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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
27th July 1995
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-[(3,3'-dimethoxy[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[3-oxo-N-phenylbutyramide]
EC Number:
229-388-1
EC Name:
2,2'-[(3,3'-dimethoxy[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[3-oxo-N-phenylbutyramide]
Cas Number:
6505-28-8
Molecular formula:
C34H32N6O6
IUPAC Name:
2,2'-[(3,3'-dimethoxy[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[3-oxo-N-phenylbutyramide]
Test material form:
solid: particulate/powder
Details on test material:
Pigment orange 16 (hereinafter abbreviated as PO16) was used as a test substance. It is also called “Disazo orange” and its name in English is C. I. Pigment Orange 16 having CAS No. 6505-28-8, molecular weight 620.66 and molecular formula C34H32N6O6.
Purity: 99 wt% or higher
The provided test substance contained a water-soluble compound (NaCl) 0.18% as impurity

For this specific material, a particle size characterization is not available.
Specific details on test material used for the study:
- Name of test material (as cited in study report): C.I. Pigment Orange 16
- Physical state: orange coloured powder
- Storage condition of test material: approximately 4 °C in the dark

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan Inc., 3-17-6, Shinyokohama, Kohoku-ku, Yokohama, Kanagawa 222-0033, Japan
- Age at study initiation: (P) 10 wks
- Weight at study initiation: (P) Males: 243.7 - 284.8 g; Females: 163.1 - 200.6 g
- Housing: 1 - 3 animals per cage during the quarantine and acclimatization period. 1 animal per cage after grouping.
- Diet: ad libitum, pellets (CRF-1, Oriental Yeast Co., Ltd.)
- Water: ad libitum, well water
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 25
- Humidity (%): 53 - 63
- Air changes (per hr): 10 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The preparation was made on a concentration-by-concentration basis. The required amount of the test substance was weighed accurately by each preparation concentration. The weighed test substance was put in a beaker containing the vehicle (60 - 70 % of final preparation volume). The liquid was stirred by to prepare a suspension. After ascertaining that the suspension condition had attained perfection, the suspension was diluted with the vehicle in a volumetric flask. The dilution was mixed by inverting the flask, and the required amounts were dispensed in vials.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item can be suspended in corn oil. It is insoluble in water.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The results of analysis by lot conducted by Japan Food Research Laboratories under a commission from Oriental Yeast Co., Ltd. were obtained to verify that impurities mixed in the feed met the acceptable reference value determined by Panapharm Laboratories Co., Ltd.
Duration of treatment / exposure:
males for total 49 days including 14 days before mating and 35 days thereafter
females for 14 days before mating, during the mating period (up to 11 days until successful copulation), the gestation period and the period until day 3 of lactation.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
Mating started from when both male and female had attained 12 weeks of age. One male and one female each in the same group were cohabitated overnight, and the copulation of the female showing the presence of a vaginal plug or sperm on a vaginal smear the following morning was confirmed to be successful, and the day was designated as day 0 of pregnancy. While mating period was up to two weeks, copulation was confirmed in all cases before day 11 of mating period, therefore re-mating (one week) was not conducted. From the examination results, number of days required for copulation, copulation index and fertility index were calculated.

Examinations

Parental animals: Observations and examinations:
During the administration period, the observation of the general conditions and the confirmation of life and death were carried out, every day twice, before and after dosing.
Throughout the administration period, the body weight was measured at a frequency of twice a week for males. For females, the body weight was measured twice a week during pre-mating dosing period, on gestation days 0, 7, 14 and 20 during pregnancy, and on lactation days 0 (day of parturition) and 4 during lactation period.

Throughout the administration period, the food consumption was measured at a frequency of twice a week for males. However, it was not measured during mating period. The feeder to put the feed was weighed and set in a cage in the morning, and the next day morning, the remaining amount of the feeder removed from the cage was weighed. The display of food consumption was set to be the measurement date of the remaining amount.
For females, food consumption was measured twice a week during pre-mating dosing period, on gestation days 1, 7, 14 and 20 during pregnancy, and on lactation days 1 (day after parturition) and 4 during lactation period.
Oestrous cyclicity (parental animals):
For females during the administration period, vaginal smears were collected to monitor estrous cycle with a cotton swab every day at a certain time in the morning. The estrous cycle was classified into diestrus (D), proestrus (P), estrus (E) and metestrus (M), and the average number of days from estrus (E) to estrus (E) during the examination period (estrous cycle) and the estrus count were calculated.
Litter observations:
On lactation day 0, the numbers of newborns, of live newborns, of stillborns, the sex of newborns, the body weight of newborns and the external appearance of newborns were checked. The birth index was also calculated. The life and death of newborns was confirmed every day, and on lactation day 4, the body weight was measured and the viability index was calculated.
Postmortem examinations (parental animals):
After necropsy, the testes and epididymides were weighed, and the organ weight to body weight was calculated based on the weight of the necropsy day.

The testis and epididymis were pre-fixed with Bouin’s solution and then preserved after fixation with 10vol% neutral buffered formalin solution. The paraffin sections of the testis and epididymis of the animals in the control and high dose groups were prepared and stained with hematoxylin and eosin and then microscopically examined. Since the results of the examination showed that the effects of the test substance administration were not observed in the high dose group, microscopic examination was not carried out in the medium and low dose groups. However, microscopic examination was carried out on the testis and epididymis of one male (No.36) in the medium dose group because the mating partner female was infertile.


Mother animals were sacrificed on day 4 of lactation with exsanguination by cutting the external iliac artery under ether anesthesia and promptly necropsied, and all the organs and tissues were closely examined for the presence or absence of abnormalities and also the ovaries and uterus were excised. The number of corpus luteum and the number of implantation sites were calculated. The animals having no parturition were sacrificed on day 24 of gestation with exsanguination by cutting the external iliac artery under ether anesthesia and necropsied to confirm the presence or absence of pregnancy.
After necropsy, the ovaries were weighed, and in addition, the organ weight to body weight was calculated based on the weight of the necropsy day.
The ovaries were fixed with 10 vol% neutral buffered formalin solution and preserved. The ovaries of the animals in the control and high dose groups were changed into paraffin sections and then stained with hematoxylin and eosin and microscopically examined. Since the results of the examination showed that the effects of the test substance administration were not observed in the high dose group, microscopic examination was not carried out in the medium and low dose groups. However, microscopic examination was carried out on the ovaries of one case (No.86) in the medium dose group because the female was infertile.
Postmortem examinations (offspring):
On lactation day 4, pups were sacrificed with exsanguination by cutting the external iliac artery under ether anesthesia, and macroscopic observation was performed on organs and tissues. The organs and tissues showing abnormalities were fixed with 10 vol% neutral buffered formalin solution and preserved. After the necropsy, the organs and tissues of the chest and abdomen of offspring were removed, and the carcasses were fixed and preserved in pure ethanol in litter units. Stillborn pups and dead pups were also necropsied, and the carcasses were fixed and preserved in pure ethanol
Statistics:
Statistical analysis was performed as described below, and the significance level was set to 1 and 5 % in both cases. Infertility cases were excluded from the evaluation.
Reproductive indices:
gestation length
delivery index
live birth index
Offspring viability indices:
live birth index
viability index
sex ratio

Results and discussion

Results: P0 (first parental generation)

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
Histopathological findings: non-neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

Animals had orange-colored feces which indicated passage of the orange test material through the gastrointestinal tract.

It is referred to the attached table for details on the indeces and key results.

Effect levels (P0)

Dose descriptor:
NOEL
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 effects were observed at the limit dose.

Target system / organ toxicity (P0)

Critical effects observed:
no

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:
no effects observed
Histopathological findings:
not examined

Effect levels (F1)

Dose descriptor:
NOEL
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: This study only covers treatment until the first 4 days after birth.

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

It is referred to the attached table (illustration/graph).

Applicant's summary and conclusion

Conclusions:
No indication of reproductive toxicity was observed in this screening study at the limit dose.