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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002

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)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Docosanoic acid
EC Number:
204-010-8
EC Name:
Docosanoic acid
Cas Number:
112-85-6
Molecular formula:
C22H44O2
IUPAC Name:
docosanoic acid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Hino, Tokyo
- Age at study initiation: 8 weeks
- Weight at study initiation: male: 312.1 - 363.7 g; female: 205.3 - 230.8 g
- Housing: metal wire floor cages
- Diet (ad libitum): CE-2, CLEA Japan
- Water (ad libitum): tap water
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1
- Humidity (%): 50 - 65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
prepared more frequently than once a week; aliquots were kept by each concentration refrigerated in airtight conditions

VEHICLE
- Justification for use and choice of vehicle (if other than water): due to insolubility in water
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): V6H2050
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: up to two weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further mating after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
- males: 42 days
- females: from 14 days prior to mating to day 3 of lactation
Frequency of treatment:
once daily
Details on study schedule:
- Age at mating of the mated animals in the study: 10 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
13
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on preliminary result in a 14 day-repeated dose toxicity study, where no signs of toxicity were found

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: males: days 1, 8, 15, 22, 29, 36, 42; pregnancy females: premating days 1, 8, 15; pregnancy days: 0, 7, 14, 20; lactation days: 0, 4; non pregnancy females: day 1, 8, 15, 22, 25

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

HAEMATOLOGY: Yes (males only)
- Time schedule for collection of blood: prior to autopsy
- Anaesthetic used for blood collection: Yes (identity): pentobarbital sodium
- Animals fasted: 18 - 24 hours before sacrifice
- How many animals: all surviving animals
- Parameters checked: red blood cell count (RBC), white blood cell count (WBC), haemoglobin content (Hb), mean corpuscular volume (MCV), ahematocrit (Ht), mean corpuscular haemoglobin content (MCH), mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes (males only)
- Animals fasted: 18 - 24 hours before sacrifice
- How many animals: all surviving animals
- Parameters checked: total protein, albumin, total cholesterol concentration, glucose, urea nitrogen, creatinine, alkaline phosphatase activity, GOT, GPT, γ-GTP, triglyceride concentration, inorganic phosphorus, total bilirubin, calcium, sodium, potassium, chlorine, A/G ratio

URINALYSIS: No
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
testis weight, epididymis weight
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, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals
- Maternal animals: all surviving animals

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights:
male: heart, liver, kidneys, thymus, testes, epididymides
female: heart, liver, kidneys, thymus

Pathological findings and Histopathology (control and 1000 mg/kg bw/day group):
male: heart, liver, spleen, kidney, adrenal, testis, epididymis, brain, thymus, bladder
female: brain, liver, spleen, thymus, kidney, adrenal, bladder, heart, ovary, uterus
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows: external malformations, visceral malformations
Statistics:
Yates test, Mann-Whitney U test, Fisher exact test, Bartlett test, Dunnett test, Scheffe test, Kruskal-Wallis test
Reproductive indices:
- Copulation index = (number of copulated pairs / number of pairs mated) x 100
- Fertility index = (number of pregnant animals / number of copulated pairs) x 100
- Gestation index = (number of pregnant females with pups alive / number of pregnant females) x 100
- Implantation index = (number of implantation sites / number of corpora lutea) x 100
Offspring viability indices:
- Delivery index = (number of pups born / number of implantation sites) x 100
- Birth index = (number of pups alive on day 0 / number of implantation sites) x 100
- Live birth index = (number of pups alive on day 0 / number of pups born) x 100
- Sex ratio on day 0 = (number of males pups alive on day 0 / number of female pups alive on day 0) x 100
- Viability index = (number of pups alive on day 4 / number of pups alive on day 0) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
ignificant increase was observed in 100 mg/kg bw/day group, not considered to be related to treatment
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
significant decrease was observed in 100 mg/kg bw/day group during lactation, not considered to be related to treatment
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
significant decrease of mean corpuscular haemoglobin concentration in the 300 and 1000 mg/kg bw/day groups; fndings are not dose-dependent
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
significant decreased ALP level in all dose groups; significant decreased glucose level in the 1000 mg/kg bw/day group; signifant increase in chloride, decrease in calcium content and in total protein in the 300 mg/kg bw/day group; fndings are not dose-dependent and considered incidental
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
observations were considered incidential
Histopathological findings: neoplastic:
no effects observed

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)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- Males: no deaths or abnormalities in general condition were observed in any of the treated groups
- Females: no deaths were observed in any treated group

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males: significant increase (p<0.01) compared to control was observed in 100 mg/kg group between 8 - 15 days and between 15 - 29 days. However, since no change was observed in other groups, the effect was not considered to be related to the dosing of compound. No changes in food consumption related to the dosing of compound were observed.
- Females: no significant changes in body weight were noted and there were no changes related to the dosing of compound in body weight gain and food consumption. A significant decrease (p<0.01) compared to control was observed during lactation in 100 mg/kg dosing group. However, since no other changes in food consumption were noted in 300 mg/kg and 1000 mg/kg dosing groups, the effect was not considered to be related to the dosing of compound.

HAEMATOLOGY
- Males: a significant decrease (p<0.01) of mean corpuscular haemoglobin concentration (MCHC) compared to control was found in the 300 and 1000 mg/kg bw/day dose group, respectively (control: 34.7 ± 0.5%; 100 mg/kg bw/day: 34.4 ± 0.2%; 300 mg/kg bw/day: 33.9 ± 0.4%**; 1000 mg/kg bw/day: 33.9 ± 0.3%**; **p<0.01). No other differences were noted.

CLINICAL CHEMISTRY
- Males: a significant decreased ALP level (p<0.05) compared to control was found in all dosing groups (control: 237 ± 45 U/L; 100 mg/kg bw/day: 200 ± 35 U/L*; 300 mg/kg bw/day: 195 ± 35 U/L*; 1000 mg/kg bw/day: 197 ± 41 U/L*; *p<0.05). A significant decreased glucose level (p<0.05) compared to control was found in the high dose group (control: 152 ± 16 mg/dL; 1000 mg/kg bw/day: 135 ± 14 mg/dL*; *p<0.05). While a significant increase in chloride (p<0.05) was found in the 300 mg/kg bw/day group, a significant decrease in calcium content (p<0.01) and in total protein (p<0.05) was noted in this group. However, all the findings lack a dose-dependency and were regarded as incidental.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no significant differences in number of corpora lutea, implantation rate, number of implantations and all other reproductive parameters in all treated groups compared to the control group.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Males: in 100 mg/kg bw males, the actual weight ratio of liver weight (p <0.05) compared to control values were increased. No other significant differences were found in all groups. Thus, this finding lacks a dose-dependency and was regarded as incidental.
- Females: kidney weight was significantly reduced (p<0.05) in the 300 mg/kg bw group. No other weight changes were noted. Thus, this finding lacks a dose-dependency and was regarded as incidental.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Males:
- Heart: myocardial degeneration was noted in one animal of the control group and the 1000 mg/kg bw group, respectively
- Liver: periportal fatty change of the liver was found in all animals of the control and the high dose group. Focal necrosis was noted in one animal of the high-dose group.
- Spleen: all animals of the control and the high-dose group showed brown pigment deposits of the spleen as well as extramedullary haematopeiosis
- Kidney: while fibrosis was only found in 1/13 animals of the control group, eosinophilic bodies and basophilic tubuli in the cortex were noted in animals of both control and high dose groups, respectively
- Adrenal gland: one animal of the high-dose group showed fibrosis
- Testis: atrophy of the seminiferous tubules was observed in two animals of the 1000 mg/kg dose group, with one animal affected on both sides and one side affected in the other animal. No other abnormalities were noted.
- Epididymis: abnormal sperm granuloma was found in one animal of the control group
- Brain: no abnormal findings noted
- Thymus: no abnormal findings noted
- Bladder: no abnormal findings noted

Females:
- Brain: one animal of the high-dose group showed abnormal mineral deposition in the thalamus
- Liver: focal necrosis and fibrosis was found in one animal of the control group, while one animal of the high-dose group showed only focal necrosis
- Spleen: brown pigmentation and extramedullary haematopeiosis was found in animals of the control and the high-dose group, but no varying degrees of frequency was observed
- Thymus: atrophy and bleeding was observed in animals of both groups, control and high-dose group, respectively
- Kidney: basophilic tubuli of the cotex and a dilatation of the renal pelvis was seen in the control and high-dose group
- Adrenal gland: cortical necrosis was observed in one animal of the control group
- Heart: no abnormal findings noted
- Bladder: no abnormal findings noted

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 substance -related systemic effects and findings on fertility were noted

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
No change in number of birth, birth rate, infant live birth rate, infant survival and birth rates at day 4 was found in all dose groups compared to the values of the control group.

BODY WEIGHT (OFFSPRING)
No difference in bodyweight of pups of the dose groups and the control group was found.

GROSS PATHOLOGY (OFFSPRING)
No morphological abnormalities were found in all groups.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental
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: no adverse substance-related findings were noted

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Table 1. Summary of development up of pups from dams treated orally with docosanoic acid in the combined repeat dose and reproductive/developmental toxicity screening test: Mean ± SD (N)

Dose group (mg/kg bw/day)

0

100

300

1000

Number of pregnant females

13

12

12

13

Number of pregnant females with live pups

13

12

12

13

Gestation index

100

100

100

100

Gestation length in days

22.2±0.4 (13)

22.4±0.5 (12)

22.3±0.5 (12)

22.2±0.4 (13)

Number of corpora lutea

16.6±1.6 (13)

16.7±1.8 (12)

16.8±2.3 (12)

16.2±1.5 (13)

Number of implantation sites

16.1±1.5 (13)

15.8±2.1 (12)

16.0±1.5 (12)

15.2±3.1 (13)

Implantation index

96.9±5.1 (13)

94.8±7.1 (12)

96.1±6.0 (12)

93.1±15.2 (13)

Day 0 of lactation

 

 

 

 

Number of pups born

15.2±1.6 (13)

14.8±2.2 (12)

15.2±1.5 (12)

14.3±2.7 (13)

Delivery index

94.5±8.0 (13)

93.5±3.9 (12)

94.9±4.3 (12)

94.9±6.2 (13)

Number of live pups

14.9±1.6 (13)

14.3±2.2 (12)

15.1±1.6 (12)

14.1±2.7 (13)

Birth index

93.1±8.8 (13)

90.7±8.7 (12)

94.3±5.1 (12)

93.5±7.2 (13)

Live birth index

98.5±2.8 (13)

97.0±8.5 (12)

99.4±2.2 (12)

98.5±4.0 (13)

Sex ration on day 0

50.0±11.3 (13)

46.7±9.8 (12)

54.8±12.3 (12)

48.9±13.4(13)

Day 4 of lactation

 

 

 

 

Number of live pups

14.7±1.4 (13)

13.0±4.7 (12)

15.1±1.6 (12)

14.1±2.7 (13)

Viability index

98.6±2.7 (13)

89.4±28.8 (12)

100.0±0.0 (12)

100.0±0.0 (13)

Sex ration on day 4

49.8±11.5 (13)

46.3±10.3 (11)

54.8±12.3 (12)

48.9±13.4(13)

Applicant's summary and conclusion