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Diss Factsheets

Administrative data

developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
december 2010 - april 2011
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD guidelines and according to GLP principles.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
Limit test:

Test material

Constituent 1
Reference substance name:
Details on test material:
- Name of test material (as cited in study report): E-C104
- Physical state: blue colored powder
- Lot/batch No.: MB-1
- Expiration date of the lot/batch: five years from shipment day (November 4, 2010)
- Stability under test conditions: the test substance was confirmed to be stable during the experimental period based on infrared absorption spectra
- Storage condition of test material: room temperature (permissible range 1°-30°C) in an air-tight container in a dark place

Test animals

other: Crl:CD(SD)
Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories Japan, Inc. (Hino Breeding Center)
- Age at study initiation: 9 weeks
- Weight at receipt (8 weeks old): 251.6 to 280.8 g for males and 164.6 to 193.3 g for females
- Housing: stainless-steel cages, except during gestation and lactation period (polymethylpentene cages). One male and one female were housed in a cage during the mating period. One dam and its litter were housed in a cage during the lactation period. As for the other periods, animals were housed individually. Bedding material: satirized wood chip
- Diet (e.g. ad libitum): ad libitum autoclave-sterilized pellet diet (CRF-1, Oriental Yeast Co., Ltd)
- Water (e.g. ad libitum): ad libitum well water admixed with NaClO (about 2 ppm)
- Acclimation period: 9 days

- Temperature (°C): 22.8° to 23.9°C
- Humidity (%): 52.0 to 79.6%
- Air changes (per hr): 10 to 20 times
- Photoperiod (hrs dark / hrs light): 12h/12h
IN-LIFE DATES: From: January 7,2011 To: April 18, 2011

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
Dosing solutions were prepared once every 5 or 7 days and stored in a cool [place shielded from light.
Analytical verification of doses or concentrations:
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear] referred to as
- After successful mating each pregnant female was caged in polymethylpentene cages during the gestation and lactation period. One dam and its litter were housed in a cage during the lactation period.
Duration of treatment / exposure:
males: from 14 days before mating until necropsy through the mating period (42 days).
females: from 14 days before mating until day 4 of lactation through the mating and pregnancy periods and delivery.
recovery females (satellite group): for 42 days without mating.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
once daily
Duration of test:
42 days, with additionally a 14-day recovery period
Doses / concentrations
Doses / Concentrations:
50, 250, 1000 mg/kg bw/day
actual ingested
No. of animals per sex per dose:
Each test group consisted of 12 males (including 5 males for recovery test) and 12 females (5 females each were added for recovery test in the control and 1000 mg/kg bw/day groups).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on results obtained in "Fourteen days repeated oral dose toxicity study of E-C104 in rats (No. P101038; dose levels 0, 100, 300 and 1000 mg/kg bw, with 5 rats/sex/dose). As no toxicologically relevant effects were observed, the highest dose was set at 1000 mg/kg bw/day as stated in the guideline and two lower doses were established.
- Post-exposure recovery period in satellite groups: 14 days


Maternal examinations:
- Time schedule: twice a day during the dosing period; once a day during recovery period.

- Time schedule: once before dosing; once a week until week 6

- Time schedule for examinations: males were weighed on days 1, 8, 15, 22, 29, 36, 42 and 43. The recovery males were also weighed on days 50 and 56. The satellite females were weighed in the same manner as the recovery males. The test females were weighed on days 1, 8 and 15, once every 7 days after initiation of copulation, and days 0, 7, 14 and 20 of gestation and days 0 and 4 of lactation for females after parturition. The final body weight was also measured on the day of necropsy.

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Food consumption was measured between days 1 to 8, 8 to 15, 22 to 29, 29 to 36 and 36 to 40 for test and recovery males and between days 43 to 50 and 50 to 54 for recovery males only. For females it was measured between days 1 to 8, 8 to 15, 15 to 22, 22 to 29, 29 to 36, 36 to 42, 43 to 50 and 50 to 56 for the satelite females. Food consumption of the test females was measured at the same time as the body weights. Food consumption was not measured for either sex during the mating period. Gross weight of each feeder was weighed, and the main daily food consumption of each animal was calculated for each period.

- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes, sodium pentobarbital (i.p., 30 mg/kg bw)
- Animals fasted: Yes, at least 18 hours
- How many animals: 5 animals with smaller animal numbers for the test males, all animals for the recovery males and satellite females, and 5 animals from the earlier parturition date with small numbers for the test females.

- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes, at least 18 hours
- How many animals: same as for haematology

- Time schedule for collection of urine: day 40
- Metabolism cages used for collection of urine: No, fresh urine samples excreted spontaneously on a washed try under the cage were collected.
- Animals fasted: Yes
- Parameters checked: PH, protein, glucose, ketone body and occult blood were determined by means of reagent strip method.

- Time schedule for examinations: week 6
- Dose groups that were examined: 5/sex/dose group
- Battery of functions tested: sensory activity / grip strength / motor activity
Ovaries and uterine content:
Vaginal smears were collected with swabs from all test females in teh morning from teh initial dosing day to the day of mating or end of mating period to confirm the estrous cycle. The obtained smears were colelcted on a plate for each animal and thionine-eosin-stained. The estrous cycle was classified into diestrus, proestrus, estrus and metestrus. The mean estrous cycle and the number of the estrous period during the test period were calculated. The data obtained during the mating period were treated as referential data.
All copulated females were allowed natural delivery. The observation of delivery was from days 21 to 24 of gestation. The females that did not deliver by 24 days after copulation were regarded as non-delivered females. The delivered dams were allowed to nurse offsprings until day 4 postpartum (day 4 of lactation) and postpartum behavior such as lactation, nesting, and presence or absence of cannibalism was observed every day.
Gestation length, delivery index and implantation index were recorded.
Fetal examinations:
Observation for offspring included: live birt index, stillborne rate, viability index on day 4, sex ratio, individual body weight of all live offsprings on days 0 and 4 postpartum, external anomaly index and external anomaly typing index.
Statistical analysis was performed with computer system, MiTOX-PPL with significant levels of 1% and 5% (two-tailed). The data of offspring were handled on a litter-basis. The body weight and food consumption of the female not pregnant, as well as the body weight after initiation of mating in the females failed mating were excluded from evaluation.
For grip strength, motor activity, body weights, food consumption, urinalysis, hematology, blood chemistry, absolute and relative organ weigh, the group mean and standard deviation of the following items were calculated and homogeneity of the variance was tested by the Bartlett's test (significant level 5%). When the variance was homogeneous, the Dunnett's multiple comparison was applied, and when it was not homogeneous, the Steel's multiple comparison was applied for control group and each test substance group.

For Histopathological examination, the grades were converted to numerical values, and the Mann-Whitney U test was applied to compare between the control and each test substance group.

Copulation index, fertility index, destation index and sex ratio were tested with the chi-square test for comparison between the control and each test substance group.
Implantation index and stillborne rate were tested with the Wilcoxon's rank sum test for comparison between the control and each testsubstance group.

External anomaly index, external anomaly typing index, live birth index and viability index on day 4 were tested with the Wilcoxon's rank sum test for comparison between the control and each test substance group.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Effect levels (maternal animals)

open allclose all
Dose descriptor:
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:
Dose descriptor:
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Remarks on result:
other: no effects

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

In an OECD 422 study in rats orally exposed to 50, 250, 1000 mg/kg bw/day E-C104, no substance related effects in the offspring was observed. As a result, the NOAEL offspring is considered to be at least 1000 mg/kg bw/day.
Executive summary:

E-C104 was repeatedly administered to rats (7/sex/dose) by oral gavage at dose level 0, 50, 250 and 1000 mg/kg bw/day from 14 days before mating for 42 days in males and satellite females, and 14 days before mating through gestation and parturition until day 4 of lactation in females to determine the effects of repeated exposure and reproductive and developmental toxicity. Reversibility was studied in satellite groups for controls and high dose (5/sex/dose). As a result of the test substance administration, substance-mixed feces (dark blue color) was noted in all dose groups. Haematological examination showed increases in platelet count and numbers of neutrophils and lymphocytes. At the end of the recovery period, increase in reticulocyte number was observed in males and females showed increase in neutrophils and decrease in lymphocytes. Clinical biochemistry showed in high dose animals decreased levels of total bile acids, total cholesterol and ALT. High dose males showed decreased plasma glucose levels, whereas high dose females showed a decreased plasma triglyceride level. Gross pathology showed blue coloration in the GI contents, mesenteric, submaxillary, cervical and bronchial lymph nodes, lung and kidneys (with no reversibility in coloration in teh lymph nodes and kidney in the mid- and high dose). Histopathological examination showed pigment-laden macrophages in the GI-tract, mesenteric lymph nodes and lungs in rats dosed at and above 250 mg/kg bw/day, but the toxicological relevance is unclear.

No reproduction toxicity and no developmental toxicity was observed.