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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD 414 and GLP compliant study

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 414 (Prenatal Developmental Toxicity Study)

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)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: About 10-12 weeks
- Weight at study initiation: 146.5 – 189.0 g
- Housing: single caging
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: six days

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

IN-LIFE DATES: From: 2013-06-25 To: 2013-07-16

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The oily test substance preparations were prepared at the beginning of the administrationperiod and thereafter at maximum intervals of 7 days, which took into account the period of established stability. For the test substance preparations, the specific amount of test substance were weighed, topped up with corn oil in a graduated flask and intensely mixed by shaking until it is dissolved.



VEHICLE
- Justification for use and choice of vehicle (if other than water): the test item is soluble in the vehicle. It is not soluble in water.
- Amount of vehicle (if gavage): 4 ml/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC-method

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
The day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory.

Duration of treatment / exposure:

gestation days 6- 19

Frequency of treatment:

daily

Duration of test:

14 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 30 ,100 and 300 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

25

Control animals:

yes

Details on study design:

- Dose selection rationale: Based on range-finder study with pregnant rats

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality/Morbidity, pertinent behavioral changes and/or signs of overt toxicity. were checked twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays (GO 0 to 20).

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: GO 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.


POST-MORTEM EXAMINATIONS: Gross pathology
- Sacrifice on gestation day: GD 20

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Site of implantations in the uterus

Fetal examinations:

- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [ half per litter ]
- Skeletal examinations: Yes: [ half per litter ]

In the present study the glossary of WISE et al. (1997) and its updated version of MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD et al. (1999) and SOLECKI et al. (2001, 2003):

Malformation
A permanent structural change that is likely to adversely affect the survival or health.

Variation
A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect the survival or health. This includes delays in growt or morphogenesis that have otherwise followed a normal pattern of development.

The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations.

Statistics:

DUNNETT's test: Food consumption, body weight, body weight change, DUNNETT's test
corrected body weight gain, carcass weight, weight of
the unopened uterus, weight of the placentas and
fetuses, corpora lutea, implantations, pre- and
postimplantation losses, resorptions and live fetuses

FISHER's exact test
Number of pregnant animals at the end of the study, FISHER's exact test mortality rate (of the dams) and number of litters with fetal findings

WILCOXON test
Proportion of fetuses with findings per litter

Indices:

sex ratio
conception rate (in %)
preimplantation loss (in %)
postimplantation loss (in %)

Historical control data:

Included in the final report.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
High-dose female No. 94 (300 mg/kg bw/d) died on GD 15. The gross pathological examination of this animal revealed findings indicative of a gavage error. There were no further substance-related or spontaneous mortalities in any females of all test groups (0, 30, 100 or 300 mg/kg bw/d).

All females of the high-dose group (300 mg/kg bw/d), nearly all females (23 out of 25) of the mid-dose group (100 mg/kg bw/d) and 16 females of the low-dose group (30 mg/kg bw/d) showed transient salivation during major parts of the treatment period. Salivation persisted in the respective animals for some minutes after daily gavage dosing (i.e. up to 10 minutes) and was initially observed on GD 7. No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 100, 300 or 1000 mg/kg bw/d during the entire study period.

The mean food consumption of the high-dose dams (300 mg/kg bw/d) was statistically significantly reduced on GD 6-10 and GD 15-17. If calculated for the entire treatment period (GD 6-19) or the entire study period (GD 0-20), the dams of test group 3 consumed 12% or 7% less food, respectively, in comparison to the concurrent control group. Furthermore, the mean food consumption of the mid-dose dams (100 mg/kg bw/d) was statistically significantly reduced on GD 6-8. If calculated for the entire treatment period (GD 6-19) or the entire study period (GD 0-20), the dams of test group 2 consumed 4% less food, respectively, in comparison to the concurrent control group. The mean food consumption of the dams in test group 1 (30 mg/kg bw/d) was generally comparable to the concurrent control throughout the entire study period.

The mean body weights of the high-dose females (300 mg/kg bw/d) were statistically significantly reduced from GD 8 onwards until terminal sacrifice on GD 20. At the beginning of treatment these dams even lost weight, thus, the average body weight gain was statistically significantly reduced on GD 6-8 and GD 17-19. If calculated for the entire treatment period (GD 6-19) or entire study period (GD 0-20), these dams gained 21% or 13% less weight than the concurrent control animals, respectively.
The mean body weights of the mid-dose group (100 mg/kg bw/d) were slightly lower than the concurrent control values (without attaining statistical significance), mainly caused by the statistically significantly reduced mean body weight gain value on GD 6-8. If calculated for the
entire treatment period (GD 6-19) or entire study period (GD 0-20), these dams gained 8% or 7% less weight than the concurrent control animals (without attaining statistical significance). The impairments of body weights in the mid- and high-dose groups were considered to be treatment-related, whereas the statistically significantly increased body weight gain value on GD 1-3 (test group 3) was an incidental occurrence.
The mean body weights and the average body weight gains of the low-dose rats (30 mg/kg bw/d) were in general comparable to the controls throughout the entire study period. This includes the statistically significantly reduced body weight gain value on GD 17-19 which was considered as an accidental finding.

The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) was distinctly and statistically significantly lower in test groups 2 (100 mg/kg bw/d – about 20% below the concurrent control value) and 3 (300
mg/kg bw/d – about 29% below the concurrent control value). Furthermore, the carcass weight of the high-dose dams was statistically significantly lower in comparison to the control group (about 5% below controls).
These effects are assessed as direct, test substance-related signs of maternal toxicity.
The corrected body weight gain of test group 1 (30 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Mean carcass weights remained also unaffected by the treatment.

No necropsy findings which could be attributed to the test substance were seen in any dam. One finding indicative for misgavaging, i.e. thoracic cavity filled with serous fluid, was recorded for dam No. 94 (300 mg/kg bw/d), which died on GD 15.

Although without attaining statistical significance, the mean gravid uterus weight of the animals of test group 3 (300 mg/kg bw/d) was reduced in comparison to the control group (about 10% below controls), which is likely to be a subsequent effect of the increased number of resorptions in this test group (see 4.2.2.3. Reproduction data). The mean gravid uterus weights of the low- and mid-dose animals (30 and 100 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were statistically significant differences in the values calculated for the postimplantation loss (5.2%/4.5%/4.0%/13.8%** [p<=0.01] in test groups 0-3), the number of resorptions and viable fetuses (94.8%/95.5%/96.0%/86.2%** [p<=0.01]. These differences were exclusively caused by an increased number of early resorptions in this dose group. All values were outside of the historical control range of the test facility.

There were no test substance-related and/or biologically relevant differences between the test groups 0, 1 and 2 in the values calculated for the pre- and postimplantation losses, the number of resorptions and viable fetuses.

The sex distribution of the fetuses in test groups 1-3 (30, 100 and 300 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.
The mean placental weights of the low-, mid- and high-dose groups (30, 100 and 300 mg/kg bw/d) were comparable to the corresponding control group.
The mean fetal weights of test groups 1, 2 and 3 (30, 100 and 300 mg/kg bw/d) were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

The high-dose of the test item (300 mg/kg bw/d) caused a significant increase of early resorptions and, subsequently, postimplantation loss as well as a decrease in the number of viable fetuses. All values for these parameters were outside of the historical control range of the test facility. This effect is considered to be treatment–related. However, it occurred exclusively in the presence of distinct maternal toxicity, which was most pronounced during the early phase of the treatment, i.e. in the days shortly after implantation (GD 6-8). A relationship between the profound disturbance of maternal nutritive status during this sensitive window of development and an adverse effect on the number of surviving early implants is assumed.
No influence of the test compound on fetal weight and sex distribution of the fetuses was noted at any dose. Particularly, the surviving high-dose fetuses showed a normal prenatal development in terms of fetal weight gain, which is correlated to the lower average litter size in these animals.
Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

All females of the high-dose group (300 mg/kg bw/d), nearly all females (23 out of 25) of the mid-dose group (100 mg/kg bw/d) and 16 females of the low-dose group (30 mg/kg bw/d) showed transient salivation during major parts of the treatment period. Salivation persisted in the respective animals for some minutes after daily gavage dosing (i.e. up to 10 minutes).

Table 1: Total external variations

		Test group 0 0 mg/kg bw/d	Test group 1 30 mg/kg bw/d	Test group 2 100 mg/kg bw/d	Test group 3 300 mg/kg bw/d
Litter Fetuses	N N	25 261	25 243	25 267	23 218
Fetal incidence	N (%)	2 (0.8)	0.0	0.0	0.0
Litter incidence	N (%)	2 (8.0)	0.0	0.0	0.0
Affected fetuses/litter	Mean%	0.7	0.0	0.0	0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 2: Total soft tissue malformations

		Test group 0 0 mg/kg bw/d	Test group 1 30 mg/kg bw/d	Test group 2 100 mg/kg bw/d	Test group 3 300 mg/kg bw/d
Litter Fetuses	N N	25 121	25 114	25 127	23 105
Fetal incidence	N (%)	0.0	0.0	0.0	2 (1.9)
Litter incidence	N (%)	0.0	0.0	0.0	2 (8.7)
Affected fetuses/litter	Mean%	0.0	0.0	0.0	2.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Soft tissue malformations were recorded for male high-dose fetus No. 83-06 (300 mg/kg bw/d, Tab. 2.). Female high-dose fetus No. 78-08 had more than one malformation affecting the liver, lung and heart. The total incidence of soft tissue malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.

Table 3: Fetal soft tissue variations

		Test group 0 0 mg/kg bw/d	Test group 1 30 mg/kg bw/d	Test group 2 100 mg/kg bw/d	Test group 3 300 mg/kg bw/d
Litter Fetuses	N N	25 121	25 114	25 127	23 105
Fetal incidence	N (%)	5 (4.1)	3 (2.6)	6 (4.7)	0.0
Litter incidence	N (%)	5 (20)	3 (12)	6 (24)	0.0
Affected fetuses/litter	Mean%	3.9	2.8	5.3	0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Some soft tissue variations were detected in the test groups 0-2 (0, 30 or 100 mg/kg bw/d), i.e. short innominate, dilated renal pelvis and dilated ureter. These variations were neither statistically significantly different from control nor dose-dependent and therefore, not

considered biologically relevant.

Table 4: Total fetal skeletal malformations

		Test group 0 0 mg/kg bw/d	Test group 1 30 mg/kg bw/d	Test group 2 100 mg/kg bw/d	Test group 3 300 mg/kg bw/d
Litter Fetuses	N N	25 140	25 129	25 140	25 113
Fetal incidence	N (%)	3 (2.1)	1 (0.8)	0.0	4 (3.5)
Litter incidence	N (%)	3 (12)	1 (4.0)	0.0	4 (17)
Affected fetuses/litter	Mean%	2.0	0.8	0.0	5.6

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Some skeletal malformations were detected in the test groups 0, 1 and 3 (0, 30 and 300 mg/kg bw/d) affecting the skull, sternum and forelimbs (Tab. 4). The incidences of these malformations were neither statistically significantly different from control nor dose-dependent and therefore, not considered biologically relevant.

Table 5: Total fetal skeletal variations

		Test group 0 0 mg/kg bw/d	Test group 1 30 mg/kg bw/d	Test group 2 100 mg/kg bw/d	Test group 3 300 mg/kg bw/d
Litter Fetuses	N N	25 140	25 129	25 140	25 113
Fetal incidence	N (%)	139 (99)	128 (99)	139 (99)	111 (98)
Litter incidence	N (%)	25 (100)	25 (100)	25 (100)	23 (100)
Affected fetuses/litter	Mean%	99.4	99.2	99.2	98.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeleton and appeared without a relation to dosing (Tab. 5). The overall incidences of skeletal variations were comparable to the historical control data.

Applicant's summary and conclusion

Conclusions:

The substance does not cause developmental toxicity or teratogenicity in rats.