1,4-dihydroxy-2,2,6,6-tetramethyl piperidinium-2-hydroxy-1,2,3-propanetricarboxylate

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-07-07 to 2010-08-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant Guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Version / remarks:

adopted May 12, 1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

429-370-5

EC Name:

-

Cas Number:

220410-74-2

Molecular formula:

C34H67N3O13

IUPAC Name:

tris(1,4-dihydroxy-2,2,6,6-tetramethylpiperidin-1-ium) 2-hydroxypropane-1,2,3-tricarboxylate

Test material form:

other: Ivory-peach flakes

Details on test material:

- Identity: FAT 76’042/G
- Lot/batch No.: 06087EB6
- Expiration date of the lot/batch: 18 July 2009
- Description : whitish powder
- Analytical purity: 97.3%
- Date of receipt at Test Facility: 5 January 2007
- Storage conditions : at room temperature and protected from humidity

Test animals

Species:

rat

Strain:

other: Crl:WI (GLX/BRL/Han) IGS BRO

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, l’Arbresle, France
- Age at study initiation: approximately 9 weeks on first day of treatment
- Weight at study initiation: male mean body weight 282 g (range: 255 g to 304 g) and 191 g (range: 175 g to 207 g) for the females
- Housing: individually housed in suspended wire-mesh cages
- Diet: SSNIFF R/M-H pelleted maintenance diet, batch Nos. 3916447 and 6557303 (SSNIFF Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water: tap water ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 50 ± 20%
- Air changes: 12 cycles / hour
- Photoperiod: 12h dark / 12h light

IN-LIFE DATES: From: 2007-02-21 To: 2007-05-24/25 (or 2007-06-21 for recovery groups)

Administration / exposure

Type of coverage:

open

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

TEST SITE
- Area of exposure: on the dorsum of the animals
- % coverage: 10% of body surface area (i.e. from 45 to 50 cm2 in males and from 30 to 35 cm2 in females)
- Time intervals for shavings or clipplings: whenever necessary, at least 4 hours before dosing and at least once a week (except during weeks 4, 10 and 13)

REMOVAL OF TEST SUBSTANCE
- Washing: Application sites were not wiped after dosing. In the case of excessive residues of test item on the test site prior to the application, it was cleaned with purified water.

TEST MATERIAL
- Concentration (if solution): 0, 20, 60 and 200 mg/mL
- Constant volume or concentration used: yes (2.5 mL / kg bw / day)

VEHICLE
- Concentration (if solution): 0.5% carboxymethylcellulose aqueous solution in purified water
- Lot/batch no.: 026K0141 and 035K0131, supplied by Sigma (Saint-Quentin-Fallavier, France)

USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of samples taken from each control and test item dosage form prepared for use in weeks 1, 4, 8 or 13 was determined. A detailed review of the analytical data including validation of the analytical method was conducted after the end of the study and no deviations were identified by this review procedure.

Duration of treatment / exposure:

at least 13 weeks (max.: 94 days)

Frequency of treatment:

daily (7 days per week)

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 150, 500 mg / kg bw / day
Basis:
nominal per unit body weight

No. of animals per sex per dose:

10 males and 10 females (plus 5 males and 5 females per satellite group: vehicle and high dose). Total: 100 rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Results of a preliminary 2-week toxicity study performed in the same species (CIT/Study No. 32665 TSR)
Results of a 28-day oral gavage study
Results of an in vitro skin permeation study

- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality or signs of morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day

DERMAL IRRITATION: Yes
- Time schedule for examinations: daily from day 1 to week 4, weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: once before group allocation, on the first day of treatment, and then once a week until the end of the study

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated:
- g food / kg body weight / day: No
- g food / animal / day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the beginning of the treatment period and on weeks 13 or 14; recovery groups: at the end of the treatment-free period.
- Dose groups that were examined: all animal groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: both, at the end of the treatment and the treatment-free periods
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animal groups
- Parameters investigated: erythrocytes, reticulocytes, haemoglobin, mean cell volume, packed cell volume, cean cell hemoglobin concentration, mean cell haemoglobin, thrombocytes, leucocytes, differential white cell count with cell morphology: neutrophils, eosinophils, basophils, lymphocytes and large unstained cells, monocytes; bone marrow: differential cell count
- Coagulation: prothrombin time, thromboplastin activated partial thromboplastin time, fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: both, at the end of the treatment and the treatment-free periods
- Animals fasted: Yes
- How many animals: all animal groups
- Parameters investigated: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total proteins, albumin, albumin/globulin ratio, total cholesterol, triglycerides, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to blood sampling (at the end of the treatment and the treatment-free periods)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters investigated: appearance, volume, pH, specific gravity, proteins, glucose, ketones, bilirubin, nitrites, blood, urobilinogen, leucocytes, erythrocytes, cylinders, magnesium ammonium phosphate, calcium phosphate crystals, calcium oxalate crystals, epithelial cells

Sacrifice and pathology:

On completion of the treatment or treatment-free period, after at least 14 hours fasting, all animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.

GROSS PATHOLOGY: Yes
- Parameters investigated: external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.
- Preservation of tissues: adrenals, brain (including medulla / pons cerebellar and cerebral cortex), epididymides, heart, kidneys, liver, ovaries (with oviducts), spleen, testes, thymus, thyroids with parathyroids, macroscopic lesions, aorta, cecum, colon, duodenum, esophagus, eyes with Harderian glands, femoral bone with articulation, ileum (with Peyers patches), jejunum, larynx, lungs with bronchi, lymph nodes (mandibular and mesenteric), mammary gland area, optic nerves, pancreas, pituitary gland, prostate, rectum, salivary glands (sublingual and submandibular), sciatic nerves, seminal vesicles, skeletal muscle, skin (1 level on treated area and 1 level on non treated area), spinal cord (cervical, thoracic and lumbar), sternum with bone marrow, stomach with forestomach, tongue, trachea, ureters, urinary bladder, uterus (horns and cervi), vagina

ORGAN WEIGHTS: liver, kidneys, adrenal glands, testes with epididymides, ovaries, spleen, thymus, thyroids, brain and heart of all animals. Paired organs were weighed together.

HISTOPATHOLOGY: Yes
- Parameters investigated: all tissues preserved except larynx and ureters

Statistics:

Tests for body weight, food consumption, hematology, blood biochemistry and urinalysis data: Kolmogorov-Lilliefors, Dunn, Bartlett, Mann-Whitney / Wilcoxon, Student (depending on type of variable and size of data set).

Test for organ weight data (within PathData version 6.2b5): Kolmogorov, Bartlett, F-test, Kruskal-Wallis, ANOVA, Dunn, Dunnett (depending on type of variable and size of data set).

- level of significance: 0.05 or 0.01

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Scabs were transiently noted at the application site during the treatment period

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Minimal acanthosis of the epidermis occurred in males and females in all test item groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

Scabs were transiently noted at the application site during the treatment period

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Chorioretinopathy was noted at the end of the treatment period

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Lower white blood cell count correlated with lower lymphocyte count was observed at the end of the treatment period

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, low glucose and high urea concentrations were noted in animals

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increases in splenic weights were noted in males and females

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
Scabs were transiently noted at the application site during the treatment period in 2/15 males and 3/15 females given 500 mg / kg bw / day, and in 1/10 females given 50 or 150 mg / kg bw / day. There were no unscheduled deaths or premature sacrifices during the treatment period.

BODY WEIGHT AND WEIGHT GAIN - not affected by the test item treatment

FOOD CONSUMPTION - not affected by the test item treatment

OPHTHALMOSCOPIC EXAMINATION
Chorioretinopathy was noted at the end of the treatment period in 2/15 males and 1/15 females given 500 mg / kg bw / day. This finding was spontaneously encountered in rats of this stain and age, and was consequently not considered to be an adverse effect.

HAEMATOLOGY
Lower white blood cell count correlated with lower lymphocyte count was observed at the end of the treatment period in males and females (non dose-related) from 50 mg / kg bw / day when compared to controls. At the end of the treatment-free period, these differences from controls were still observed in males and females from the high dose group.

CLINICAL CHEMISTRY
At the end of the treatment period, low glucose and high urea concentrations were noted in animals given 500 mg / kg bw / day. Low potassium concentrations were noted in males and females from 50 mg / kg bw / day. This was associated with low chloride concentrations in males and females from 150 mg / kg bw / day. Slightly higher ASAT and ALAT activities were noted in females given 500 mg / kg bw / day at the end of the treatment period when compared to controls. These findings were no longer observed at the end of the treatment-free period.

URINALYSIS
No urinalysis parameter disturbances were observed at the end of the treatment and treatment-free periods.

ORGAN WEIGHTS
Increases in splenic weights were noted in males and females from 150 and 500 mg / kg bw / day at the end of the treatment period. Splenic weights were no longer increased in the 500 mg / kg bw / day group animals following the recovery period, indicating complete reversal.

GROSS PATHOLOGY
There were no test item-related macroscopic findings at the end of the treatment and treatment-free periods.

HISTOPATHOLOGY
Minimal acanthosis of the epidermis occurred in males and females in all test item groups. Because the change observed was minimal after three months of treatment and was similar to some of the control animals, it was considered not to be adverse. Minimal splenic congestion was observed in all test item-treated males and in females given 500 mg / kg bw / day. Because the spleens lacked alterations in lymphoid components, and because a smaller number of controls had similar changes, the splenic congestion was considered not to be adverse.

Effect levels

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Primarily induced by mildly abnormal parameters (scabs, chorioretinopathy, haematology, organ weight and histopathology) at 500 mg/kg bw/day.

Target system / organ toxicity

Critical effects observed:

not specified