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Toxicological information

Toxicity to reproduction

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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:
February 2015 - August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according GLP and OECD guideline, well documented

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks
- Fasting period before study: no
- Housing: individually, following exceptions: During overnight matings, male and female mating partners were housed together. Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7d

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): water
- Storage temperature of food: RT
- applied as a suspension
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): pregnant animals and litter together
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- according GLP
- stability of the test substance in drinking water for a period of 7 days at room temperature was proven before the start of the study
- method stability of test item in drinking water: UV/VIS spectroscopy
Duration of treatment / exposure:
The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males,
and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females.
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 13-14 weeks

Doses / concentrations
Remarks:
Doses / Concentrations:
50, 150, 500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: 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).

FOOD CONSUMPTION
- weekly
- 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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the course of FOB
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of administration period
- Anaesthetic used for blood collection: Yes, anaesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany)
- Animals fasted: Yes
- How many animals: 5/sex/dose

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of administration period
- Animals fasted: Yes
- How many animals: 5/sex/dose

URINALYSIS: Yes
- Time schedule for collection of urine: males: after mating, females: 1 day before end of administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: male: postnatal day 0, female: 10d after gestation
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity
Sperm parameters (parental animals):
stages of spermatogenesis were examined in histopathology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality up to day 4, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
yes, all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
1. Adrenal glands
2. All gross lesions
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal gland
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. Target organs
43. Testes (modified Davidson’s solution)
44. Thymus
45. Thyroid glands
46. Trachea
47. Urinary bladder
48. Uterus
49. Vagina

HISTOPATHOLOGY
ORGAN WEIGHTS: Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus

HISTOPATHOLOGY: Yes, control and high dose group, gross lesions in all animals
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Liver
16. Lung
17. Lymph nodes (mesenteric and axillary lymph nodes)
18. Ovaries
19. Oviducts
20. Peyer’s patches
21. Prostate
22. Rectum
23. Sciatic nerve
24. Seminal vesicles
25. Spinal cord (cervical, thoracic and lumbar cords)
26. Spleen
27. Stomach (forestomach and glandular stomach)
28. Testes
29. Thymus
30. Thyroid glands
31. Trachea
32. Urinary bladder
33. Uterus
34. Vagina
Postmortem examinations (offspring):
SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: All pups delivered from the F0 parents were examined as soon as possible on the day of birth

GROSS NECROPSY
All surviving pups (sacrificed on PND 4 under isoflurane anesthesia with CO2), all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically.
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 (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

Urinalysis parameters: WILCOXON-test (one-sided)

Food consumption: DUNNETT-test (twosided)

fertility indices: FISHER'S EXACT test

Proportions of affected pups per litter with necropsy observations: WILCOXON-test

Weight parameters: KRUSKAL-WALLIS test
Reproductive indices:
Male reproduction data:
- Male mating index
- Male fertility index

Female reproduction and delivery data:
- Female mating index
- Female fertility index
- Gestation index
- Live birth index
- Post implantation loss
Offspring viability indices:
Pup number and status at delivery
Pup viability/mortality
Sex ratio
Pup body weight data

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
decreased food consumption during premating (up to -9%), gestation (up to -20%), and lactation (not statistically significant -10%) in females; decreased body weight during lactation (up to -10%)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
decreased food consumption during premating (up to -9%), gestation (up to -20%), and lactation (not statistically significant -10%) in females; decreased body weight during lactation (up to -10%)
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: no effects
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
clinical signs

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not examined

Details on results (F1)

CLINICAL EXAMINATIONS/ GROSS FINDINGS
• decreased live birth index (64.2%)
• increase number of stillborn pups (35.8%) leading to 88.9% dams with stillborn pups
• increased perinatal loss (36.7%)
• decreased viability index (73%)
• increased number of pups found dead (4 pups)
• increased number of pups missing (cannibalized, 13 pups)
• decreased pup weights of both sexes at postnatal day 1 (-23.0%)
• decreased pup weight change between postnatal day 1 and 4 in pups of both sexes
(-28.5%) were bases on a decreased pup weight change in male pups of -25.4%, and
in female pups of -24.1% (not statistically significant)
• increased number of runts (10 males and 12 females)
• increased number of pups with post mortem autolyzes
• increased number of pups with discolored liver
• increased number of pups with empty stomach

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: see below

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of octadecanoic acid, sulfo-, potassium salt to Wistar rats revealed no signs of general systemic toxicity in male parental animals up to 500 mg/kg bw/d. Signs of systemic toxicity were observed in female parental animals at 500 mg/kg bw/d. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 500 mg/kg bw/d in male parental animals and 150 mg/kg bw/d in female parental animals. The no observed adverse effect level (NOAEL) for reproductive performance and fertility was 500 mg/kg bw/d in male and female Wistar rats. The NOAEL for developmental toxicity in the F1 progeny was 150 mg/kg bw/d.