2-Propenoic acid, heptadecyl ester, branched

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Behenylacrylate (Acrylate 22 45%)

IUPAC Name:

Behenylacrylate (Acrylate 22 45%)

Constituent 2

Reference substance name:

2-Propenoic acid, C18-26-alkyl esters

EC Number:

285-348-3

EC Name:

2-Propenoic acid, C18-26-alkyl esters

Cas Number:

85085-17-2

Test material form:

other: solid, turbid

Details on test material:

Name of test substance: Behenylacrylate (Acrylate 22 45%)
Batch No. 300011314299
CAS Nos.: 4813-57-4, 48076-38-6, 18299-85-9
Purity: 93.7 % (corr. area % as a sum of three main compounds)
Date of production: July 2011
Expiry date: Jan 2013
Physical state/ appearance: solid / colorless, turbid
Storage conditions: 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
- Age at study initiation: 10-11 weeks (males/females)

- Weight at study initiation: (P) Males: 314.9- 319.4g; Females: 208.6-211.9 g

- Fasting period before study: no

- Housing:

During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Makrolon type M III cages.
• Pregnant animals and their litters were housed together until PND 4 (end of lactation).
• For motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material (the present supplier is documented in the raw data).

Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
Dust-free wooden bedding was used in this study. Wooden gnawing blocks (Type NGM E-022) supplied by Abed® Lab. and Vet. Service GmbH, Vienna, Austria, were added for environmental enrichment. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.

- Diet (ad libitum): ground Kliba maintenance diet mouse-rat “GLP”
- Water (ad libitum): from water bottles

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod: 12 hours light from 06.00-18.00h, 12 hours dark from 18.00-06.00h
- Acclimation period: 5-6 days before the beginning of the treatment period

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
Behenylacrylate (Acrylate 22 45%) was applied as a solution. To prepare this solution, the test substance was melted at 50°C. The appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired weight, subsequently released with a magnetic stirrer. The test substance preparations were produced at least once a week.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: max 2 weeks
- Proof of pregnancy:A vaginal smear was prepared after each mating and examined for the presence of sperm. The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The study was carried out in compliance with the Principles of Good Laboratory Practice.
The stability of the test substance in corn oil for a period of 7 days at room temperature was proven before the start of the study.
Concentration control Analysis of the test substance preparations were performed in samples of all concentrations at the start of the administration period.

Duration of treatment / exposure:

After the acclimatization period, the test substance was administered orally via gavage to the F0 generation parental animals, daily at the same time in the morning (exception: no administration to animals being in labor). The treatment lasted up to one day prior to sacrifice.

Frequency of treatment:

daily in the morning

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (vehicle alone), 100, 300 and 1000 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

10 male and 10 female per dose group

Control animals:

yes, concurrent vehicle

Positive control:

No positive control.

Examinations

Parental animals: Observations and examinations:

- Mortality
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.
- Clinical observations
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal.
The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of littering was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.
- Detailed clinical observations
Detailed clinical observations (DCO) were 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. exophthalmus
15. feces (appearance/consistency)
16. urine
17. pupil size

- Food consumption
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
• Food consumption of F0 females, which gave birth to a litter was determined for PND 4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

- Body weight data
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). The body weight change of the animals was calculated from these results.
The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 1) and on PND 4.
• Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

- Functional observational battery
A functional observational battery was performed in the first five male animals per test group, the first 5 female animals with litter of all test groups at the end of the administration period starting at about 10.00 h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations
in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

- Home cage observations:
The animals were observed in their closed home cages; during this period any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Impairment of gait
Open field observations:

The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined:
1. Behavior when removed from cage
2. Fur
3. Skin
4. Salivation
5. Nose discharge
6. Lacrimation
7. Eyes/ pupilsize
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/ stereotypes
14. Impairment of gait
15. Activity/ arousal level
16. Feces excreted within 2 minutes (number/ appearance/ consistency)
17. Urine excreted within 2 minutes (amount/ color)
18. Rearing within 2 minutes

- Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory motor or reflex tests:
1. Reaction to an object being moved towards the face (Approach response)
2. Touch sensitivity (Touch response)
3. Vision (Visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (Auditory startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Grip strength of forelimbs
12. Grip strength of hindlimbs
13. Landing foot-splay test
14. Other findings

- Motor activity assessment
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and females (with litter) per group. Motor activity was measured on the same day as FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts was counted over 12 intervals of 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.


Clinical pathology

In the morning blood was taken from the retrobulbar venous plexus from fasted animals.
The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence. The following examinations were carried out in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group:

- Hematology
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and
Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101).

Parameters: Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular
hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Differential blood count, Reticulocytes, Prothrombin time

- Clinical chemistry
Parameters: Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase; Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase; Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase; γ-Glutamyltransferase
(GGT) (γ -glutamyl) peptide: aminoacid-γ- glutamyl-transferase; Sodium (Na+), Potassium, Chloride, Calcium, Inorganic phosphorus, Glucose, Urea, Creatinine, Total bilirubin, Total proteins, Albumin,Total cholesterol, Triglycerides , Biles acids

- Urinalysis (parameters)
pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment Color, turbidity, Volume

Litter observations:

-. Pup number and status at delivery
All pups delivered from the F0 parents were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn pups in each litter. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.

- Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1 - 4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. The viability index was calculated.

- Sex ratio
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally
confirmed at necropsy. The sex ratio was calculated at day 0 and day 4 after birth.

- Pup clinical observations
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

- Pup body weight data
The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results.
The individual weights were always determined at about the same time of the day (in the morning). “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

Postmortem examinations (parental animals):

Necropsy
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs.

Weight parameters
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Epididymides
3. Testes
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations):
1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus

Organ/tissue fixation
The following organs or tissues of all parental animals were fixed in 4% buffered formaldehyde solution or modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating gland
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal glands
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Testes (modified Davidson’s solution)
43. Thymus
44. Thyroid glands
45. Trachea
46. Urinary bladder
47. Uterus
48. Vagina

- Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings from the following organs of all animals/test group, all animals affected/test group or 5 animals per sex/test group, females with litters only, same animals as used for
clinical pathological examinations.
Special attention was given to stages of spermatogenesis in the male gonads.
A correlation between gross lesions and histopathological findings was attempted.

Organs:
Adrenal glands, All gross lesions, Bone marrow (femur) , Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides, Heart,
Ileum, Jejunum, Kidneys, Live, Lungs, Lymph nodes (axillary and mesenteric, Ovaries , Oviducts, Prostate gland, Peyer’s patches , Rectum,
Sciatic nerve , Seminal vesicles, Spinal cord (cervical, thoracic, lumbar) , Spleen, Stomach (forestomach and glandular stomach), Testes , Thymus , Thyroid glands , Trachea , Urinary bladder , Uterus , Vagina

Postmortem examinations (offspring):

- Pup necropsy observations
All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically.
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-bycase basis, depending on the type of finding noted.

Statistics:

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 (twosided) 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) for the hypothesis of equal medians.

Reproductive indices:

The following indeces were determined: mating and fertility index for both males and females, gestation index, live birth index

Offspring viability indices:

The following indices were determined: sex ratio and viability index

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

Mortality
No animal died prematurely in the present study.

Detailed clinical observations
No abnormal clinical signs were observed.

Summary clinical observations for males and females
Two male animals of test group 2 (300 mg/kg bw/d), four male animals of test group 3 (1000 mg/kg bw/d) and five female animals of test group 3 (1000 mg/kg bw/d) showed salivation in the premating period. Four male animals of test group 2, six male animals of test group 3 and seven female animals of test group 3 showed salivation in the mating period. Six male animals of test group 2 and seven male animals and one female animal of test group 3 showed salivation in the postmating period. These findings were assessed as substance-related but not as an adverse effect, due to the taste of the substance.

Summary clinical observations for females during gestation
Two female animals of test group 2 (300 mg/kg bw/d) showed salivation on GD 17 and 22 and eight animals of test group 3 (1000 mg/kg bw/d) showed salivation during the gestation period. These findings were assessed as substance-related but not as an adverse effect, due to the taste of the substance. One sperm-positive F0 female of the test group 3 (Nos. 136) and 1 sperm-positive F0 female of the control group (No. 108) did not deliver any F1 pups.


Clinical observations for females during lactation
One female animal of test group 0 (No. 107) had no GD 0 but pups. One female animal (No. 116) of test group 1 (100 mg/kg bw/d) showed a palpable mass through the skin on PND 23 and 24. This finding was assessed as being spontaneous in nature and without biological relevance. Two female animals of test group 2 (300 mg/kg bw/d) and eight female animals in test group 3 (1000 mg/kg bw/d) showed salivation during the lactation period.

Food consumption
Food consumption of the male and female F0 generation parental animals in all test substance-treated groups (100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control group during the entire study period, covering premating, gestation and lactation.

Body weight data
Mean body weights and mean body weight gain of the F0 males in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control throughout the entire study period. Mean body weight and mean body weight gain of the F0 females in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control throughout the entire premating, gestation and lactation periods.

Functional observational battery
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered as incidental.
The following examinations were performed during FOB and are assessed individually:
Home cage observations
No test substance-related effects were observed.
Open field observations
No test substance-related effects were observed.
Sensorimotor tests/reflexes
No test substance-related effects were observed.
Quantitative Parameters
No test substance-related effects were observed.

Motor activity measurement
Male animals of test group 3 (1000 mg/kg bw/d) showed one significantly lower value in interval 7. There were no significant deviations concerning the overall motor activity (summation of all intervals) in male and female animals. All described findings were assessed as being incidental and not related to treatment.

Male mating index
The male mating index was 100% in test groups 0, 1 and 2 and 90% in test group 3. This finding 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.

Male fertility index
Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter.
One control male (No. 8 mated with female No. 108) and 2 males of test group 3 (Nos. 33, 36 mated with females Nos. 133, 136) did not generate F1 pups. The male fertility index was 90% in test group 0, 100% in test group 1-2 and 80% in test group 3.
The apparently infertile male animal No. 8 (control group) showed bilateral granulomata of the epididymides, which might have affected fertility in this animal. This finding was not treatment-related and 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.

Female mating index
The female mating index calculated after the mating period for F1 litter was 100% in test groups 0, 1 and 2 and 90% in test group 3. The mean duration until sperm was detected (GD 0) was 5.1, 3.0, 2.3 and 1.7 days in test groups 0-3. This finding 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.

Female fertility index
All sperm positive rats delivered pups with the exception of female No. 108 (test group 0) and No. 136 (test group 3), which were mated with male Nos. 8 and 36 did not become pregnant. Female animal No. 133 (test group 3) which was mated with male No. 33 did not get sperm in vaginal smear. The female fertility index was between 89% and 100%. Female animals Nos. 108, 133 and 136, which delivered no pups, showed no implantation sites. Female No. 107 (control group, mated with male No. 7) was not sperm-positive but delivered pups.

Gestation index
The gestation index was 100% in all test groups.

Live birth indices
The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 100% in all test groups.

Postimplantation loss
The postimplantation loss in test group was 4.7% in test group 0, 5.8% in test group 1, 6.5% in test group 2 and 4.2% in test group 3. These findings 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.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Results: F1 generation

Details on results (F1)

Pup number and status at delivery
The mean number of delivered F1 pups per dam was evenly distributed about the groups.

Pup viability/mortality
The viability index indicating pup mortality during lactation (PND 0 - 4) was 99% (test groups 1, 2 and 3) and 100% (test group 0) and was in the normal range of biological variation inherent in the strain of rats used for this study.
One female pup of test group 1 was found dead. These findings 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.

Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Pup clinical observations
The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.

Pup necropsy observations
One F1 pup of test group 2 (300 mg/kg bw/d) and one pup of test group 3 (1000 mg/kg bw/d) were cannibalized.
These findings were assessed as being spontaneous in nature and without biological relevance.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Overall reproductive toxicity

Reproductive effects observed:

not specified