4-hydroxy-2,2,6,6-tetramethylpiperidinoxyl

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-05-08 to 1992-12-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1981)

Deviations:

yes

Remarks:

(slightly limited histopathology)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: "28-day Repeated Dose Toxicity Study in Mammalian Species" prescribed in "The Notification on Partial Revision of Testing Methods Relating to New Chemical Substances" (Notification No. 700 of Kanpogyo, No.1039 of Yakuhatsu, and No.1014 of 61 Kikyoku)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-hydroxy-2,2,6,6-tetramethylpiperidinoxyl

EC Number:

218-760-9

EC Name:

4-hydroxy-2,2,6,6-tetramethylpiperidinoxyl

Cas Number:

2226-96-2

Molecular formula:

C9H18NO2•

IUPAC Name:

1-λ1-Oxidanyl-2,2,6,6-tetramethylpiperidin-4-ol

Test material form:

other: solid (unspecified)

Details on test material:

- Appearance: Orange solid
- Storage conditions: The test material was stored at ambient temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crj:CD(SD) (SPF)
- Age at study initiation: 5 weeks
- Weight at study initiation: 149.2 to 166.8 g (males); 121.6 to 142.0 g (females)
- Fasting period before study: No report of fasting
- Housing: Animals were housed individually in suspended stainless steel wire mesh cages (165 x 300 x 150 mm)
- Diet: ad libitum
- Water: ad libitum supply of chlorinated water from the municipal supply via sipper tubes from an automatic watering device
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ±3 °C
- Humidity: 55 ± 10 % (relative)
- Air changes: 10 to 15 per hour
- Photoperiod: 12 hrs dark / 12 hrs light (artificial light 07:00 to 19:00)

IN-LIFE DATES: From: 1992-06-30 To: 1992-08-06 or 1992-08-20 (recovery group)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

(purified)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
- Concentration in vehicle: 2, 0.4 or 0.08 % from 10 % (w/v) stock solution. The test material was dissolved in purified water to form the 10 % (w/v) concentration. This was further diluted with purified water.
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

STABILITY TEST
The test substance was taken (n = 1) before the dosing period, and analysed qualitatively using an infrared spectrophotometer (IR-470, Shimadzu). The test substance was stored at a room temperature in a dark place during the test period.
The IR spectrum of the test substance was identical with the IR spectrum provided. There was no difference between the IR spectra patterns before and after the dosing period. Therefore it was concluded that the test substance was stable during this period.

CONCENTRATION ANALYSIS AND STABILITY TEST
The solution for dosing was prepared prior to the start of the dosing period and the middle layer of the solution was taken (n = 3) immediately after, and at 4 and 7 days after preparation. After pre-treatment, test substance in the solution was quantitatively analysed by high performance liquid chromatography (HPLC) and subjected to the stability test. During the dosing period, the solutions for dosing were kept in a cold, dark place.

- Preparation of the standard solution
Approximately 50 mg of the test substance was accurately weighed and dissolved with purified water to make up to 50 mL volume.

- Analytical conditions
Instrument: High performance liquid chromatography (Model CCPE, Tosoh Corporation)
Column: 15 cm x 4.6 mm i.d., stainless-steel
Packing: L-column
Temperature: 40°C
Eluent: acetonitrile:water (20:80, v/v)
Flow rate: 1 mL/minute
Detector: UV detector (Model UV-8010, Tosoh Corporation)
Wave length: 262 nm
Sensitivity: 1 ABU/V
Amount injected: 10 µL
Recorder: Data Analyser (Model D-2500, Hitachi)
Output: 1024 mV
Under these analytical conditions, the linearity of detector response was confirmed for the concentration range of 0 to 1560 µg/mL test substance.

- Calculation
The total peak area of the test substance in each HPLC sample and the standard solution was measured by HPLC assay to calculate the concentration in each dosing solution using the following equation:
C (% w/v) = [C(standard) x A(sample) x D] / [A(standard) x 10 000]
where
C(standard) = Concentration (µg/mL)of the test substance in the standard solution
A(sample) = Total peak area of the test substance in each HPLC sample
A(standard) = Total peak area of the test substance in the standard solution
D = Rate of dilution in each dosing solution

- Results
Test substance in the solution was stable for 7 days. The mean concentration of the 0.05 % w/v nominal dosing solutions immediately after preparation, after 4 days and after 7 days were 0.051, 0.051 and 0.051 % w/v, respectively. The concentrations of the 10 % w/v nominal dosing solutions over the same time period were 10.02, 10.11 and 10.15 % w/v, respectively. The relative standard deviation was a maximum of 0.68 %.
The concentration after 4 days relative to the concentration immediately after preparation was 99.6 and 100.8 % for the 0.05 and 10 % nominal dose levels, respectively. The concentration after 7 days relative to the concentration immediately after preparation was 10.6 and 101.2 % for the 0.05 and 10 % nominal dose levels, respectively.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
8, 40, 200, and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

6 males and 6 females (total of 84 rats; incl. recovery groups for vehicle and high dose)

Control animals:

yes, concurrent vehicle

Details on study design:

- Post-exposure recovery period in satellite groups: 14 days
- Dose selection rationale: Based on the results of a dose range finding study at the same laboratory. The preliminary 14-day repeated-dose oral toxicity study was carried out at 3 doses of 50, 250 and 1000 mg/kg. Abnormal changes were noted in haematological parameters in the 1000 mg/kg group, and in general condition, blood chemical examinations and organ weight in the 250 and 1000 mg/kg groups. Histopathological examinations revealed increased eosinophilic bodies in the kidneys in one animal of the 50 mg/kg group.
- Rationale for selecting satellite groups: Studying reversibility of possible effects

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once per day
- Cage side observations: General condition (not specified in detail)

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: day of receipt; study days -5, -2, 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, 26, and 28; and recovery days 1, 3, 5, 8, 10, 12, and 14. Animals were also weighed immediately prior to necropsy.

FOOD CONSUMPTION Yes
- Food consumption for all animals was determined once before dosing and twice a week during the dosing and recovery periods.

FOOD EFFICIENCY: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of in life phase, blood samples were taken via the abdominal aorta. EDTA-2K was used as the anticoagulant (except for prothrombin time and activated partial thromboplastin time).
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, 16 to 20 hrs prior to blood sampling
- How many animals: all animals
- Parameters examined: Red blood cell count, white blood cell count, haemoglobin concentration, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count and reticulocytes; differential blood count (%): monocytes, basophils, eosinophils, lymphocytes, rod-shaped neutrophils, segmental neutrophils; blood coagulation: prothrombin time and activated partial prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of in life phase, blood samples were taken via the abdominal aorta
- Animals fasted: Yes, 16 to 20 hrs prior to blood sampling
- How many animals: all animals
- Parameters examined: GOT, GPT, alkaline phosphatase, γ-GTP, cholinesterase, total bilirubin, total cholesterol, triglyceride, glucose, total protein, albumin, A/G ratio, Blood urea nitrogen, creatinine, Ca, IP, Na, K and Cl.

URINALYSIS: Yes
- Time schedule for collection of urine: once during the dosing period (day 28) and once during the recovery period (day 14)
- Metabolism cages used for collection of urine: Yes. 16 hour urine samples were collected.
- Animals fasted: No data
- Parameters examined: volume, colour, pH, protein, ketone bodies, bilirubin, occult blood, glucose and urobilirubin.

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals were subjected to necropsy.
- Organ weights: brain, liver, spleen, kidneys, adrenals and testes/ovaries. All organs were weighed wet.

HISTOPATHOLOGY: Yes
- Tissues preserved in 10 % formalin fixative: brain, pituitary, eye, thyroid (with parathyroid), heart, lung, liver, kidneys, spleen, adrenals, stomach, intestine (duodenum, jejunum ileum, cecum, colon, rectum) testis/ovary, urinary bladder, bone marrow (femur) and gross lesions.
Tissues were embedded in paraffin, sectioned and stained with haematoxylin and eosin and examined under a light microscope.

Tissues examined during the dosing period:
- Vehicle and 1000 mg/kg bw/day dose group: liver, spleen, kidneys, heart, stomach, intestine (duodenum, jejunum ileum, cecum, colon, rectum) and adrenal
- 200 mg/kg bw/day dose group: liver, spleen, kidneys (males only)
- 40 mg/kg bw/day dose group: spleen (females only)

- Tissues examined during the recovery period:
Vehicle and 1000 mg/kg bw/day dose group: liver, spleen, kidneys (males only)

- Other: In addition, the spleens in the following groups were stained with Berlin's blue: 1000 mg/kg bw/day dose and recovery groups, 200 & 40 mg/kg bw/day groups, 8 mg/kg bw/day (females only) and the vehicle control and vehicle control recovery groups.

Statistics:

With regard to body weight, food consumption, haematological and blood chemical examinations, urine volume and organ weights, Bartlett’s test for homogeneity of variance was used. If the variances were homogeneous at the 5 % significance level, one way analysis of variance was performed. When there were significant differences between the vehicle control group and the dose groups in the analysis of variance, Dunnett’s test between groups with equal number of data or Scheffe’s test between groups with unequal number of data was carried out.
If the variances were not homogeneous, the Kruskal-Wallis test was used. When there were significant differences between the vehicle control group and each group in the Kruskal-Wallis test, nonparametric Dunnett’s test between groups with equal number of data or nonparametric Scheffe’s test between groups with an unequal number of data was performed.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see below

Mortality:

mortality observed, treatment-related

Description (incidence):

see below

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see below

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see below

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
- Mortality: No mortality was reported.
- Clinical signs: In all animals at 1000 mg/kg bw, and in a single male in the 200 mg/kg bw dose group, salivation was observed. In males the sign appeared in the 200 mg/kg male only after dosing on days 26 and 27; in the 1000 mg/kg bw males it appeared from day 4 or 6 and occasionally before dosing from day 16. In the 1000 mg/kg bw females it appeared from day 3 or 6 and occasionally before dosing from day 17.
One female in the 8 mg/kg bw group exhibited decreased spontaneous locomotion, increased respiratory rate, abnormal gait and subnormal temperature. As these signs were transient and not dose-related, they were not attributed to treatment with the test substance.
No abnormalities were observed during the recovery period.

BODY WEIGHT AND WEIGHT GAIN
No abnormalities in body weight development were observed in the treated groups of both sexes.

FOOD CONSUMPTION
No abnormalities in food consumption were observed in the treated groups of both sexes.

HAEMATOLOGY
High dose males and females showed decreased red blood cell count, haemoglobin concentration and haematocrit value and an increase in recticulocytes. Males showed an increase in prolonged prothrombin time and females showed a decrease in mean corpuscular haemoglobin and in lymphocytes, and an increase in segmental neutrophils was found.
In the 1000 mg/kg bw/day recovery group, at the end of the recovery period in males a decreased red blood cell count and haemoglobin concentration and an increase in platelet count was observed; in females, an increase in mean corpuscular volume and mean corpuscular haemoglobin was reported.
An additional change seen during the dosing period was the prolongation of the activated partial thromoboplastin time in the 40 mg/kg bw/day females only.

CLINICAL CHEMISTRY
Males of the 1000 and 200 mg/kg bw/d groups showed a decrease in alkaline phosphatase. Females showed an increase in glucose in the 40 mg/kg dose group and a decrease in GOT was observed in the 8, 40 and 200 mg/kg bw dose groups, but the magnitude was not dose related.
No abnormalities were observed in males of the recovery group, while females showed an increase in alkaline phosphatase as well as Na at 1000 mg/kg bw/day.

URINALYSIS
No abnormal observations were observed in the treated groups of both sexes

ORGAN WEIGHTS
Increased absolute and relative liver and spleen weights were observed in males and females of the high dose group. An increase in absolute spleen weight was also observed in the 200 mg/kg bw/day females. In males, absolute kidney weights were increased in the 1000 and 200 mg/kg bw/day dose groups.
At the end of the recovery period in males no differences from controls were observed, while females showed absolute and relative increases in spleen weights and an increase in absolute adrenal weight in the 1000 mg/kg bw/day dose group.

GROSS PATHOLOGY
Black coloration of the spleen was observed in the high dose males (5/6) and females (6/6) and in one 200 mg/kg bw/day female. Spotty surface of the kidney was noted in 2/6 males of the high dose group. At the end of the recovery period, a whitish nodule was observed in the kidney of one high dose male. No abnormalities were observed in the recovery group females.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Spleen: In the high dose group, congestion (6/6 males, females) and increased haemosiderin-laden cells (6/6 males, females) were observed; in the 200 mg/kg bw dose group, congestion (3/6 females) and increased haemosiderin-laden cells (2/6 females) were observed. At the end of the recovery period, congestion (5/6) and increased haemosiderin-laden cells (6/6) were observed in the males only.
- Kidneys: Increased eosinophilic bodies (6/6 males) were observed in the high dose group. At the end of the recovery period, there were basophilic changes of the tubular epithelium (1/6 males) and fibrosis (1/6 males) in the kidneys in the vehicle control group.
- Liver: Swelling of hepatocytes (4/6 males, 2/6 females) was observed in the high dose group. Other changes included erilobular lipid droplets (1/6 female) in the liver in the vehicle control group and perilobular lipid droplets (3/6 females) in the liver in the 200 mg/kg group. At the end of the recovery period, there were perilobular lipid droplets in the vehicle control group and 1000 mg/kg dose group (3/6 and 1/6 females, respectively).

HISTOPATHOLOGY: NEOPLASTIC
- Liver: Microgranuloma was observed in one male of the 200 mg/kg dose group. At the end of the recovery period, microgranuloma was observed in one female of the vehicle control group.
- Kidneys: Nephroblastoma was observed in one male of the 1000 mg/kg recovery group.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

2

Dose descriptor:

LOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Summary of Findings

Sex	Males					Females
Dose (mg/kg bw/day)	0	8	40	200	1000	0	8	40	200	1000
	Clinical signs
				Salivation	Salivation					Salivation
	Body weight (n = 12 control and high dose, n = 6 intermediate groups)
Day 1	157.9	158.7	157.5	159.7	158.1	130.2	129.3	131.4	128.6	130.2
Day 28 (end of treatment)	321.8	335.5	315.1	328.9	307.7	217.2	214.8	226.7	219.5	222.5
Day 42 (end of recovery)	372.5				361.8	249.0				256.7
	Haematology (n = 6)
RBC (x 10⁴/mm³)	803	801	800	794	738**	769	760	749	789	694**
Recovery	845				797**	813				793
Hb (g/dL)	15.4	15.5	15.5	15.3	14.1**	15.2	15.2	14.8	15.4	13.5**
Recovery	15.5				14.7*	15.1				15.4
Hct (%)	45.3	45.3	45.2	44.9	42.2*	42.8	42.5	42.0	43.7	39.4*
Recovery	45.8				44.3	43.8				45.1
Reticulocytes (‰)	10	9	10	10	18*	9	9	10	9	23**
Recovery	5				5	5				6
PT (sec)	14.9	15.2	14.9	15.9	20.2**	11.9	12.2	12.2	11.8	11.8
Recovery	13.3				12.7	11.2				11.3
APTT (sec)	26.6	27.7	30.0	28.8	28.2	20.2	20.9	24.2	19.6	21.7
Recovery	27.3				28.1	20.7				21.7
	Organ weights (n = 6)
Spleen (g)	0.58	0.55	0.59	0.61	0.79**	0.38	0.40	0.44	0.50*	0.81**
Recovery	0.80				0.84	0.46				0.58*
Relative (R) to bw (g/100 g)	0.20	0.16	0.20	0.20	0.28**	0.19	0.20	0.22	0.25	0.30**
R (g/100 g) Recovery	0.18				0.19	0.20				0.25*
Liver (g)	8.41	9.34	8.20	9.56	10.11*	6.03	6.21	6.66	8.53	7.12
Recovery	9.44				9.57	6.74				7.10
Relative (R) to bw (g/100 g)	2.88	3.01	2.80	3.10	3.54**	3.06	3.12	3.24	3.21	3.53**
R (g/100 g) Recovery	2.70				2.87	2.92				3.00
Kidney (g)	2.25	2.43	2.21	2.52*	2.40	1.74	1.65	1.70	1.66	1.66
Recovery	2.54				2.67	1.75				1.75
Relative (R) to bw (g/100 g)	0.75	0.79	0.76	0.82	0.84*	0.88	0.83	0.83	0.82	0.83
R (g/100 g) Recovery	0.73				0.80	0.76				0.74
Adrenal Gland (mg)	51.6	49.9	44.6	46.5	55.5	64.0	68.0	71.8	71.2	73.6
Recovery	50.8				52.6	63.7				78.6*
Relative (R) to bw (mg/100 g)	17.5	16.1	15.4	15.1	19.3	32.6	34.2	34.9	35.0	36.4
R (g/100 g) Recovery	14.8				15.9	27.8				33.3
	Gross pathology
Spleen Blackish change	0/6	0/6	0/6	0/6	1/6	0/6	0/6	0/6	1/6	6/6
Recovery	0/6				0/6	0/6				0/6
Kidney Apparent spotty pattern on surface	0/6	0/6	0/6	0/6	2/6	0/6	0/6	0/6	0/6	0/6
Recovery	0/6				0/6	0/6				0/6
	Histopathology
	Liver
Swelling of hepatocytes	0/6	-	-	0/6	4/6	0/6	-	-	0/6	2/6
Recovery	0/6	-	-	--	0/6	0/6	-	-	-	0/6
Microgranuloma	0/6	-	-	1/6	0/6	0/6	-	-	0/6	0/6
Recovery	0/6				0/6	1/5				0/6
	Spleen
Congestion	0/6	-	-	0/6	6/6	0/6	-	0/6	3/6	6/6
Recovery	0/6				5/6	0/6				0/6
Increased hemosiderin in laden cells	0/6	-	-	0/6	6/6	0/6	-	0/6	2/6	6/6
Recovery	0/6				6/6	0/6				0/6
	Kidney
Increased Eosinophilic Bodies	0/6	-	-	0/6	6/6	0/6	-	-	-	0/6
Recovery	0/6				0/6	0/6				0/6

*P<0.05

**P<0.01

– not examined

Blank cell indicates incidence not specifically listed in tables on the basis on absence of effect

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the NOAEL was determined to be 40 mg/kg bw/day. The liver and spleen were identified as target organs.

Executive summary:

The repeated dose toxicity of the test substance was investigated in accordance with the standardised guideline OECD 407 and the Japanese guidelines detailed in Notification No. 700 of Kanpogyo, No.1039 of Yakuhatsu, and No.1014 of 61 Kikyoku under GLP conditions.

Male and female Sprague-Dawley rats were exposed to the test substance at dose levels of 0 (vehicle control), 8, 40, 200 and 1000 mg/kg bw/day for 28 days followed by a 14 day recovery period. Six animals per sex per group were dosed, with an additional group of males and females included in both the vehicle control and high dose groups to be used as the recovery group. 

Animals were examined once daily for clinical signs and body weight and food consumption were measured throughout the study period. Prior to sacrifice, haematology, clinical chemistry and urinalysis investigations took place. At the end of the study animals were sacrificed and subjected to necropsy.

No mortality was observed. No effects of test substance administration were observed in body weight, food consumption, blood chemical examinations and urinalysis.

Salivation was observed in all high-dose animals and one male of the 200 mg/kg/day dose group during the dosing period. There were no abnormal clinical observations during the recovery period.

The 1000 mg/kg/day dose group revealed decreases in red blood cell count, haemoglobin concentration and haematocrit value and an increase in reticulocytes in both sexes, and a decrease in mean corpuscular haemoglobin concentration and an increase in segmental neutrophils in females at the end of the dosing period. Decreased red blood cell count and haemoglobin concentration persisted during the recovery period.

At the end of the dosing period, absolute and relative spleen and liver weights were increased in both sexes of the 1000 mg/kg/day dose-group. The increased spleen weight effect persisted in the females during the recovery period.

At necropsy, blackish change of the spleen was noted in males and females of the 1000 mg/kg/day dose group, which were apparently reversible during the recovery period.

Upon histopathologic examination, the following dose-dependent findings were evident at the end of the dosing period: congestion and increased hemosiderin-laden cells in the spleen of females in the 200 mg/kg/day dose group; and swelling of hepatocytes in both sexes, congestion and increased hemosiderin-laden cells in the spleen in both sexes of the 1000 mg/kg/day dose group.

Under the conditions of this study, the NOAEL was determined to be 40 mg/kg bw/day. The liver and spleen were identified as target organs.