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Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from J-check database

Data source

Reference
Reference Type:
other: Authoritative database
Title:
Combined Repeated Dose and Reproductive/Developmental Toxicity Screening Test of test material by Oral Administration in Rats
Author:
Ministry of Health, Labour and Welfare", "Ministry of the Environment" and "National Institute of Technology and Evaluation
Year:
2013
Bibliographic source:
Japan chemicals collborative knowledge database (J-check)

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
Combined Repeated Dose and Reproductive/Developmental Toxicity study of test material by Oral Administration in Rats
GLP compliance:
not specified
Limit test:
no
Justification for study design:
No data available

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Name: Docosanoic acid
Mol. Formula:C22H44O2
Mol. wt.:340.5876g/mol
Smiles:C(CCCCCCCCCCCCCC)CCCCCCC(=O)O
InChI:1S/C22H44O2/c1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-18-19-20-21-22(23)24/h2-21H2,1H3,(H,23,24)
Physical state:solid: crystalline
Substance type:Organic

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
No data available
Sex:
male/female
Details on test animals and environmental conditions:
Details on test animals and env. conditions
TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 8 weeks old
- Weight at study initiation: male 312.1 to 363.7 g, female 205.3 -230.8 g).
- Fasting period before study:
- Housing: individually housed in a wire mesh floor cage (Nihon Cage ), raised, and allowed to take solid feed
(CE - 2, Japan Clea )
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period:7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1 ° C.
- Humidity (%):50 to 65%,
- Air changes (per hr): about 15 times / hour
- Photoperiod (hrs dark / hrs light): a lighting condition set at 12 hours of lighting (7 am to 7 pm)

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
corn oil
Details on exposure:
Details on exposure
PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared by suspending it in corn oil and used as a test sample
DIET PREPARATION
- Rate of preparation of diet (frequency):No data available
- Mixing appropriate amounts with (Type of food )
- Storage temperature of food: No data available
VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Concentration in vehicle: 0 ,100, 300 and 1,000 mg/kg bw/day
- Amount of vehicle (if gavage): 5ml/kg bw
- Lot/batch no. (if required): (Lot No. V 6 H 2050, Nacalai Tesque ),
- Purity: No data available
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: at the most 14days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy : until proof of pregnancy (formation of vaginal closing or sperm detection in vagina)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Males: 42 days
Females: from 14 days prior to mating to day 3 of lactation
Frequency of treatment:
Daily
Details on study schedule:
No data available
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (vehicle), 100, 300 or 1000 mg/kg/day.
Basis:

No. of animals per sex per dose:
Total :104
0 (Vehicle): 13 male and 13 female
100 mg/kg/day: 13 male and 13 female
300 mg/kg/day: 13 male and 13 female
1000 mg/kg/day: 13 male and 13 female
Control animals:
yes, concurrent vehicle
Details on study design:
No data available
Positive control:
No data available

Examinations

Parental animals: Observations and examinations:
Parental animals observation and examinations
CAGE SIDE OBSERVATIONS: yes

DETAILED CLINICAL OBSERVATIONS:

Time schedule: Daily

BODY WEIGHT: Yes
Time schedule for examinations: For all males, measurements were made on 1, 8, and 15 days of administration 1 (administration start date), 8, 15,
22, 29, 36, 42 days and postmenopausal days and females for all cases,
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes For all males, measurements were made on 1, 8, and 15 days of administration (administration start date), 8, 15,22, 29, 36, 42 days and postmenopausal days and females for all cases,


Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available
Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data: No data available


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
Time schedule for examinations
Oestrous cyclicity (parental animals):
Estrous cycle length and pattern not performed because the test was Conducted by the TG adopted in 1990.
Sperm parameters (parental animals):
Sperm examination were not performed because the test was Conducted by the TG adopted in 1990.
Litter observations:
litter size at birth, neonatal death, sex ratio of pups were observed
Postmortem examinations (parental animals):
Postmortem examinations (Parent Animal)
SACRIFICE : males were killing under pentobarbital sodium deep anesthesia the next day (day corresponding to 43 days of administration). Females delivered were sacrificed on the fourth day of nursing, females that did not deliver to the 25th day of pregnancy, females who did not copulate were exsanguinated and lethal under pentobarbital sodium anesthesia at the end of the mating period,
GROSS NECROPSY: yes
HISTOPATHOLOGY / ORGAN WEIGHTS: yes
Weights of the heart, liver, kidney, thymus, testis and epididymis were measured for all cases ,the testes and epididymis were fixed and stored in Bouin's solution, and other organs and brain, spleen, adrenal gland and bladder were fixed and fixed in 10% formalin. For these high-dose groups and control groups, paraffin sections were prepared according to a conventional method, and hematoxylineosin staining was performed to perform histopathological examination.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Statistics:
Dunnett’s or Scheffe’s test for continuous data, Chi square test for copulated index and fertility index, and Mann-Whitney U test or Fisher’s test for histopathological examination data.
Reproductive indices:
No data available
Offspring viability indices:
No data available

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
In males, no abnormalities in general conditions were observed. In females, there was only one change from day 42 of administration (day of delivery) to the one in 100 mg / kg administration group (administration 42 days: piloerection, blood-like fluid discharge from the vaginal opening; administration 43 to 44 days: coat contamination) But it recovered on day 45 of administration. In other females, no abnormalities in the general condition were observed
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
No deaths in male and female was observed.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In male, the body weight gain increased from 8 to 15 days in the 100 mg / kg administration group significantly (p <0.01) compared with the control group, However, in the group administered with 300 mg / kg or more, there was no significant difference between the control group and treated group. In females, there was no significant difference in body weight and body weight gain between control group and treated group before breeding, pregnancy period and nursing period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no significant difference between the control group and treated group for males and female at any time of feeding intake
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
For male In the 100 mg / kg administration group, the actual weight and the specific body weight value of the liver increased significantly (p <0.05) compared to the control group, but not the dose-dependent change. For female In the 100 mg / kg administration group, the actual weight of the kidney decreased significantly (p <0.05) compared to the control group, but not the dose-dependent change. For other organs in male and female, no significant difference was observed between the control group and treated group.
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Atrophy of seminiferous tubules was found in 2 animals in the 1000 mg / kg administration group, one on both sides and one on one side, but both were localized and extremely mildly changed. There were no other abnormalities. Sperm granuloma was observed in one of the control group, and there was no abnormality. There were no abnormalities in the ovaries of the unexpanded and infertile cases.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There was no significant difference between the control group and treated group in the mating rate, conception rate, the number of days taken from the start of cohabitation to mating and the number of estrus periods recurred during that period.
An abnormality in labor condition (not treated with placenta) and abnormality in nursing condition (not collecting children, poor protrusion of nipple, poor protrusion of infant, child's fetus) was administered to one of 100 mg / kg administration group, The decrease in body surface temperature) was observed, and all children died on nursing day 2, but no abnormality was observed in other mother animals.
Birth rate was 100% in all administration groups, and no significant difference was observed between gestation period between control group and treated group. No significant difference was observed between the control group and treated group for the number of luteinism, the number of implantation and the implantation rate of pregnant animals.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive performance
Remarks on result:
other: overall no toxic effects on reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no significant difference between the control group and treated group for the number of births, delivery rate, birth rate, fertility rate, and survival rate on 4th day of newborn.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no significant difference between the control group and the treated group on body weight at 0 and 4 days of nursing
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
No abnormalities were found in all pups examined in either the external observation at day 0 or the autopsy performed at day 4 after birth.
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology
Remarks on result:
other: overall no developmental toxicity was observed

Target system / organ toxicity (F1)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified
Treatment related:
not specified
Relation to other toxic effects:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Table: Body weight of female rats treated orally with test material in the combined repeat dose and reproductive/ developmental toxicity screening test

Dose (mg/kg)

0

100

300

1000

Days of administration

(Pre mating period)

 

 

 

 

 

1 (init. Wt.)

216.2 ± 6.4 (13)

216.7 ± 7.0 (13)

216.6 ± 6.8 (13)

216.3 ± 6.7 (13)

 

8

221.4 ± 7.3 (13)

221.9 ± 8.4 (13)

219.5 ± 8.4 (13)

221.4 ± 5.8 (13)

15

233.7 ± 9.6 (13)

232.0 ± 10.6 (13)

234.9 ± 10.9 (13)

231.2 ± 8.8 (13)

Days of pregnancy

 

 

 

 

0

240.4 ± 8.8 (13)

238.3 ± 13.1 (12)

236.7 ± 9.2 (12)

238.1 ± 10.4 (13)

7

273.8 ± 15.0 (13)

273.5 ± 12.4 (12)

270.9 ± 11.3 (12)

269.3 ± 12.1 (13)

14

312.2 ± 19.7 (13)

310.4 ± 11.4 (12)

309.1 ± 14.5 (12)

301.9 ± 13.4 (13)

20

391.5 ± 24.0 (13)

383.1 ± 17.3 (12)

386.2 ± 18.2 (12)

374.6 ± 16.1 (13)

Days of lactation

 

 

 

 

0

278.7 ± 16.7 (13)

270.3 ± 21.0 (12)

271.8 ± 20.4 (12)

270.0 ± 24.5 (13)

4

303.2 ± 17.3 (13)

287.3 ± 15.2 (11)

297.0 ± 11.1 (12)

290.7 ± 15.4 (13)

 

Values are expressed as mean ± S. D. In grams

Parenthesis indicates number of animals.

Table: Summary of reproductive performance in parental rats treated orally with test material in the combined repeat dose and reproductive/developmental toxicity screening test

Dose (mg/kg)

0

100

300

1000

No. Of mated pairs

13

13

13

13

No. Of copulated pairs

13

12

13

13

Copulation indexa)

100

92.3

100

100

No. Of pregnant animals

13

12

12

13

Fertility indexb)

100

100

92.3

100

Pairing days until copulation

(Mean ± S. D.)

1.5 ± 1.0

2.3 ± 1.2

2.6 ± 3.8

2.5 ± 2.4

Frequency of vaginal estrus

(Mean ± S. D.)

1.0 ± 0.0

1.0 ± 0.0

1.1 ± 0.3

1.1 ± 0.3

 

A) Copulation index= (number of copulated pairs/number of mated pairs) X100; %

B) Fertility index= (number of pregnant animals/number of copulated pairs) X100;

 

Table: Summary of development of pups from Dams treated orally with test material in the combined repeat dose and reproductive/developmental toxicity screening test

Dose (mg/kg)

0

100

300

1000

Number of pregnant females

13

12

12

13

Number of pregnant females with pups alive

13

12

12

13

Gestation indexA)

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 indexB)

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 indexC)

94.5 ± 8.0 (13)

93.5 ± 3.9 (12)

94.9 ± 4.3 (12)

94.9 ± 6.2 (13)

Number of pups alive

14.9 ± 1.6 (13)

14.3 ± 2.2 (12)

15.1 ± 1.6 (12)

14.1 ± 2.7 (13)

Birth indexD)

93.1 ± 8.8 (13)

90.7 ± 8.7 (12)

94.3 ± 5.1 (12)

93.5 ± 7.2 (13)

Live Birth indexE)

98.5 ± 2.8 (13)

97.0 ± 8.5 (12)

99.4 ± 2.2

98.5 ± 4.0

Pups in grams

 

 

 

 

Male

6.1 ± 0.3 (13)

6.3 ± 0.4 (12)

6.2 ± 0.5 (12)

6.2 ± 0.5 (13)

Female

5.9 ± 0.3 (13)

5.9 ± 0.5 (12)

5.9 ± 0.4 (12)

5.9 ± 0.5 (13)

Sex ratioF)

1.09 ± 0.46 (13)

0.94 ± 0.36 (12)

1.37 ± 0.65 (12)

1.22 ± 1.18 (13)

Day 4 of lactation

 

 

 

 

Number of pups alive

14.7 ± 1.4 (13)

13.0 ± 4.7 (12)

15.1 ± 1.6 (12)

14.1 ± 2.7 (13)

Viability indexG)

98.6 ± 2.7 (13)

89.4 ± 28.8 (12)

100.0 ± 0.0 (12)

100.0 ± 0.0 (13)

Pup weight in grams

 

 

 

 

Male

9.6 ± 0.7 (13)

9.6 ± 1.5 (11)

9.9 ± 0.9 (12)

9.7 ± 1.1 (13)

Female

9.4 ± 0.6 (13)

9.1 ± 1.3 (11)

9.5 ± 0.8 (12)

9.4 ± 1.1 (13)

Values expressed as Mean ± S.D.

Parenthesis indicates the number of litters evaluated

a): gestation index= (Number of pregnant females with pups alive/ Number of pregnant females) X100; %

b): Implantation index= (Number of implantation sites / Number of Corpora lutea) x100; %

c): Delivery index= (Number of pups born/ Number of implantation sites) x100; %

d): Birth index= (Number of pups alive on day 0/ Number of implantation sites) X 100; %

e): Live Birth index (Number of pups alive on day 0/ Number of pups born) X 100; %

 f): Sex ratio= (Number of male pups alive on day 0/ Number of female pups alive on day 0)

g): Viability index= (Number of live pups on day 4/ Number of live pups on day 0) X 100; %            

Applicant's summary and conclusion

Conclusions:
No Observed Adverse Effect Level (NOAEL) for reproductive and developmental toxicity was considered to be 1000mg/kg bw .When male and female Sprague Dawley rats were treated with test material orally.
Executive summary:

Combined Repeat Dose and Reproductive / Developmental Toxicity Screening Test of test material were performed on male and female Sprague Dawley rats. The test material was suspended in corn oil in dose concentration0 ,100, 300 and 1,000 mg/kg bw/day and administered via oral gavage route in dose volume 5ml/kg bw. Dose selected on the bases ofpreliminary test by the previously repeated 14-day oral administration. 13 male and 13 female each were placed in each group. Administration period For each males, 2 weeks before mating, and 42 days from the end of the mating period to the day before the necropsy for males, and for female 2 weeks before mating and a maximum of 2 weeks mating period (Until mating) and once daily for the entire gestation period and 3 days after nursing (delivery day = nursing 0 day) in mating females. Matings were made with the same sex of the same group living together within the same group for up to two weeks from the evening on 15th day of administration. Mating was confirmed every morning by examining the presence of sperm in the vaginal plug and vaginal smear and females confirmed to be mated were separated from male starting from that day as the 0th day of pregnancy and raised individually. All the animals were observed forClinical signs, body weight and food consumption.

No deaths in male and female were observed. In males, no abnormalities in general conditions were observed. In females, there was only one change from day 42 of administration (day of delivery) to the one in 100 mg / kg administration group (administration 42 days: piloerection, blood-like fluid discharge from the vaginal opening; administration 43 to 44 days: coat contamination) But it recovered on day 45 of administration. In other females, no abnormalities in the general condition were observed. In male, the body weight gain increased from 8 to 15 days in the 100 mg / kg administration group significantly (p <0.01) compared with the control group, However, in the group administered with 300 mg / kg or more, there was no significant difference between the control group and treated group. In females, there was no significant difference in body weight and body weight gain between control group and treated group before breeding, pregnancy period and nursing period. There was no significant difference between the control group and treated group for males and female at any time of feeding intake. There was no significant difference between the control group and treated group in the mating rate, conception rate, the number of days taken from the start of cohabitation to mating and the number of estrus periods recurred during that period.

An abnormality in labor condition (not treated with placenta) and abnormality in nursing condition (not collecting children, poor protrusion of nipple, poor protrusion of infant, child's fetus) was administered to one of 100 mg / kg administration group, The decrease in body surface temperature) was observed, and all children died on nursing day 2, but no abnormality was observed in other mother animals. For male In the 100 mg / kg administration group, the actual weight and the specific body weight value of the liver increased significantly (p <0.05) compared to the control group, but not the dose-dependent change. Atrophy of seminiferous tubules was found in 2 animals in the 1000 mg / kg administration group, one on both sides and one on one side, but both were localized and extremely mildly changed. There were no other abnormalities. Sperm granuloma was observed in one of the control group, and there was no abnormality. There were no abnormalities in the ovaries of the unexpanded and infertile cases. For female In the 100 mg / kg administration group, the actual weight of the kidney decreased significantly (p <0.05) compared to the control group, but not the dose-dependent change. For other organs in male and female, no significant difference was observed between the control group and treated group. Birth rate was 100% in all administration groups, and no significant difference was observed between gestation period between control group and treated group. No significant difference was observed between the control group and treated group for the number of luteinism, the number of implantation and the implantation rate of pregnant animals.There was no significant difference between the control group and treated group for the number of births, delivery rate, birth rate, fertility rate, and survival rate on 4th day of newborn. In addition, no significant difference was observed between the control group and treated group for sex ratio.There was no significant difference between the control group and thetreated grouponbody weight at 0 and 4 days of nursing. Births that showed morphological abnormalities were not observed in the external table observation on thebirthday, necropsy of the dead child, and necropsy on the 4th day of nursing. HenceNo Observed Adverse Effect Level (NOAEL) for reproductive and developmental toxicity was considered to be 1000mg/kg bw .When male and female Sprague Dawley rats were treated with test material orally.