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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
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 from J-check database

Data source

Reference
Reference Type:
other: Authoritative database
Title:
Reproductive and Developmental toxicity screening test of the test chemical.
Author:
MHLW
Year:
2013
Bibliographic source:
JECDB (2013)

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD guideline 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test" (adopted 22 March 1996)
Principles of method if other than guideline:
Reproductive toxicity study of test material was performed on Sprague Dawley rats.
GLP compliance:
not specified
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 material form:
liquid
Details on test material:
- Name of test material: Docosanoic Acid
- IUPAC name: Docosanoic Acid
- Molecular formula: C22H44O2
- Molecular weight: 340.5876 g/mol
- Smiles: C(CCCCCCCCCCCCCC)CCCCCCC(=O)O
- InChl: 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)
- Substance type: Organic
- Physical state: liquid
Specific details on test material used for the study:
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:
Sprague-Dawley
Remarks:
(Crj: CD, SPF)
Details on test animals or test system 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 chemical 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
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No Data Available
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: at the most 14days
- Further matings after two unsuccessful attempts: [no / yes (explain)] : no
- Verification of same strain and source of both sexes: [yes / no (explain)] : no
- 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)

Duration of treatment / exposure:
Males: 43 days; Females: from 14 days prior to mating to day 4 of lactation.
Frequency of treatment:
Daily
Duration of test:
43 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day (in corn oil)
Basis:
nominal conc.
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:
- Dose selection rationale: As a result of the preliminary test by the previously repeated 14-day oral administration, no signs of toxicity were observed even in the group administered with the critical dose of 1000 mg / kg prescribed in the OECD Chemical Test Method Guideline
- Rationale for animal assignment (if not random):
- Other: No Data Available

Examinations

Maternal 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:

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: No Data Available
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes:
- Skeletal examinations: Yes:
- Head examinations: No data
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.
Indices:
Copulation index, fertility index, implantation index, gestation index, delivery index, birth index sex ratio, viability index
Historical control data:
No data available

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
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
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:
effects observed, non-treatment-related
Description (incidence and severity):
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:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no abnormalities in the ovaries of the unexpanded and infertile cases.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
No Data Available

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
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.
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
Birth rate was 100% in all administration groups.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No significant difference was observed between gestation period between control group and treated group.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): No significant difference was observed between gestation period between control group and treated group.
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Details on maternal toxic effects:
Maternal toxic effects:no effects. Remark: No abnormalities were found.

Details on maternal toxic effects:
No abnormalities were found in all reproductive parameters (fertility index, number of Implantations and implantation index) in each dose group. No statistically significant difference from controls in the case of Body weight,Food/water consumption and Reproductive responses.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
changes in number of pregnant
changes in pregnancy duration
clinical biochemistry
clinical signs
dead fetuses
early or late resorptions
effects on pregnancy duration
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss
Remarks on result:
other: overall no effects on reproductive performance

Maternal abnormalities

Abnormalities:
not specified
Localisation:
not specified

Results (fetuses)

Fetal body 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.

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified
Reduction in number of live offspring:
no effects 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 and survival rate on 4th day of newborn.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
no significant difference was observed between the control group and treated group for sex ratio.
Changes in litter size and weights:
not specified
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
There was no significant difference between the control group and treated group for survival rate on 4th day of newborn.
External malformations:
no effects observed
Description (incidence and severity):
No morphological abnormalities in pups were observed in any of the treated groups.
Skeletal malformations:
not specified
Visceral malformations:
not specified
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: No abnormalities were found in all pups.

Details on embryotoxic / teratogenic effects:
The compound did not demonstrate any adverse effects on the sex ratio, body weights or viability of pups. Also, no morphological abnormalities in pups were observed in any of the treated groups.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
changes in litter size and weights
changes in postnatal survival
external malformations
Remarks on result:
other: overall no developmental toxic effects observed

Fetal abnormalities

Abnormalities:
not specified
Localisation:
other: not specified

Overall developmental toxicity

Developmental effects observed:
not specified
Treatment related:
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:
The oral administration of 100, 300 and 1000 mg/kg of docosanoic acid to SD rats produced no reproductive and developmental toxicity therefore, the No Observed Adverse Effect Level (NOAEL) for reproductive and developmental toxicity was considered to be 1000mg/kg.
Executive summary:

A combined repeated dose toxicity and reproductive/developmental screening test of Docosanoic acid was performed on Sprague-Dawleyrats. Groups of 13 rats/sex/dose were orally administered (gavage) 0, 100, 300 and 1000 mg of docosanoic acid per kg. The test chemical was suspended in corn oil and used in volume of 5 ml/kg bw. The doses were selected on the bases of a preliminary, 14-day repeated dose toxicity study. In male rats, the administration period was two weeks prior to mating, 2 weeks of mating and 2 weeks after the completion of the mating period (42 days). In females, in addition to maximum four weeks pre-mating and mating period, they were exposed through pregnancy until day 3 of post-delivery (PDN 3). Mating was confirmed by the presence of sperm in the vaginal plug or in the vaginal smear. Females were allowed to deliver spontaneously and the conditions of parturition such as labour difficulty or delay and nursing behaviour were observed. No deaths or clinical signs of toxicology were observed in any male and female animals. The body weight and food consumption of treated animals, both males and females, were comparable with controls. At day 43 male animals were necropsied, most of the organs were weighed including reproductive organs (testis and epididymis) then tissues were fixed, and histopathological examination were performed. Before necropsy blood was collected for clinical biochemistry and haematology. The mean red blood cell haemoglobin concentration (MCHC) decreased significantly compared to the control group in the 300 and 1000 mg/kg dose groups, but no further significant difference was observed. The alkaline phosphates activity significantly decreased in all docosanoic acid administration groups and the glucose level was significantly lower in 1000 mg/kg group than in the control. However, alterations of blood parameters were not accompanied by any changes found in other examination including pathological examination, therefore they are considered to be accidental changes and have no toxicological significance. At necropsy there were no obvious changes that could be attributed to administration of docosanoic acid. No significant differences in organ weights were found between the control group and dosed animals. No treatment-related abnormalities were observed in adrenal gland, testis and epididymis in any treated males. Atrophy of the seminiferous tubule was observed in two animals at 1000 mg/kg, but the lesions were localized and very mild. There were no other abnormalities in male reproductive organs. Female animals (both mated and infertile) were also necropsied and organ weights were measured for heart, liver, kidney, and thymus. The ovaries and uterus were removed and the number of corpora lutea, the number of implantation and the implantation rate were calculated. Organs were fixed and tissue samples were prepared for further histopathological analysis. No obvious changes attributable to administration of docosanoic acid were seen in autopsy findings and organ weights. There were no significant differences in the number of corpora lutea, the number of implantations and the implantation rate of the pregnant animals between the control group and each administration group of docosanoic acid. The copulation and fertility indexes of each dose were comparable to controls. There were no significant differences between the control group and each dose of docosanoic acid in the mating rate, conception rate, the number of days required from the start of cohabitation to mating, and the number of oestrus returned during that period. The birth rate was 100% in all treatment groups. The number of births, delivery rate, live birth rate, and 4-day survival rate of newborns were not significantly different between the control and dosed groups. No significant difference in sex ratio of pups between the control and treated groups were seen. Similarly, there were no significant differences in body weight on day 0 and 4 between the control and docosanoic acid groups. No offspring with morphological abnormalities were observed at PND 0 and 4 following external surface observation and autopsy. In summary, no abnormality was found in fertility, reproductive function and parturition of parental animals and the prenatal and early postnatal exposure to the test chemical did not produced adverse effects on neonatal survival and growth, when male and female SD rats were orally administered of 100, 300 and 1000 mg/kg of docosanoic acid. Hence, the NOAEL for reproductive and developmental toxicity is considered as 1000 mg/kg under the certain experimental conditions.