2,2,6,6-Tetramethylpiperidin-4-yl dodecanoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline conform study with well characterised material

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2,6,6-tetramethylpiperidin-4-yl dodecanoate

EC Number:

700-503-1

Cas Number:

101238-01-1

Molecular formula:

C21 H41 N O2

IUPAC Name:

2,2,6,6-tetramethylpiperidin-4-yl dodecanoate

Details on test material:

Physical state: Liquid/ yellowish, clear
Expiry date: Jan 2022
Purity: > 95%
Storage conditions: Room temperature
Stability und er storage conditions: stable

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Weight at study initiation: 130g females and 160 g males
- Fasting period before study: no
- Housing: 5 animals per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr):15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2 July 2013 To: 11 October 2013

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

For each concentration, the test substance was weighed out and mixed with a small amount of food. Then corresponding amounts of food, depending on test group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability in the diet at room temperature for a period of 32 days was proven before the start of the administration period in a separate GLP study.

Duration of treatment / exposure:

92 days (male animals) / 93 days (female animals).

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
500, 2500 and 10000 ppm
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
32, 170 and 722 mg/kg bw (males)
Basis:
other: average calculated from food consumption

Doses / concentrations 3

Remarks:

Doses / Concentrations:
39, 193 and 961 mg/kg bw (females)
Basis:
other: average calculated from food consumption

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on result of the dose-range finding studies.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund and dead animals was made twice daily on working day and once daily on Saturday, Sunday and public holidays. If animals were in a moribund state, they were sacrificed and dissected.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All rats were checked daily for any abnormal clinical signs prior administration, as well as, within 2 hours and 5 hours post administration. Detailed clinical observation (DCO) was performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined:

1. abnormal behavior during “handling”
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalmos
15. feces (appearance/ consistency)
16. urine
17. pupil size


BODY WEIGHT: Yes
- Time schedule for examinations:
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between body weight on the respective day of weighing and body weight on day 0 was calculated as body weight change.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: At the end of the administration period, on study day 91.
- Dose groups that were examined: control and highest dose group animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the administration period, on study day 92.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: 5 animals per test group and sex
- Parameters checked below were examined.
Leukocyte count
(WBC)
Erythrocyte count
(RBC)
Hemoglobin
(HGB)
Hematocrit
(HCT)
Mean corpuscular volume
(MCV)
Mean corpuscular hemoglobin
(MCH)
Mean corpuscular hemoglobin concentration
(MCHC)
Platelet count
(PLT)
Differential blood count
Reticulocytes
Prothrombin time (Hepato Quick’s test) (HQT)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the administration period, on study day 92.
- Animals fasted: Yes
- How many animals: 5 per sex and dose
- Parameters checked below were examined.

Alanine aminotransferase
(ALT)

Aspartate aminotransferase
(AST)

Alkaline phosphatase
(ALP)

g-Glutamyltransferase
(GGT)

Sodium
(NA)
Potassium
(K)
Chloride
(CL)
Inorganic phosphate
(INP)
Calcium
(CA)
Urea
(UREA)
Creatinine
(CREA)
Glucose
(GLUC)
Total bilirubin
(TBIL)
Total protein
(TPROT)
Albumin
(ALB)
Globulins
(GLOB)
Triglycerides
(TRIG)
Cholesterol
(CHOL)
Magnesium
(MG)
Bile acids
(TBA)




URINALYSIS: Yes
- Time schedule for collection of urine: study day 85
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (For urinalysis the individual animals were transferred to metabolism cages
(withdrawal of food and water) and urine was collected overnight.)
- Parameters checked below were examined.
pH
Protein
Glucose
Ketones
Urobilinogen
Bilirubin
Blood
Specific gravity
Sediment
Color, turbidity
Volume


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: days 86 - 91
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / (FOB)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

The following weights were determined in all animals sacrificed on schedule:

1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Ovaries
9. Spleen
10. Testes
11. Thymus
12. Thyroid glands
13. Uterus with cervix



HISTOPATHOLOGY: Yes (see table)

Statistics:

Blood parameters For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
For parameters with unidirectional changes:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians * for p < 0.05
** for p < 0.01 SIEGEL, S. (1956):
Non-parametric statistics for the behavioural sciences.
McGraw-Hill New York Holm (1979): A Simple Sequentially Rejective Multiple Test Procedure. Scand. J. Statist. 6, 65-70


Urinalysis parameters (except pH, urine volume, specific gravity, color and turbidity Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians * for p < 0.05
** for p < 0.01 SIEGEL, S. (1956):
Non-parametric statistics for the behavioural sciences.
McGraw-Hill New York


Urine pH, volume, specific gravity, color and turbidity Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians. Urine color and turbidity are not evaluated statistically. * for p < 0.05
** for p < 0.01 SIEGEL, S. (1956):
Non-parametric statistics for the behavioural sciences.
McGraw-Hill New York

Weight parameters Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians * for p ≤ 0.05
** for p ≤-0.01 HETTMANNSPERGER, T.P. (1984): Statistical Inference based on Ranks, John W

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Palpable mass in abdomen was observed in male animal Nos. 32 and 37 as well as in female animal Nos. 71, 72, 73 and 77 of test group 3 (10000 ppm) during the last third of the administration period for a limited time.

Mortality:

mortality observed, treatment-related

Description (incidence):

Palpable mass in abdomen was observed in male animal Nos. 32 and 37 as well as in female animal Nos. 71, 72, 73 and 77 of test group 3 (10000 ppm) during the last third of the administration period for a limited time.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced at 2500 and 10000 ppm for males (see figure)

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):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

hemoglobin and hematocrit values were slightly increased in rats of both sexes of test group 3 (10000 ppm).

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

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

caecum at 10000 ppm

Histopathological findings: neoplastic:

not examined

Details on results:

With regard to clinical examinations, signs of general systemic toxicity in Wistar rats of either sex were observed in test group 3 (10000 ppm) indicated by decreased mean body weight values below the historical control data. In addition, masses in the abdomen were palpable in several animals during the last third of the treatment period. A clear reason for the occurrence of this finding could not be evaluated.
No signs of toxicological relevance were observed for male and female animals in test groups 1 and 2 (500 and 2500 ppm).

Regarding clinical pathology in rats of both sexes of test group 3 (10000 ppm), higher hemoglobin and hematocrit values were due to hemoconcentration in these individuals. Higher albumin levels in males of the mentioned test group were also due to this effect. No hint of a renal dysfunction as cause of hemoconcentration could be found. The reason for isolated higher calcium levels in males and females of test group 3 (10000 ppm) and lower urea levels in males of this test group could not be elucidated.

Regarding pathology, the cecum was the target organ. In the cecum of three males and three females of test group 3 (10000 ppm) slight inflammatory cell infiltrates and edema were present, which was interpreted to be an inflammation of the cecum. This inflammation was regarded to be tre tment related and adverse.
Furthermore, a body weight decrease in male animals of test group 3 (10000 ppm) was observed. This was also regarded to be test substance-related and adverse. Whether or not there was a relation between the cecum findings and the body weight could not be determined. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

During numerous time intervals, food consumption in male animals of test group 1-3 (500, 2500 and 10000 ppm) was lower when compared to the control group. As no dose-response relationship occurred the changes were assessed to be incidental and not related to treatment.
In female animals, mean food consumption in female animals of test group 3 (10000 ppm) was higher during the entire study period. The higher values were caused by the animals of cage No. 16, most likely related to spilling of food out of the tray. The values of cage No. 15 were within the range typical for animals of this strain and age. Therefore, the effect was assessed as being incidental and without toxicological relevance.




Specifically. three males (Nos. 31, 32 and 39) and three females (Nos. 72, 73 and 75) of test group 3 (10000 ppm) revealed a slight infiltration of the mucosa of mixed cells (granulocytes, macrophages and lymphocytes). The same animals also showed a submucosal edema, with occasionally inflammatory cells of the mucosa infiltrating the edematous submucosa. This finding was regarded to be an inflammation of the cecum and treatment related.

Other: No test substance-related effects on estrous cycle length and the number of cycles were obtained. Concerning motility of the sperms and incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Dose-dependent reduction in body weight gain at higher doses.

2

Dose descriptor:

NOAEL

Effect level:

2 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: local inflammation of the caecum at 10000 mg/kg bw

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

	Males				Females
Test group	0	1	2	3	0	1	2	3
(ppm)	0	500	2500	10000	0	500	2500	10000
No. of animals	10	10	10	10	10	10	10	10
Infiltrates, mixed cell, diffuse	0	0	0	3	0	0	0	3
Grade 2				3				3
Edema, submucosal				3				3
Grade 2				3				3

Table 1: Histopathology findings

	Male animals			Female animals
Test group	1	2	3	1	2	3
(ppm)	500	2500	10000	500	2500	10000
Terminal body weight	93%	90%*	83%**
Heart	93%	95%	88%**
Kidneys	95%	94%	86%**
Liver	90%	88%*	83%**
Spleen				114%*	123%*	111%

Table 2: Relevant effects on absolute organ weights and terminal body weight,*: p≤0.05, **: p≤0.01

	Male animals
Test group	1	2	3
(ppm)	500	2500	10000
Brain	105%	107%*	117%**
Epididymides	112%*	108%	118%**
Testes	110%*	111%	117%**

Table 3: Relevant effects on relative organ weights, *: p≤0.05, **: p≤0.01

Applicant's summary and conclusion

Conclusions:

The substance caused a local inflammation of the caecum at the highest dose group of 10000 ppm as shown by histopathology. Body weight gain was affected in males only. The NOAEL for 90-day feed exposure was identified as 2500 ppm (ca 170 - 193 mg/kg bw).