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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to or similar to guideline study OECD 421:GLP.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1996

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Undecane is a colorless and clear liquid insoluble in water. It was stored in an air-tight and light-shielding container placed in a refrigerator. In the tests, undecane manufactured by Tokyo Chemical Industry Co., Ltd., Lot # FBQ041 (purity of 99% or higher) was used. The substance was confirmed to be stable during the administration term.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Number of Animals: Males, 48; females, 48 (total)
Crj:CD (SD strain) SPF male and female rats 8 weeks old were purchased from Nippon Charles River Co., Ltd., and quarantined for 14 days. The administration was started when the animals were 10 weeks old, and the mean body weight (body weight range) of males at the start of administration was 400.3 g (374-431 g), and that of females was 226.5 g (192-255 g).

The animals were individually placed in metal bracket cages with a metal mesh floor before mating, one male and one female per cage during mating, one mother animal per cage during gestation, and a mother and her newborns after birth.

Animals were maintained in a barrier-isolated room with a temperature of 23 +/- 3°C, humidity of 55 +/- 10%, ventilation changes of 10-15 times per hour, and an illumination of 12 hr/day. After the 17th day of gestation, the metal mesh floor was changed to a stainless steel pan spread with a floor-covering material for experimental animals. Solid feed (CRF-1, manufactured by Oriental Yeast Industry Co., Ltd.), and tap water (tap water of the City of Sapporo) were given freely.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
The administration of the test substance was carried out by oral gavage. The volume administered was 5 mL per kg of body weight, and it was calculated based on the results of body weight measured on the day closest to the day of administration. The administration period was 46 days, which including 14 days before mating and during the mating period for males. The administration period for the female rats began 14 days before mating and continued until after the first 3 days of nursing. The administration was started when the animals were 10 weeks old, and the mean body weight (body weight range) of males at the start of administration was 400.3 g (374-431 g), and that of females was 226.5 g (192-255 g).
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
The administration period was 46 days, which including 14 days before mating and during the mating period for males. The administration period for the female rats began 14 days before mating and continued until after the first 3 days of nursing.
Frequency of treatment:
once per day
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (vehicle), 100, 300, 1000 mg/kg/day
Basis:
actual ingested
oral gavage
No. of animals per sex per dose:
Males, 12 ; females, 12 per dose
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
General conditions, body weight and feed intake:
For all cases, the general conditions were observed once a day throughout the study term. The body weight measurements were carried out on the 1st (before administration), 2nd, 5th, 7th, 10th and 14th day after administration and subsequently, every 7 days (including the last day of administration) as well as 0th, 1st, 3rd, 5th, 7th, 10th, 14th, 17th and 20th day of gestation and the 0th, 1st and 4th day of nursing for females. In addition, the body weight gain and rate were calculated from the 1st day of administration to the 46th day for males and for females, from the 1st day to the 14th day, 0th day to the 20th day of gestation and 0th day to the 4th day of nursing. The amount of feed intake was measured on the same days as those of body weight measurements except mating period and dissection day for males and 0th day of gestation and 0th day of nursing for females. Incidentally, with respect to the number of day of pregnancy, the successful copulation day was set as the 0th day of gestation, and in the case of lactation, the day of delivery completion was set as the 0th day of nursing.

Urinary tests:
In the final week (43rd-44th day of administration) during the administration term, 6 male cases of each group were placed in metabolism-measurement cages, and their urine samples were collected under non-starvation conditions. For urine samples, collected in about 3 hr, pH, protein, glucose, ketone, urobilinogen, bilirubin, occult blood reaction, and sedimentation (microscopic observation) were tested, and for urine samples collected for 21 hr, volume, and specific gravity were measured. In addition, the amount of drinking water (weight) in urine samples was also measured.

Hematological tests:
Before dissection, all male animals were starved for about 16 hr, blood samples were collected from the femoral vein under ether anesthesia, and EDTA•2K-treated blood samples were used to measure the erythrocyte count, mean erythrocyte volume, platelet count, leucocyte count, hemoglobin content (cyanmethemoglobin method), hematocrit (calculated from erythrocyte count and mean erythrocyte volume), mean erythrocyte hemoglobin content (calculated from erythrocyte count and hemoglobin content), mean erythrocyte hemoglobin concentration (calculated from hematocrit and hemoglobin content), reticulocyte proportion (Brecher method) and leucocyte fraction (microscopic observation). In addition, untreated blood samples were used to measure coagulation time (fluid viscosity change air pressure measurement, Gryner Microcogulometer). Furthermore, plasma samples, which were prepared by collecting blood samples from the abdominal aorta, treating with sodium citrate and subsequently carrying out centrifugation at 3,000 rpm for 10 min, were used to measure prothrombin time (thromboplastin method) and activated partial thromboplastin time (ellagic acid method).

Hematobiochemical test:
After the hematological tests, serum samples of all male cases, which were prepared by collecting from the abdominal aorta, were used to measure GOT, GPT (IFCC method), GPT (glutamyl-p-nitroanilide substrate clathrate method), choline esterase (butyrylthiocholine iodide substrate method), blood glucose (hexokinase method), total cholesterol and phospholipids (enzymatic method), triglyceride (free glycerol deduction method), total bilirubin (azobilirubin method), urea nitrogen (urease-indophenol method), creatinine (Yaffe method), calcium (OCPC method), inorganic phosphorus (Fiske-Subbarow method), total protein (Biuret method) and albumin (BCG method) (Hitachi automated analyzer, Model 7150); sodium and potassium (flame photometry: Corning flame photometer, Model 480); chlorine (coulometric titration method: Hiranuma chloride counter, Model CL-6M); A/G ratio (calculated from total protein and albumin); and protein fraction (cellulose acetate electrophoresis).
Litter observations:
The viability of the newborns was confirmed once a day from delivery completion to the 4th day of nursing. The viability of newborns for 4 days [(No. of live pups on the 4th day/No. of pups born) x 100] was calculated. In addition, the general condition and appearance of the pups was observed. The body weight was measured on the 0th, 1st and 4th day of nursing, and the body weight gain and the rate of body weight gain were calculated. Dead pups were dissected immediately after discovery. All pups were sacrificed and dissected on the 4th day of nursing.
Postmortem examinations (parental animals):
Dissection and organ weight measurement:
After the 46th day of administration, all males were sacrified ex sanguine under ether anesthesia after blood sampling and dissected. Any newborns that died were dissected immediately after discovery. Females that successfully copulated were sacrificed on the 4th day of nursing. Female rats who did not successfully copulate on the 25th day of gestation (infertile cases) on the 26th day of gestation were sacrified. The implantation sites in the uterus and corpus luteum of pregnancy in the ovaries were counted. In addition, the weight measurements were carried out for the liver, kidney, thymus gland, adrenal gland, testes, epididymis and ovary, and the ratio to body weight was calculated.

Histopathological observation:
The following tissues were embedded in paraffin and stained with hematoxylin-eosin or with oil red O staining/ luxol fast blue-Bodian double stain to conduct a histopathological examination: the liver, kidney, spleen, heart, lung, brain (cerebrum and cerebellum), hypophysis, thymus gland, adrenal gland, thyroid gland, stomach (anterior stomach and glandular stomach), duodenum, jejunum, ileum, cecum, colon, rectum, testes, epididymis, prostate gland, ovary and abnormal sites.
Statistics:
Fisher’s accuracy probability test was carried out to compare the control and undecane-administered groups for the sexual cycle, copulation index, fertility index, gestation index and nursing index. Other test parameters were analyzed by using Bartlett’s homogeneity of variance and subsequently single dimensional configuration variance analysis or Kruskal-Walls method. If the results were found to be significant, the Dunnett ‘s method or Mann-Whitney U-test method was used to compare the undecane-administered groups from the control group. However, those qualitative items in urinary tests were analyzed by using the Kruskal-Walls method and Mann-Whitney U-test method. The viability of newborns on the 4th day and body weight were analyzed using the litter as a unit and the results were compared with the control group. A significance level of less than 5% was considered to be statistically significant.
Reproductive indices:
Reproductive ability test:
For female rats, vaginal smear specimens were prepared every day starting from 10 days before administration. Specimens were prepared until successful copulation was confirmed in order to evaluate any abnormalities in the sexual cycle.

On the 14th day of administration, males and females rats (1 to 1 pairs; same dose) were allowed to cohabit for a maximum of 14 days. Successful copulation was confirmed when spermatozoa were detected in the vaginal smear of the female. Gestation was confirmed through the detection of implantation sites in the uterus. In addition, the copulation index [(No. of pairs with successful copulation/No. of pairs mated) x 100] and fertility index [(No. of pregnant animals/No. of pairs with successful copulation) x 100] were calculated.
Offspring viability indices:
The viability of the newborns was confirmed once a day from the delivery completion day to the 4th day of nursing. The viability of newborns for 4 days [(No. of live pups on the 4th day/No. of pups born) x 100] was calculated. In addition, the general condition and appearance of the pups was observed. The body weight was measured on the 0th, 1st and 4th day of nursing, and the body weight gain and the rate of body weight gain were calculated. Dead pups were dissected immediately after discovery. All pups were sacrificed and dissected on the 4th day of nursing.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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:
not examined

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)

In the repeat dose toxicity test, salivation was observed in males and females given 300 and 1000 mg/kg. Body weight gain was suppressed in males given 1000 mg/kg, and body weights were increased in females given 1000 mg/kg during the lactation period. Food consumption was decreased in males given 300 and 1000 mg/kg in the first half of the administration period, increased in males given 1000 mg/kg in the second half of the administration period, and increased in females given 1000 mg/kg in the second half of pregnancy and during the lactation period. Hematological and blood chemical examinations revealed a decrease in hemoglobin concentration, an increase in the white blood cell count, a decrease in albumin, and increases in a2-globulin, GPT, cholinesterase and total cholesterol in males given 1000 mg/kg. Relative liver weights and absolute and relative thymus weights were increased in males given 1000 mg/kg, and absolute and relative liver weights were elevated in females given 1000 mg/kg. No effects were detected in the autopsy or histopathology findings. Due to the lack of effects in both sexes and the lack of corresponding histopathology, these effects are determined not to be toxicologically relevant. The NOAEL for repeat dose toxicity is considered to be 1000 mg/kg/day for both sexes.

Reproductive and developmental toxicity
No effects of undecane administration were observed on the sex cycle of females and copulation and conception of males and females. In addition, no effects of undecane administration were observed on the weights of reproductive organs (testis, epididymis and ovary) and there were no abnormalities noted in the dissection and histopathological examination. Incidentally, the reproductive organs of infertile cases showed no histopathological findings suggesting the causes. Abnormal cases observed in this study were confirmed to be spontaneous when compared to historical controls. Those cases observed in the present study were considered to be unrelated to undecane.

The number of live or dead pups delivered per each dam in the undecane-administered group showed no apparent difference from that in the control group if those dead and unknown pups in those cases of abnormal delivery or death of all of the litter during nursing were excluded. The body weight gain rates of both males and females in the 1,000 mg/kg group were observed to be reduced, but no effects of undecane were observed as a result of general condition observation or dissection. The NOAEL for reproductive performance is considered to be 1000 mg/kg/day.

Effect levels (P0)

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Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No effects noted at highest dose tested.
Dose descriptor:
NOAEL
Remarks:
repeat dose
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No effects noted at highest dose tested.

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

No effects were detected with regard to reproductive ability, reproductive organ weights, or autopsy or histopathology findings in either sex, and there was no apparent influence on delivery or maternal behavior of dams. Body weight gain was decreased in male and female offspring of the 1000 mg/kg group. This was not considered a toxicological effect. No effects were noted in terms of viability, general condition or autopsy findings of offspring.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No effects noted at highest dose tested.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL for repeated dose toxicity is considered to be >=1000 mg/kg/day for both sexes. The NOAELs for reproductive performance is considered to be >=1000 mg/kg/day.
Executive summary:

In the repeated dose toxicity test, male and female rats were given 0, 100, 300 and 1000 mg/kg. Male rats were dosed for 46 days (14 days prior to mating and then during the mating period) and female rat from 14 days before mating to day 3 of lactation. The NOAEL for repeated dose toxicity is considered to be >=1000 mg/kg/day for both sexes. The NOAEL for reproductive performance is considered to be >=1000 mg/kg/day.