µm in diameter. Pigment granules were not observed in epithelial cells that did not have eosinophilic protein droplets. The pigment's appearance was consistent with lipofuscin. Similar incidences of PDN and pigmentation were not noted in male rats given 0 or 5 mg/kg/day. A variable incidence and severity of tubular degeneration/regeneration occurred in the cortex and outer stripe of the outer zone of the medulla of male rats irrespective of dose level. These differences were not attributed to SYMTET because of a lack of a dose response in the incidence of moderate renal tubular degeneration/regeneration. Kidney lesions of a different appearance occurred in female rats given 150 mg/kg/day. Very slight to slight, unilateral and bilateral subacute to chronic inflammation of the renal papilla(e) was histologically observed in seven of the 15 female rats given 150 mg/kg/day. Papillary inflammation occurred in conjunction with very slight to slight distension of the tubules, and slight to severe tubular degeneration/regeneration. These latter effects were attributed to the extension of the inflammation up the nephron, with resultant secondary tubular lesions. Two female rats given 150 mg/kg/day also had very slight pigment noted in tubular epithelial cells, which was considered to be a nonspecific finding associated with the secondary degenerative lesions in the kidneys. There were no histopathologic treatment-related alterations in either the testes or epididymides of male rats given 150 mg/kg/day which could be attributed to be the cause of the slightly lower epididymides weights.

Reproductive Indices, Pup Survival.
No treatment-related effects were observed on the male or female fertility indices, reproductive parameters, length of gestation, time to mating, pup survival indices or pup sex ratio.

Litter Size, Neonate Body Weights and Observations.
No treatment-related effects were observed on litter size or pup body weight at any dose level at any time during the study. Similarly, no effects attributed to treatment were observed on clinical observations or physical alterations in pups from any dose group.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: This NOAEL is for subchronic systemic toxicity.

2

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: This NOAEL is for subchronic systemic toxicity.

3

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: This NOAEL is for reproductive and developmental toxicity.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The no-observed-adverse-effect level (NOAEL) for subchronic systemic toxicity was 150 mg/kg/day for males and 25 mg/kg/day for females. The NOAEL for reproductive and developmental toxicity was 150 mg/kg/day.

Executive summary:

2,3,5,6-tetrachloropyridine (SYMTET) was evaluated for subchronic, reproductive and developmental toxicity in a GLP compliant combined repeat dose and reproductive/developmental toxicity screening study conducted in accordance with OECD guidelines. Groups of 15 adult male and 15 adult female Sprague-Dawley rats were given the test material by oral gavage at dose levels of 0, 5, 25 or 150 mg/kg/day for approximately 52 days. The animals were dosed 14 days prior to breeding, during a 14-day mating period and throughout the gestation and lactation periods. All parental animals and litters were euthanatized following lactation Day 4. Parental parameters evaluated included clinical observations, feed consumption, body weight, body weight gain, clinical chemistry, hematology, organ weights, gross and histopathology, and fertility. Neonatal parameters evaluated included clinical/morphological alterations, body weight and survival.

 

Females given 150 mg/kg/day had slight to severe renal tubular epithelial celldegeneration/regeneration, and inflammation of the renal papilla(e). Males given 25 or 150 mg/kg/day had protein droplet nephropathy which was not considered relevant to human risk assessment, as this effect is a male rat­specific response. Increased liver weights and centrilobular hypertrophy of hepatocytes occurred in males and females given 150 mg/kg/day. Liver weight and histologic differences were interpreted to be an adaptive change, secondary to the metabolism of SYMTET, and were not considered toxicologically significant. Alterations in a number of clinical chemistry parameters were also observed in females given 150 mg/kg/day and were considered secondary to renal and hepatic effects.

No effects attributed to treatment were observed on the pregnancy rate, time to mating, gestation length, number of corpora lutea or implantations, preimplantation or postimplantation loss, fertility indices, litter size, neonatal growth or survival, or testes and epididymides weights at any dose level. In addition, no gross or histopathologic alterations of the testes, epididymides or ovaries were observed at 150 mg/kg/day.

The no-observed-adverse-effect level (NOAEL) for subchronic systemictoxicity was 150 mg/kg/day for males and 25 mg/kg/day for females. TheNOAEL for reproductive and developmental toxicity was 150 mg/kg/day.