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

Endpoint summary

Description of key information

NOAEL oral (rat, 28 d) = 100 mg/kg/d (OECD 407; Springborn Laboratories, 1999)
NOAEL dermal (90 d, rat) = 150 mg/kg/d (OECD 411, CIT, 2010)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-02-16 to 1999-1999-05-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant OECD Guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted July 27, 1995

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, Michigan, USA
- Age at study initiation: approximately seven weeks
- Weight at study initiation: ranging from 211 to 250 g for males and 151 to 193 g for females
- Fasting period before study: no
- Housing: 2 or 3 males or females per cage for six days following receipt to adjust to the automatic watering system. During remainder of acclimation and while on study: individually in suspended stainless steel cages
- Diet: PMI Certified Rodent Chow #5002 (Purina Mills, Inc.) ad libitum
- Water: municipal tap water ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 65 to 79°F (18.3 °C to 26.1 °C)
- Humidity: 30 to 70%
- Air changes: 10 to 15 air changes per hour
- Photoperiod: 12-hour light / 12-hour dark cycle

IN-LIFE DATES: From: 1999-03-04 To: 1999-04-01 or 1999-04-14 (recovery groups)

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Prior to weighing, the test article was ground using a mortar and pestle, then weighed into a beaker. Reverse Osmosis Deionized (RODI) water was added to the beaker to achieve the desired concentration, and stirred for 15 minutes. Prepared fresh weekly, dispensed into daily aliquots and stored refrigerated. The physical state of the vehicle and each test article dosing mixture was recorded during each preparation. Daily aliquots of the dosing mixtures were removed from the refrigerator, stirred continuously and allowed to equilibrate to room temperature prior to dispensing.

VEHICLE
- Amount of vehicle: 10 mL / kg bw
The vehicle control material used in the preparation of dosing mixtures and for administration to control animals was Reverse Osmosis Deionized (RODI) water.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration verification analysis was performed on the vehicle and each test article dosing mixture during weeks 1, 2, 3 and 4.

Prior to initiation of the 28-day study, homogeneity and stability analyses were performed on concentrations of the test article in the vehicle which bracketed the low- and high-doses administered in this study. Homogeneity analyses were performed on duplicate samples taken from the top, middle and bottom of the two mixtures. Stability of the test article in the vehicle was evaluated on duplicate samples at 0, 3 and 8 days following preparation and refrigerated storage.
The analytical results indicated that the doses were accurately formulated during the toxicity study. The results also confirmed that the formulations were homogeneous and stable from the time of preparation to completion of dosing.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily (7 days per week)

Remarks:

Doses / Concentrations:
0, 100, 500, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

five males and five females, in addition 5 males and 5 females per recovery group (vehicle and high dose)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dosage levels were selected based on data from the range-finding study to produce graded responses to the test article.
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: during weeks -1, 1, 2 and 3 (days - 1 , 7, 14 and 21, respectively) for each animal.

BODY WEIGHT: Yes
- Time schedule for examinations: -Day 2, 8, 15, 22 and 28 (during the treatment phase) and day 35 (during the recovery phase). In addition, a terminal body weight was recorded on the day of scheduled euthanasia (day 29 or 42) for calculation of relative organ weight data

FOOD CONSUMPTION: Yes
- Time schedule for examinations: -Day 2, 8, 15, 22 and 28 (during the treatment phase) and days 35 and 41 (during the recovery phase).

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: near the end of the treatment phase (day 26) and the recovery phase (day 37)
- Dose groups that were examined: all dose groups (surviving animals)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment phase (day 29) or the recovery phase (day 42)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving rats (incl. recovery groups)
- The following parameters were examined:
Erythrocyte count (RBC), hematocrit (Hct), hemoglobin concentration (Hgb), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), platelet count, total and differential leukocyte counts, reticulocyte count (reticulocyte slides were prepared, however, reticulocyte counts were not considered necessary by the Study Director)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment phase (day 29) or the recovery phase (day 42)
- Animals fasted: Yes
- How many animals: all surviving rats (incl. recovery groups)
- The following parameters were examined:
Alanine aminotransferase (ALT), albumin, albumin/globulin ratio (calculated), alkaline phosphatase, aspartate aminotransferase (AST), calcium, cholesterol, blood creatinine, gamma glutamyl transpeptidase (GGT), globulin (calculated), glucose, electrolytes (sodium, potassium and chloride), phosphorus, total bilirubin, total serum protein, triglycerides, urea nitrogen

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment phase (day 29) or the recovery phase (day 42), overnight, prior to scheduled euthanasia
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (food withheld when in metabolic cage, water provided)
- The following parameters were examined:
Overnight volume, color and appearance, pH, specific gravity, protein, glucose, ketones, urobilinogen, nitrites, bilirubin, occult blood, leukocytes, microscopy of spun deposit

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during weeks 4 and 6 (days 27/28 and 40/41, respectively).
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength and motor activity

Sacrifice and pathology:

- Euthanized by carbon dioxide Inhalation followed by exsanguination

GROSS PATHOLOGY: Yes, on all animals (incl. recovery groups)
- Parameters: external surfaces of the body and all viscera
- Fresh organ weights: liver, kidneys, adrenal glands, testes with epididymides, ovaries, spleen, thymus, thyroids, brain and heart of all animals. Paired organs were weighed together.
- Preserved from all animals: accessory genital organs (epididymides, seminal vesicles and prostate or uterus and vagina), adrenals, all gross lesions, aorta, brain (including sections of medulla/pons, cerebellar cortex and cerebral cortex), cecum, colon, duodenum, esophagus, exorbital lachrymal glands, eyes with optic nerve, femur (including articular surface) and bone marrow, heart, ileum, jejunum, kidneys, liver (3 sections collected), lungs (infused with formalin) with bronchi, mammary gland, mandibular lymph node, mediastinal lymph node, mesenteric lymph node, pancreas, peripheral nerve (sciatic) pituitary, rectum, skeletal muscle (thigh), skin, spinal cord (cervical, midthoracic and lumbar), spleen, sternum with bone marrow, stomach (glandular/nonglandular), submaxillary salivary gland, testes/ovaries, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder


HISTOPATHOLOGY: Yes
Investigated: All tissues and organs collected at necropsy (days 29 and 42) from all animals in the control and high-dose groups

Statistics:

- One-way analysis of variance (ANOVA): body weights, weight gain, food consumption, hematology, coagulation, biochemistry, organ weights and appropriate urinalysis parameters
- Tukey-Kramer Test (group comparisons when significance was observed)
- Kruskal-Wallis Test (rank and count data)
- Fisher's Exact Test (Descriptive (categorical) and quantal data)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mild, dose-dependent clinical abnormalities

Mortality:

mortality observed, treatment-related

Description (incidence):

Mild, dose-dependent clinical abnormalities

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A slight, dose-dependent decrease in mean body weights

Food efficiency:

no effects observed

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:

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):

A dose-dependent trend towards increased spleen weights was noted primarily in the 500 and 1000 mg/kg bw / day males at the end of the dosing phase of this study.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Mild, dose-dependent clinical abnormalities were noted following test article administration in the 500 and 1000 mg / kg bw / day males and females (salivation and apparent blood around the facial area, neck and forelimbs). The clinical abnormalities resolved during recovery phase; no mortality occurred.

BODY WEIGHT AND WEIGHT GAIN
A slight, dose-dependent decrease in mean body weights (i.e., 3%, 4% and 6% for the 100, 500 and 1000 mg / kg bw / day groups, respectively) was noted in treated males by the end of the dosing phase. The biological impact of this change was considered marginal as the decrease in mean body weights for the 1000 mg / kg bw / day males (i.e., 6%) was well below 10%.

FOOD CONSUMPTION - no notable abnormalities

OPHTHALMOSCOPIC EXAMINATION - no notable abnormalities

HAEMATOLOGY and CLINICAL CHEMISTRY
Slight treatment-related effects: decreased RBC counts, hemoglobin concentration and hematocrit, increased serum bilirubin in the 500 and 1000 mg / kg bw / day groups.

URINALYSIS -no notable abnormalities

NEUROBEHAVIOUR - no notable abnormalities

ORGAN WEIGHTS
A dose-dependent trend towards increased spleen weights was noted primarily in the 500 and 1000 mg/kg bw / day males at the end of the dosing phase of this study.

HISTOPATHOLOGY:
A reversible minimal to mild increase in the congestion of the red pulp of the spleen was observed microscopically in several of the 1000 mg / kg bw / day males and females at the end of the dosing phase.

GROSS PATHOLOGY - no notable abnormalities

HISTORICAL CONTROL DATA: no notable abnormalities when compared to results of study

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Primarily induced by mildly and sporadically abnormal parameters regarding clinical signs, haematology, organ weight and histopathology at 500 and / or 1000 mg/kg bw/day.

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and guideline compliant study.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

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.

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

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)

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)

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

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

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.

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.

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and guideline compliant study.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

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.

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

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)

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)

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

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

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.

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.

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and guideline compliant study.

Additional information

Oral

The purpose of this study according to OECD TG 407 adopted 1995-07-27 was to evaluate the potential toxicity of the test item when administered orally, by gavage, to rats for 28 consecutive days followed by a 14-day recovery phase. The study design consisted of a control group and three treatment groups with ten animals per sex in the control and high-dose groups and five animals per sex in the low- and mid-dose groups. The test item was administered as a single daily dose at dosage levels of 100, 500 and 1000 mg/kg bw/day for 28 consecutive days. Control animals received reverse osmosis deionised tap water under the same experimental conditions.

Individual clinical observations were made on all animals daily. Detailed clinical observations were made outside the home cage on all animals once, prior to the first exposure and once on week 4. Body weight and food consumption were measured weekly. Blood and urine sampling for clinical pathology and gross pathology were conducted at the end of the treatment period. Selected organs were weighed. Histological examination was performed on the preserved organs or tissues of control group and high does group (1000 mg/kg bw/day).

Oral administration of the test article to rats did not produce any mortality during this study. However, mild, dose-dependent clinical abnormalities were noted following test article administration in the 500 and 1000 mg/kg bw/day males and females. These clinical findings became more prominent towards the end of the dosing period and generally included evidence of salivation and apparent blood around the facial area, neck and forelimbs. The clinical abnormalities subsequently resolved during the recovery phase as generally no notable abnormalities were observed in any animal after cessation of dosing. Weekly neurotoxicological evaluations of the animals did not reveal any notable abnormalities.

A slight, dose-dependent decrease in mean body weights (i.e., 3%, 4% and 6% for the 100, 500 and 1000 mg/kg bw/day groups, respectively) was noted in treated males by the end of the dosing phase of this study. However, the biological impact of this change was considered marginal as the decrease in mean body weights for the 1000 mg/kg bw/day males (i.e., 6%) was well below the level that is commonly suggestive of a notable biological effect in rodent toxicological studies (i.e., 10%).

A slight dose-dependent decrease was observed in the RBC counts, haemoglobin concentration and hematocrit of the treated males and females at the end of the dosing period. In addition, a slight, dose-dependent increase in bilirubin was noted in the treated males during the same period. These observations were primarily noted in the 500 and 1000 mg/kg bw/day dose groups and are suggestive of a slight, nonregenerative anemia. However, the biological impact of these findings appears to be marginal since other related hematological parameters such as MCV, MCH, MCHC and NRBCs appeared unaffected, there was no bilirubin observed in the urine and there was no related microscopic pathology noted in the spleen or liver.

No abnormalities were noted in the amount of food consumed during this study and no test article-related ocular abnormalities were observed in any animals.

A dose-dependent trend towards increased spleen weights was noted primarily in the 500 and 1000 mg/kg bw/day males at the end of the dosing phase of this study. In addition, a minimal to mild increase in the congestion of the red pulp of the spleen was observed microscopically in several of the 1000 mg/kg bw/day males and females at the end of the dosing phase. The findings in the spleen may be associated with the slight, nonregenerative anemia suggested previously. However, the toxicological significance of this finding may be limited since congestion of the red pulp is a common background lesion in the rat. In addition, there were no indications of pathologic abnormalities in this organ such as extramedullary hematopoiesis, macrophage activity or hemosiderin deposition that may be observed following a hemolytic event. By the end of the recovery phase, no treatment-related abnormalities were observed in any animal. All other microscopic changes were considered spontaneous and unrelated to treatment or within the limits of normal variation.

No significant treatment-related effects were observed in the 100 mg/kg bw/day males and females during this study. In the 500 and 1000 mg/kg bw/day groups, a slight treatment-related effect was observed and primarily included abnormal clinical signs, decreased RBC counts, haemoglobin concentration and hematocrit, increased serum bilirubin and increased spleen weights. Microscopic evidence of congestion in the red pulp of the spleen was also noted in the 1000 mg/kg bw/day group. Based on these results, a dosage level of 100 mg/kg bw/day was considered to be the no-observed-effect level (NOEL) following 28-day oral administration of the test item to rats.

Dermal

The objective of this study according to OECD TG 411 adopted 1981-05-12 was to evaluate the potential toxicity of the test item following daily cutaneous application to rats for 13 weeks. On completion of the treatment period, designated animals were held for a 4-week treatment-free period in order to evaluate the reversibility of any findings.

Three treated groups of 10 (low- and mid-dose groups) or 15 (high-dose groups) males and females Wistar Han rats received the test item by daily cutaneous application for 13 weeks at the dose-levels of 50, 150 or 500 mg/kg bw/day. The test item was administered as a suspension in 0.5% carboxymethylcellulose. A group of 15 males and 15 females received 0.5% carboxymethylcellulose only under the same experimental conditions and acted as a control group. The analysis of the suspension showed a satisfactory agreement between the theoretical and achieved concentration of the test item in the dosage forms. The animals were checked daily for mortality and clinical signs. Detailed clinical examinations were performed once a week. Body weight and food consumption were recorded once a week during the study. Ophthalmological examinations were performed on all the animals before the beginning of the treatment period, and at the end of the treatment and treatment-free periods. Hematology, blood biochemical and urinalysis investigations were performed at the end of the treatment and treatment-free periods. At the end of the treatment or treatment-free periods, animals were killed and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from designated animals.

Results were interpreted comparing treatment groups to the control group. There were no unscheduled deaths or premature sacrifices during the treatment period. 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.

Body weight, body weight gain and food consumption were not affected by the test item treatment.

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.

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 group 4 males and females.

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.

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

Increases in splenic weights were noted in males and females from 150 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.

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

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.

Based on the results of this study the No Observed Adverse Effect Level (NOAEL) of the test item administered by cutaneous application in rats during 13 weeks was determined to be at 150 mg/kg bw/day.

 

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only one GLP and guideline compliant study available.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Only one GLP and guideline study available.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Only one GLP and guideline study available.

Justification for classification or non-classification

Based on the results of the repeated dose testing, the test item was not classified and labelled for long-term toxicity according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP).