Calcium di(octanoate)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

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

Test material information

1

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
60805X
- Purity:
85.9 wt%
- Impurities: C14-C20 Fatty acids:10.9%, C24fatty acids: 2.3%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
avoid direct sunlight, store at room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crj: CD,SPF

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:

corn 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 / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

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
Animals were fasted for 24 hours after the final administration.
- Male animals: all surviving animals
- Maternal animals: all surviving animals

Gross Pathology
Organ weights:
male: heart, liver, kidneys, thymus, testes, epididymides
female: heart, liver, kidneys, thymus, ovaries and uterus

Pathology and histopathology (control and 1000 mg/kg bw/day group):
male: heart, liver, spleen, kidney, adrenal, testis, epididymis, brain, thymus, bladder
Testes and epididymides were stored by fixing in buan solution, all other organs were fixed in 10% formalin. Paraffin sections were produced and histopathological examination was performed using hematoxylin-eosin staining.

female: brain,heart, liver, spleen, kidney, bladder, adrenal glands, thymus, uterus, ovaries
Ovaries were stored by fixing in buan solution, all other organs were fixed in 10% formalin. Sections were prepared and histopathological examination was performed.

Blood chemistry:
Males: blood was taken from a vein in the abdomen under sodium pentobarbital anesthesa.
The numbder of red blood cells (RBC), leukocytes (WBC), blood pigment (Hb), average red blood cell volume (MCV) were calculated. Platelet count was carried out using a Coulter Counter Model S-PLUS IV.
Average erythrocyte blood pigment (MCH), average erythrocyte blood pigment concentration (MCHC) were also examined. The leukocyte percentile ratio was calculated by staining the specimens with Geisma and Wright stain and examined under and optical microscope.
Following blood collection the presence of peptides was determined using the Biuret method. Albumin concentration (BCG), total cholesterol levels (COD), glucose concentration (Glucokinase G6PD), urea nitrogen concentration (UreaseGt.DH), creatinine concentration (Jaff), alkaline phosphatase activity (Paranitophenyl phosphate substrate method), GOT/GPT activity (SSCC) - (y-GTPActivityCGlutamyl-3-Carboxy-4-Nitroanirid substrate method), triglyceride concentration (GPO), inorganic phosphorus concentration (Molybdate Direct method), total bilirubin concentration (Bilirubin [Roche' KitSSeries), calcium concentration (OCPC), potassium concentration (ion electrode method), chlorine concentration (ion electrode method) were examined.

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):

Significant 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)

Key result

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)

Key result

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:

not examined

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)

Key result

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)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

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

Conclusions:

No deaths were observed in any group, and no changes in general conditions, weight and feeding were observed. After 42 doses there were some decreases noted in the average red blood pigment concentration in males in the 300 mg/kg group, glucose levels decreased significantly in the 1000 mg/kg group. However, since both changes were mild and there were no associated changes in other test items, including pathological examinations, these were considered to be incidental changes and of no toxicological significance. In histopathological examination, two of the animals in the1000 mg/kg group showed very mild microtubule atrophy. As these were very mild and localized, and there were no other changes, these were note considered to be of concern.
These results conclude that under these test conditions, the NOAEL for repeated administration of docosanic acid in regard to reproduction and fertility is 1000 mg/kg/day.

Executive summary:

Docosanic acid is a mixture ofhigh-grade saturated fatty acids contained intheconstituent fatty acids of carasi oil and rapeseedoil. Industrially, it is distilled and purified after the rapeseed oil is hydrolyzed mainly as a raw material, but it has recently come to be used as a raw material for cosmetics. There is very little information in regard to the toxicity and hazards associated with docosanic acid. A study was conducted to evaluate docosanoic acid. 0 (solvent control), 100, 300 and 1000 mg/kg of docosanoic acid was administered to

Sprague-Dawley (Crj:CD)rats, male andfemale, (13 animals / group), started 2weeksbefore matingand 42 days in total for males. For females administration started 2 weeks before mating and continued until 3 days after the start of lactation. Parent animals and infants were examined for effects of repeated administration on the toxicity, fertility, as well as development of the next generation infants.

No abnormalities or death were observed in any docosanic acid administration group in males. In addition, there was no observed changes in weight and feeding . Autopsy results from histopathological, hematology, hematology and hemobiochemistry tests showed no indications of toxicity or abnormal values attributed to the administration of docosanoic acid.

In females, there were no deaths in any of the docosanic acid administration groups. In addition, changes in general condition, weight, and feeding amount were not observed. In the autopsy and histopathological examination four days after delivery, there were no findings suggesting of effects as a result of docosanic acid administration.

Furthermore, docosanic acid administered in doses up to 1000 mg/kg did not affect mating rate and conception rate. In addition, there were no changes to suggest effects of docosanic acid administration on pregnancy, birth rate, delivery, and baby-care bythe mother.

There were no changes in the sex ratio, weight, and survival rate of birth children attributed to the effects of docosanic acid administration. In addition, morphological abnormalities in birth children were not observed in any docosanic acid administration group. These results conclude that under these test conditions, the NOAEL for repeated administration of docosanic acid in regard to reproduction and fertility is 1000 mg/kg/day.