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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993-1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Test Item: Cetylpyridinium Chloride
Chemical Name: Cetylpyridinium Chloride, Monohydrate
Identification: D1470.01
Purity was assumes to be 100%.
Physical State: Powder, white

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., Raleigh, NC. USA
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 190-247g (males) and 140-188 g (females)
- Fasting period before study:
- Housing: stainless steel wire mesh cages, suspended above excretia trays
- Diet (e.g. ad libitum): ad libitum, Purina Certified Rodent Chow #5002
- Water (e.g. ad libitum): ad libitum. quality analysis available
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 66-79 F
- Humidity (%):24-70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
deionized
Details on oral exposure:
Dose formulations were prepared weekly, and aliquoted into 7 daily doses. Formulations were stored refrigerated, allowed to warm to r oom temperature before dosing, sonicated for 30 minutes prior to use, and stirred with a magnetic stirrer during the dosing procedure. Rats were weighed weekly and doses adjusted accordingly.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate samples (10 ml) were taken from each dose level of each weekly mix for a reserve sample and for analysis. The stablility of the test formulations was established for the samples after they were stored up to 10 days under refrigeration and also at room temperature for 0 and 10 days. Analyses were in duplicate. The concentration of dosing solutions was analyzed in duplicate, using RP-HPLC.
Duration of treatment / exposure:
6 months (26 weeks)
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 15, 40, 75 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
Animals were administered the test solution via oral gavage, once daily, at a volume of 10 ml/kg bw, for 28 days.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily at 1 hour post dosing; thorough physical examination was conducted at each weighing interval.

BODY WEIGHT: Yes
- Time schedule for examinations: measured weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Assessed weekly
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

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: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes; weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to initiation and at termination, using 1% Mydriacyl as the mydriatic agent.
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes. CO2 inhalation anesthetic at termination. Obtained via orbital plexus venipuncture.
- How many animals: all, 20 per sex per dose group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: Yes, fasted overnight
- How many animals: all, 20 per sex per group

URINALYSIS: Yes
- Time schedule for collection of urine: at termination.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, fasted overnight

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Samples for hematology and serum chemistry were also collected from animals sacrificed in the moribund condition.
Sacrifice and pathology:
All animals which were found dead or sacrificed in extremis during the study were subjected to a gross postmortem examination. All surviving animals were fasted overnight, weighed and injected ip with sodium pentobarbital, and exsanguinated. Necropsies were performed on all animals by appropriately trained personnel using procedures approved by board-certified pathologists.

Organs were weighed after trimming of fat and other continguous tissue: adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, stomach, testes with epididymides,thymus, thyroid/parathyroids.

GROSS PATHOLOGY: Yes; all animals
HISTOPATHOLOGY: Yes, on groups 1 and 4 (control and 75 mg/kg/d) and any gross lesions from animals in any group. Tissues preserved in 10% netural buffered formalin were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically.

Tissues preserved were: adrenals, aorta, brain, colon with cecum and rectum, duodenum, jejunum and ileum, esophagus, exorbital lacrimal glands, eyes, femur, heart, kidneys, liver, lung, mammary gland, mesenteric lymph node, ovaries, pancreas, pituitary, prostate, salivary glands, sciatic nerve, seminal vesicles, skin, spleen, sternum with bone marrow, stomach, spinal cord, testes with epididymides, thigh musculature, thyroid/parathyroids, thymus, trachea, urinary bladder, uterus with vagina and cervix.
Other examinations:
Hematology: activated partial thromboplastin time, erythocyte count, hemoglobin, hematocrit, leukocyte count, leukocyte differential and cell morphology, mean cell volume, mean cell hemoglobin, platelet count, prothrombin time, reticulocyte count.
Serum chemistry: alanine aminotransferase, albumin, albumin/globulin rtio, alkaline phosphatase, aspartate aminotransferase, calcium, chloride, creatinine, gamma glutamyl transferase, globulin, glucose, inorganic phosphorus, potassium, sodium, total bilirubin, total protein, urea nitrogen.
Urinalysis: appearaance/color, glucose, ketone, microscopic examination of sediment, occult blood, pH, protein, specific gravity, total volume.
Statistics:
Groups 1-4 were analyzed statistically, as groups 5 and 6 did not survive. Analysis used ANOVA (one-way), with Dunnett's t-Test for control vs. treatment comparison or Bartlett's test. If variances of untransformed data were heterogeneous, a series of transformations were performed according to Levene. If not successful, analyses were performed on rank-transformed data. Group comparisons were performed at the 5% two-tailed probability level. Statistical significance was at P< 0.05.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
1 unexplained death at 75 mg/kg/d
Mortality:
no mortality observed
Description (incidence):
1 unexplained death at 75 mg/kg/d
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant decreases in body weight and/or body weight gain at 40 and 75 mg/kg
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Decreased food consumption in males, and occasionally females, at 75 mg/kg/d
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
characteristic of dehydration
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Increases in AST and ALT at 40 and 75 mg/kg; may be adaptative.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increases in absolute weight of stomach
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
acanthosis and irritation/erosion in the non-glandular stomach and distention of the cecum
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
inflammation and necrosis
Histopathological findings: neoplastic:
no effects observed
Details on results:
Analysis of dose concentrations indicated that the formulations were stable for 10 days at both room temperature and refrigeration. With a few exceptions, values (stability and dose samples) were within 10% of the initial/target concentration. One was within 12% and 3 were within 11-21% of target.

Mortality: There were 3 nonaccidental deaths during the test: 2 to non-treatment related chronic disease, and 1 high dose male to unknown causes. This animal displayed gastric irritation. Adjusted survival rates were 100, 95, 100, 94 and 95% for group 1-5 males, and 100% for each of the group 1-5 females. The following general observations were noted in group 5 (75 mg/kg/d): salivation, soft feces and abnormal respiratory sounds.

Mean body weights were significantly decreased in group 5 males at most weekly weighing sessions, and sporadically in group 5 females and Group 4 (40 mg/kg/d) males. The body weights of group 3 (15 mg/kg/d) females were nonsignificantly lower than that in controls during the final 15 weeks of the study. Total food consumption was significantly decreased in both sexes at 75 mg/kg/d, and sporadically in females at lower doses.
Significant alterations in hematology were only noted in high dose (75 mg/kg/d) females, consistent with dehydration, and were of no biologic consequence. Increases in AST and ALT were observed in group 5 (75 mg/kg/d) animals and in males receiving 40 mg/kg/d at week 27. Glucose decreased in a dose-dependent manner in groups 4 and 5 animals. Neither sex had alterations in urinalysis.

There was a high incidence of thickened non-glandular stomach sections in group 5 animals These changes also correlated with increases in stomach weights, which occurred in groups 3-5. There were increased incidences of abnormal respiratory sounds noted in treated rats (groups 3-5 males and groups 4-5 females), due to apparant irritation. Treatment-related morphologic alterations also included a distended cecum in males of groups 3-5 and in about a third of group 5 females. There was increased histopathology finding noted in the stomach. Hyperplasia (acanthosis) occurred with the greatest incidence in groups 4 and 5. Gastric necrosis, erosion and edema was sometimes seen in this group. Nontreatment related findings of spontaneous diseases included chronic inflammation of the liver, chronic progressive nephropathy in the kidney, and lymphoid infiltration in the lungs.

In conclusion, localized gastrointestinal irritation was observed in rats receiving CPC at dose levels of 15 mg/kg/d and higher. These symptoms included salivation, soft feces, decreased food consumption, decreased body weight, thickened appearance of the nonglandular region of the stomach, increased stomach weights, and/or hyperplasia/necrosis/erosion/edema of the nonglandular stomach. There was no histological evidence of systemic toxicity at any dose as high as 75 mg/kg/d. The NOEL for CPC (D1470.01) based on the absence of local irritation effects in the stomach is 5 mg/kg/d.

Effect levels

Dose descriptor:
NOEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Local irritation (with necrosis and hyperplasia) of the stomach occurred at higher doses.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Distention of the cecum may not be a direct effect of the test material or the result of gastric irritation. It may be caused by alterations in the microflora of the gut due to the chronic feeding of the test material.

Changes in seum AST and ALT may be an adaptive response to the oral administration of the test material. Other changes in hematology and clinical chemistry are likely due to dehydration partially as a result of increased salivation.

There were increased incidences of abnormal respiratory sounds noted in treated rats (groups 3-5 males and groups 4-5 females). This was thought to be due to the irritating effects of the material in the stomach, and possibly due to aspiration of the material.

Applicant's summary and conclusion

Conclusions:
In conclusion, localized gastrointestinal irritation was observed in rats receiving CPC at dose levels of 15 mg/kg/d and higher. These symptoms included salivation, soft feces, decreased food consumption, decreased body weight, thickened appearance of the nonglandular region of the stomach, increased stomach weights, and/or hyperplasia/necrosis/erosion/edema of the nonglandular stomach. There was no histological evidence of systemic toxicity at any dose as high as 75 mg/kg/d. The NOEL for CPC (D1470.01) based on the absence of local irritation effects in the stomach is 5 mg/kg/d.