Bis(1,2,2,6,6-pentamethyl-4-piperidyl) [[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]butylmalonate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD testing guideline compliant study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Preliminary Reproduction Toxicity Screening Test (Precursor Protocol of GL 421)

Deviations:

yes

Remarks:

extra investigations were performed for endpoints known to be affected (haematology, histopathology of liver, spleen and intestine)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(1,2,2,6,6-pentamethyl-4-piperidyl) [[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]butylmalonate

EC Number:

264-513-3

EC Name:

Bis(1,2,2,6,6-pentamethyl-4-piperidyl) [[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]butylmalonate

Cas Number:

63843-89-0

Molecular formula:

C42H72N2O5

IUPAC Name:

bis(1,2,2,6,6-pentamethylpiperidin-4-yl) 2-butyl-2-[(3,5-di-tert-butyl-4-hydroxyphenyl)methyl]propanedioate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Purity: HPLC: approx. 99 area-% (220 nm)
Homogeneity: given (visually)
Expiry date: 21 Feb 2014
Physical state/ appearance: solid/ white
Storage conditions: ambient (room temperature)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld
- Age at study initiation: 11-12 weeks; males/ females
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: none
- Housing: Individually, except during mating and females with litters
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 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: Oct 11, 2011 To: Dec 6, 2011

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% aqueous CMC and 5% Cremophor EL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
To prepare the test item suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then,
drinking water containing 1% carboxymethylcellulose and 5 mg/ 100 mL Cremophor EL was filled up to the desired volume, subsequently released with a magnetic stirrer. During administration of the test substance preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least once a week and were stored at room temperature.


VEHICLE
- Justification for use and choice of vehicle (if other than water): A homogenous suspension could be prepared.
- Concentration in vehicle: 0.05 - 1 g/L
- Amount of vehicle (if gavage): ca 10 ml (The calculation of the administered volume was generally based on the most recent individual body weights)

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in drinking water containing 1% carboxymethylcellulose and 5 mg/ 100 mL Cremophor EL for a period of 7 days at room temperature was proven during the study

Duration of treatment / exposure:

males: 36 days
females: 45, 49 or 50 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 0.5, 2 and 10 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest dose group was selected to cause parental toxicity (based on range-finder study).

Positive control:

not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays.
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO; including palpation) were performed in all animals prior to the administration period and thereafter at weekly intervals.

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. exophthalmus
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 body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).


OTHER: In the morning blood was taken from the retroorbital venous plexus from fasted animals. The animals were anesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany). Parameters determined were Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular
volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Differential blood count,

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities,

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was - if possible - determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

ORGAN WEIGHTS:
Epididymides, Testes

HISTOPATHOLOGY and weights:
All gross lesions
Epididymides (modified Davidson’s solution)
Jejunum
Liver
Lymph nodes (axillary and mesenteric lymph nodes)
Ovaries (modified Davidson’s solution)
Spleen
Testes (modified Davidson’s solution)

Postmortem examinations (offspring):

SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic) as follows: all F1 pups

GROSS NECROPSY
- Gross necropsy consisted of gross necropsy.

HISTOPATHOLOGY / ORGAN WEIGTHS
None

Statistics:

Organ weights: Non-parametric one-way analysis using KRUSKALWALLIS 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.
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) forthe hypothesis of equal medians

Food consumption2 (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter: Simultaneous comparison of all dosegroups with thecontrol group using the DUNNETT-test(two-sided) for the hypothesis of equa lmeans.

Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions

Proportions of affected pups per litter with necropsy observations: Pairwise comparison of each dose group with the control group using the WILCOXONtest (one-sided) for the hypothesis of equal medians

Reproductive indices:

Female mating index (%) = number of females mated (defined as the number of females with vaginal sperm or with implants in utero) / number of females placed with males x 100

Female fertility index (%) = number of females pregnant (defined as the number of females with implants in utero)/ number of females mated (defined as the number of females with vaginal sperm or with implants in utero) x 100

Gestation index = number of females with live pups on the day of birth / number of females with implants in utero x 100

Live birth index = number of liveborn pups at birth / total number of pups born X 100

Offspring viability indices:

Viability index (number of live pups on day 4 after birth/number of live pups on the day of birthx100)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

at the high dose group of 10 mg/kg bw

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

at the high dose group of 10 mg/kg bw

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

at the high dose group of 10 mg/kg bw

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

granulomatous inflammation in mesenteric lymph nodes, spleen and liver in some animals of the high dose group

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

The following test substance-related, adverse findings were noted:

Test group 3: 10 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examinations
• Body weight loss in 4 male animals during towards the end of the administration period
• Poor general condition in 2 male animals towards the end of the administration period
• Swelling of limbs and unsteady gait in 2 male animals towards the end of the administration period
• Piloerection in 1 male animal towards the end of the administration period

Reproductive Performance
• No test substance-related, adverse findings were noted.
Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. One male of test group 2 (No. 24 mated with female No. 124 – 2 mg/kg bw/d) and 1 male animal of test group 3 (animal No. 39 mated with female No. 139 – 10 mg/kg bw/d) did not generate F1 pups. Thus, the male fertility index ranged between 90% and 100% . These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
All sperm positive rats delivered pups with the exception of female No. 124 (test group 2) and female No. 139 (test group 3), which were mated with male Nos. 24 and 39 but did not become pregnant. The female fertility index varied between 90% and 100% . Female animals Nos. 124 and 139, which delivered no pups had no implantation sites.
The mean duration of gestation was between 22.0 and 22.2 days and did not show significant differences.

The female gestation index reached 100% in all test groups including the control.

The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 98% (test group 1), 99% (test group 2) and 100% (test groups 0, 3). Single stillborn pups were only seen in test groups 1 and 2 (0.5 and 2 mg/kg bw/d).



Clinical Pathology
• Decreased hemoglobin and hematocrit values in both sexes
• Decreased red blood cell (RBC) counts in females
• Decreased mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) in males
• Increased platelet counts in females
• Increased total white blood cell (WBC) counts, absolute and relative neutrophil, absolute monocyte and absolute large unstained (LUC) counts in both sexes
• Decreased relative lymphocyte counts in both sexes

Pathology
• Minimal to moderate necrosis of the liver in 5 of 10 male animals
• Granulomatous inflammation with necrosis of the mesenteric lymph node in 9 of 10 male (graded marked to massive) and 9 of 10 female (graded moderate to marked) animals
• Granulomatous inflammation with necrosis of the spleen in 2 of 10 male (graded moderate to marked) and 2 of 10 female (graded slight to moderate) animals



Test group 2: 2 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examinations, Reproductive Performance, Clinical Pathology and Pathology
• No test substance-related, adverse findings were noted.
A few animals showed foamy aggregates of macrophages in the intestinal mucosa. This was reported to be related to the treatment, but not adverse.



Test group 1: 0.5 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examinations, Reproductive Performance, Clinical Pathology and Pathology
• No test substance-related, adverse findings were noted.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Higher doses could not be tested because they would result in mortality of parental animals.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

F1 PUPS


• No test substance-related, adverse findings were noted.

The mean number of delivered pups per dam and the rate of liveborn and stillborn pups, i.e. each 1 pup of female Nos. 115 and 118 in test group 1 (0.5 mg/kg bw/d) and 1 pup of female No. 121 in test group 2 (2 mg/kg bw/d), reflect the normal range of biological variation inherent in the strain used in this study.
The viability index as indicator for pup mortality between PND 0 and 4 was 99% for the control (test group 0; 1 pup of female No. 101 cannibalized), 98% for test group 1 (0.5 mg/kg bw/d; each 1 pup of female Nos. 113 and 115 cannibalized) and 100% for test groups 2 and 3 (2 and 10 mg/kg bw/d). The remaining pups which belonged to female Nos. 101, 113 and 115 did not show any findings until sacrifice.
The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not show biologically relevant differences between test groups.
The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.
Mean pup body weights and pup body weight changes of all pups in all test groups were comparable to the concurrent control values. No runts were seen in any test group.
During the necropsy of all F1 pups no test substance-related findings were observed.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Limited parameters on the F1 generation are investigated in this type of study.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Histopathology findings in jejunum

Jejunum	Male animals				Female animals
Test group (mg/kg bw/d)	0 (0)	1 (0.5)	2 (2)	3 (10)	0 (0)	1 (0.5)	2 (2)	3 (10)
No. of animals	10	10	10	10	10	10	10	10
Foam cell aggregates, diffuse			4	10				10
Grade 1			2
Grade 2			2	6				7
Grade 3				4				3
Inflammation, granulomatous								3
Grade 1								3

Table 2: Histopathology findings in liver

Liver	Male animals				Female animals
Test group (mg/kg bw/d)	0 (0)	1 (0.5)	2 (2)	3 (10)	0 (0)	1 (0.5)	2 (2)	3 (10)
No. of animals	10	10	10	10	10	10	10	10
Necrosis, multifocal				5
Grade 1				3
Grade 2				1
Grade 3				1
Inflammation, granulomatous				2				8
Grade 1				2				3
Grade 2								1
Grade 3								3
Grade 4								1

Table 3: Histopathology findings in mesenteric lymph nodes

Mesenteric lymph nodes	Male animals				Female animals
Test group (mg/kg bw/d)	0 (0)	1 (0.5)	2 (2)	3 (10)	0 (0)	1 (0.5)	2 (2)	3 (10)
No. of animals	10	10	10	10	10	10	10	10
Aggregates, histiocytic			10	10			8	10
Grade 1			2	4			3	4
Grade 2			5	4			4	5
Grade 3			3	1			1	1
Grade 4				1
Inflammation, granulomatous, with necrosis				9				9
Grade 3								1
Grade 4				7				8
Grade 5				2

Table 4: Histopathology findings in ovaries

Ovaries	Female animals
Test group (mg/kg bw/d)	0 (0)	1 (0.5)	2 (2)	3 (10)
No. of animals	10	10	10	10
Inflammation, granulomatous				5
Grade 1				5

Table 5: Histopathology findings in spleen

Spleen	Male animals				Female animals
Test group (mg/kg bw/day)	0 (0)	1 (0.5)	2 (2)	3 (10)	0 (0)	1 (0.5)	2 (2)	3 (10)
No. of animals	10	10	10	10	10	10	10	10
Hematopoiesis, extramedullary		1	2	9	10	6	5	10
Grade 1		1			10	5	4
Grade 2			2	4		1	1	6
Grade 3				3				4
Grade 4				1
Grade 5				1
Inflammation, granulomatous, with necrosis				2				2
Grade 2								1
Grade 3				1
Grade 4				1				1
Foam cell aggregates, diffuse				9
Grade 2				7
Grade 3				2

Applicant's summary and conclusion

Conclusions:

No toxicity to reproduction was identified at the screening level.