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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:
2010-02-25 to 2010-07-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
, adopted 1996-03-22
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
700-380-4
EC Number:
700-380-4
Molecular formula:
A: C25H50O2 B: C27H54O2
IUPAC Name:
700-380-4
Details on test material:
- Name of test material (as cited in study report): SymMollient S (Cetearyl nonanoate)
- Chemical name: A: Hexadecyl nonanoate; B: Octadecyl nonanoate
- Physical state: colourless liquid to solid
- Storage condition of test material: room temperature (20 +/- 5 °C), dark, dry and in original container
- Water solubility: a) 1.1 x 10^-6 mg/L (QSAR, WSKOW v1.41); b) 1.1 x 10^-8 mg/L (QSAR, WSKOW v.1.41)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst/Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: males: 292 to 339 g; females: 185 to 218 g
- Housing: individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding ('Lignocel' J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg/Germany). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet (ad libitum): pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst/Switzerland)
- Water (ad libitum): community tap-water from Füllinsdorf
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22+/- 3 °C
- Relative humidity: 30 - 70 %
- Air changes: 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (music was played during the light period)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly using the test item as supplied. SymMollient S was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Insoluble particles observed in the dose formulation for the high dose group were discarded from the solution by decanting of the dose formulation. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Based upon the results of stability analyses performed within a non-GLP Harlan Laboratories study (C76932), dose formulations were stable for at least one week.

VEHICLE
- Batch no.: 369109168 (Carl Roth GmbH & Co. KG, 76185 Karlsruhe)
Details on mating procedure:
- M/F ratio per cage and length of cohabitation: main group females were housed with sexually mature main group males (1:1) until evidence of copulation was observed.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear; the day of mating was designated day 0 post coitum.
- After copulation each female was caged individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day, samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability. During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were stored at -20 ± 5 °C until analysis.

The samples were analyzed by GC-FID. The test item was used as the analytical standard.

Results:
The SymMollient S peaks were assigned in sample chromatograms by comparison to that of working standards. In blank sample chromatograms, no peak appeared at the retention time of SymMollient S and, therefore, the absence of the test item in the vehicle control samples (peanut oil) was confirmed.

The formulations investigated during the study were found to comprise SymMollient S in the range of 91.6% to 109.1% and, thus, the required content limit of ±20% with reference to the nominal concentration was met. The homogeneous distribution of SymMollient S in the preparations was approved because single results found did not deviate more than 3.1% (<15%) from the corresponding mean.

In addition, the test item was found to be stable in formulations when kept 7 days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item SymMollient S and peanut oil as vehicle during this study. Formulations were found to be homogeneously prepared and sufficient formulation stability at room temperature was approved.

For the results of the analysis please see also Section 8 Analytical methods (Entry: s_Morgenthal_2011).
Duration of treatment / exposure:
- Males: 14 days pre-pairing, throughout pairing (14 days maximum) and until the day before sacrifice (necropsy: after treatment of at least 28 days).
- Females: 14 days pre-pairing, throughout the pairing (14 days maximum) and gestation period (approx. 21 days) and until day 4 post partum (day 0 post partum: day when dam delivered all her pups) for a total of approx. 7 weeks of treatment.
Frequency of treatment:
once daily, at approximately 24 hour intervals
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg /day
Basis:
other: actual administered
No. of animals per sex per dose:
0 mg/kg/day: 10 males / 10 females
100 mg/kg/day: 10 males / 10 females
300 mg/kg/day: 10 males / 10 females
1000 mg/kg/day: 10 males / 10 females

Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on a dose range finding toxicity study in Han Wistar Rats. Four groups of 3 males and 3 females were treated by gavage with SymMollient S once daily over a 14-day treatment period. The following dose levels were applied: 0, 100, 300 and 1000 mg/kg/day. Control animals were dosed with the vehicle alone (peanut oil).

Results:
- all animals survived until the scheduled necropsy.
- no clinical signs or observations were noted at any dose level in males and in females.
- no test item-related effects on food consumption, body weights or body weight gain were observed in males or females at any dose level.
- no test item-related macroscopical findings were noted during the necropsy in males or in females at any dose level.
Based on these data dose levels of 0, 100, 300, and 1000 mg/kg bw/day were selected for the subsequent combined repeated dose toxicity study with the reproduction/development toxicity screening test in the rat.
Positive control:
No data

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Viability/mortality were recorded twice daily. Also, cage-side clinical observations were conducted once daily (during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioural abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration of the test item and weekly thereafter, detailed clinical observations were performed outside the home cage in all animals. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes or bizarre behaviour were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
males: weekly during pre-pairing and after pairing periods
females: pre-pairing period days 1 - 8 and 8 - 14; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and days 1 - 4 post partum.
No food consumption was recorded during the pairing period.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
- The testes and epididymides of all males were weighed separately.
- During histopathology: stages of spermatogenesis.

Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- The litters were examined for litter size, live births, still births and any gross anomalies.
- The sex ratio of the pups was recorded.
- Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: males were sacrificed after treatment of at least 28 days, when no longer needed for assessment of reproductive effects.
- Maternal animals: dams were sacrificed on day 5 post partum (day 0 post partum: delivery of pups). If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum (day 0 post coitum: day of mating).

GROSS NECROPSY
- All animals sacrificed were subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.
- All parental animals were sacrificed by an injection of sodium pentobarbital and exsanguinated and were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.
- Special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites [see Salewski (1964)].

ORGAN WEIGHTS
- From 5 males and 5 females selected from each group, the following organs were trimmed from any adherent tissue and their wet weight taken: adrenal glands (weighed as pairs), brain, heart, kidneys (weighed as pairs), liver, thymus and spleen

HISTOPATHOLOGY
The following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Males: prostate, seminal vesicles with coagulating gland, testes (in Bouin’s fixative) and epididymides (in Bouin’s fixative)
- Females: ovaries
- From the five males and five females per group selected for organ weights, the following tissues were reserved in neutral phosphate buffered 4% formaldehyde solution: gross lesions, brain, spinal chord, small and large intestines (incl. Peyer’s patches), stomach, liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and lungs (preserved by inflation with fixative and then immersion), uterus (with vagina), urinary bladder, lymph nodes (mesenterial, mandibular), peripheral nerve (sciatic) and bone marrow

-All organ and tissue samples to be examined were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion.
- Slides of all organs and tissues collected at terminal sacrifice from the animals in the control and high-dose main groups were examined . the same applied to all occurring gross lesions.
- Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
- Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.
Postmortem examinations (offspring):
GROSS NECROPSY
- Pups were sacrificed on day 4 post partum and were subjected to detailed macroscopic examination.
- Dead pups, except those cannibalized, were examined macroscopically.
- All pups were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.
Statistics:
The following statistical methods were used to analyze functional observational battery, food consumption, body weights and reproduction data, clinical laboratory investigations and organ weight and ratios:
- Means and standard deviations of various data were calculated.
- The Dunnett-test [see Dunnett (1955)] (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test [see Miller (1981)] (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test [see Fisher (1950)] was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
The following parameters were calculated:
- Fertility indices, mean precoital time, post-implanatation loss, mean litter size, and pup sex ratio.
Offspring viability indices:
The following parameters were calculated: viability indices
- Group mean values were calculated both on a litter basis and on a percentage per group basis.
- Mean pup weights were calculated from the individual weights both on a per group and on a per litter basis.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS (daily) AND MORTALITY
- All males and females survived until the scheduled necropsy.
- No clinical signs were noted in males at any dose level.
- Treatment with the test item at the dose level of 1000 mg/kg bw/day caused salivation in two females during the gestation period and in two females during the lactation period. No further clinical signs which were considered to be test item-related were noted during daily observations in females at any dose level. The finding was considered a test-item related sign of discomfort and not an adverse effect.
- In the control group, hair loss was observed in two females.
- During the detailed weekly clinical observation, salivation was confirmed in two females at the dose level of 1000 mg/kg bw/day. No further observations were noted at any dose level in males and females.

BODY WEIGHT AND WEIGHT GAIN
Males/Females:
- no effects on body weights or body weight gain were observed at any dose level.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Males/Females:
- no test item-related effects on food consumption were observed at any dose level.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- No effects were observed.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- No effects on mating performance or fertility were observed at any dose level.
- Gestation index (number of females with living pups as a percentage of females pregnant) was 100% in all groups.
- No effects on duration of gestation were observed at any dose level.
- No effects on corpora lutea were observed at any dose level.
- No effects on implanatation rate were observed at any dose level.
- No test item-related effects on post-implanatation loss were observed at any dose level.
- No test item-related effects on litter size were observed at any dose level.
- Birth index (number of pups borne alive as a percentage of implantations) was 94.4%, 86.5%, 91.0% and 94.1% in order ot ascending dose levels.
- No test item-related effects on postnatal loss were observed at any dose level.
- Viability index (number of pups alive on day 4 post partum as a percentage of pups born alive) was 100.0%, 91.7%, 99.0% and 100.0%, in order of ascending dose levels.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- No test item-related effects on organ, organ/body and organ/brain ratios were observed at any dose level in males or in females.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Males/Females:
- no test item-related findings were found during pathological examination at any dose level.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Males/Females:
- no test item-related findings were found during histopathological examination at any dose level.

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Since no test item related effects were noted during the study, the NOEL was established at the highest dose level.

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 specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
not specified

Details on results (F1)

VIABILITY (OFFSPRING)
- Pup sex ratios at first litter check were not affected by the exposure to the test item at any dose level.
- At first litter check, percentage of male pups was 51%, 43%, 52% and 54%, in order of ascending dose level.

CLINICAL SIGNS (OFFSPRING)
- No findings were noted at first litter check or during the first 4 days post partum in pups at any dose level.

BODY WEIGHT (OFFSPRING)
- No effects on pup body weights and body weight gain during the first four days post partum were observed at any dose level.

GROSS PATHOLOGY (OFFSPRING)
- No findings were noted at macroscopic examination of pups at any dose level.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Since no test item related effects were noted during the study, the NOEL was established at the highest dose level.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
During the study, no test item-related effects were observed on parental animals and on their offspring. The NOEL for reproduction/developmental toxicity was considered to be ≥1000 mg/kg/day.

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