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Toxicity to reproduction

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Administrative data

screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 26, 2016 to January 23, 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
Limit test:

Test material

Constituent 1
Test material form:
solid: particulate/powder
Details on test material:
Direct Red 83:1

Test animals

Details on species / strain selection:
The rat was chosen since it is an appropriate rodent experimental species for reproduction/developmental toxicity studies with documented susceptibility to a wide range of toxic substances.
Details on test animals or test system and environmental conditions:
- Source: BioLASCO Taiwan Co., Ltd., Taipei, Taiwan
- Age at study initiation: about 9-week old
- Weight at study initiation: Males: 322-375 g; Females: 206-249 g
- Housing: Except for the mating period, animals were housed individually in polycarbonate cages. While mating, animals were pair-housed in stainless steel wire mesh cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 days

- Temperature (°C): 21 ± 2 °C
- Humidity (%): 50 ± 20%
- Photoperiod: 12-hrs dark / 12-hrs light

Administration / exposure

Route of administration:
oral: gavage
corn oil
Details on exposure:
Vehicle: corn oil
- Supplier Sigma
- Lot no.:MKBV2080V

Dose Formulations Preparation (SOP: CTPS-TE00463)
The test article was suspended in corn oil to provide the designed concentrations of 20, 60 and 200 mg/mL and kept stirring prior to dosing. The stability of dose formulations at room temperature and 2-8 °C was 24 hours and 11 days, respectively. The dosing volume was 5 mL/kg bw.

The oral route was selected since it is the proposed exposure route to human.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: The day a vaginal plug or sperm was found was determined as Gestation Day 0 or G0; subsequent days were consecutively numbered.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility for 4 days
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): single
- Any other deviations from standard protocol:
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The homogeneity and concentration of respective formulations prepared on 01 August 2016 (all), 12 September 2016 (for high dose formulation), 18 September 2016 (for control formulation) and 19 September 2016 (for low and mid dose formulations) were verified. While stirring, duplicate samples (0.5 mL per sample) were collected. Samples collected from the top, middle and bottom layers of low and high dose formulations were used for homogeneity analysis. Samples collected from the middle layer of vehicle, low, mid and high dose formulations were used for concentration verification. The collected sample was placed into a 20-mL glass vial and stored at -15 to -25 °C. One set of samples was transferred to the Test Site for analysis and the other set was used as a backup.
The homogeneity and concentration of dose formulations were verified and determined by Test Site using a validated HPLC-UV method.
Concentration results were considered acceptable if the actual concentration was within 15.0% of the target concentration. Homogeneity results were considered acceptable if the coefficient of variation of the mean of the average top, middle, and bottom concentrations was <= 10.0%.
Duration of treatment / exposure:
All animals received vehicle or test article formulation via oral gavage once daily from pre-mating (14 days), mating (up to 14 days), gestation (female only - ca. 22 days) until four days after delivery (female only). The dosing period for males was 28 days and for females was 40-53 days, depending on the time of copulation and gestation.
There was a 2-week per-mating period followed a 14-day mating period. In mating period, all animals were copulated at a ratio of 1:1. The female was placed with the same male until pregnancy occurred. When pairing was unsuccessful in a continued 4 days, re-mating of females with proven males of the same group was conducted.
The gestation period was 21 to 23 days. Males were sacrificed after a dosing period of four weeks (D29). Maternal females with pups were sacrificed on P4. Presumed pregnant but non-delivering females were sacrificed on G25, and non-copulated females were sacrificed on D54.
Frequency of treatment:
once daily
Details on study schedule:
in concordance with OECD 421
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The test article was likely low- or non-toxic based on the LD50 in an acute oral toxicity study in rats, which was above 5000 mg/kg. The high dose (1000 mg/kg/day) for this study was the limit dose recommended in the OECD Guideline #421. The mid (300 mg/kg/day) and low (100 mg/kg/day) doses represented half-log decrements from the high dose and were included to better define the dose-response of test article effects and assure that a non-severely toxic dose was identified with the extension to repeated dosing.
- Rationale for animal assignment (if not random): random
Positive control:


Parental animals: Observations and examinations:
Observations of animals
From pre-mating to the end of lactation, all adult animals were observed twice daily for mortality check, once daily for cage side observation and once weekly for detailed clinical observation. The maternal nursing behavior including pup cleaning and warming, pups grouping together, evidence of nursing activity or milk in the pup stomach were observed on P0.

Body weights
Males: D1, weekly thereafter and at termination
Females: D1, weekly before presumed pregnancy; G0, G6, G13, and G20; P0 and P4; and necropsy day.

Food consumption
The food was measured on the same day as body weights were measured. During the mating period, food consumption was not measured.
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning starting from two weeks before pairing until a positive identification of copulation was made.

The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight, microscopic examination with emphasis on spermatogenesis staging and histopathology of interstitial testicular cell structure
Litter observations:
Litter and live pups examination
Each litter was examined as soon as possible after delivery to measure the number and sex of pups, stillbirths, live births and the presence of gross abnormalities. Live pups were counted, sexed and weighed together by litter on P0 and P4.
Postmortem examinations (parental animals):
Males were sacrificed on D29. Females with pups were sacrificed on P4. Females presumed pregnant without delivery were sacrificed on G25. The un-copulated females were sacrificed on D54. The animals were anesthetized by carbon dioxide exposure followed by exsanguination. A gross examination of the thoracic, abdominal and pelvic viscera was performed. The number of implantation sites and number of corpora lutea of all females were

The organs listed in following were sampled, weighed and fixed in appropriate fixative.

Male: Adrenal, Epididymis, Testis
Female: Adrenal, Liver, Lung (including bronchi), Mesenteric lymph node, Ovary

After fixation, representative sections of the tissues listed above of control and high dose animals were trimmed, processed, embedded, sectioned and H&E-stained, followed by microscopic examination (with emphasis on spermatogenesis staging and histopathology of interstitial testicular cell structure). The rest of the tissues were fixed and preserved in 10% neutral buffered formalin.

Organ weights: Epididymis, Testis
Paired organs were weighed together

Postmortem examinations (offspring):
Dead pups and pups euthanized by an intraperitoneal injection of overdosed pentobarbital at P4, were examined externally for gross abnormalities.
Body weight, including body weight change, food consumption and organ weights were analyzed for statistical analysis by utilizing Pristima® (see Section 4.8). The specific reproductive parameters were analyzed by utilizing SigmaStatTM Statistical Software for WindowsTM, Release 3.0 (Jandel Scientific Inc., USA). A p value of < 0.05 was used for the determination of statistical significance. Statistical analysis was performed separately for each sex of study animals. Summary statistics (mean, standard deviation and number) were calculated.
The homogeneity of data were assessed first by Bartlett’s test or Equal Variance Test depending on Pristima® or SigmaStatTM was used. When the data was homogeneous, a one-way analysis of variance (ANOVA) was applied, then Dunnett’s LSD test was performed to compare each test article-treated group (Groups 2 to 4) against control group (Group 1) if the result of one-way ANOVA was significant. Otherwise, Kruskal-Wallis one way ANOVA on ranks was used when data were heterogeneous.
Reproductive indices:
See below
Offspring viability indices:
See below

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

Key result
Dose descriptor:
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
One dead pup was observed at birth in Groups 2 and 3.
On P4, a decreased number of live pups was observed in all groups. The 4-day survival index was 99.3%, 96.7%, 96.6% and 98.1% in Groups 1 to 4, respectively, showing no dose-dependency.
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Gross pathological findings:
no effects observed

Details on results (F1)

Litter data, implantation, pre- and post-implantation losses and sex ratios did not differ between groups. Necropsy findings in pups did not show any
findings related to treatment.

Effect levels (F1)

Dose descriptor:
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

Overall reproductive toxicity

Reproductive effects observed:

Any other information on results incl. tables

Dose Formulation Analysis

Test article was not detected in vehicle samples. On the first preparation, the difference between the mean value and the targeted concentration was 10.0%, 17.9% and 0.0% for the 100, 300, and 1000 mg/kg/day dose group formulations, respectively, and 14.3%, 7.1% and -13.7% on the last preparation. Exception of the 60 mg/mL prepared on the first dosing day, all formulations were within the acceptance range (± 15.0%). The measured concentration (49.5 mg/mL) was higher than the nominal concentration (42.0 mg/mL). The dose level in Group 3 on this day was calculated as 354 mg/kg and higher than the study design (300 mg/kg/day).


Mortality and Clinical Observations

All animals survived the study period. Test article-related clinical observations were purple or red-brown colored feces, brown, pink or purple colored hair stained on forelimbs/jaw/mouth and salivation. This is due to the staining properties of the test article, which is a dye.


Body Weights

There was no significant difference in body weight and body weight gain changes in treated groups compared to control animals.


Food Consumption

There was no significant difference between treated and control groups.


Cohabitation and Pregnancy

In males, a positive indication of mating was obtained for 9, 8, 9 and 9 of 10 animals in Groups 1 to 4, resulting in a mating index of 90%, 80%, 90% and 90%, respectively. In females, a positive indication of mating was obtained for 10 of 10 animals in Groups 1 and 4 and for 9 of 10 animals in Groups 2 and 3. The mating index was 100%, 90%, 90% and 100% for Group 1 to 4, respectively. There was one animal in Group 1, 2 and 4, respectively, that showed a pre-coital interval longer 5 days. In each of these females, the mating with the first male failed but the copulation with another male was successful. In Groups 1 to 3, 1 animal with positive indications of mating was later found not to be pregnant. Thus, the fertility index was 90%, 80%, 80% and 100% in the control, low, mid and high dose group, respectively. The average length of gestation was 21.9 − 22.1 days without dose-dependency.


Litter Observation

All pregnant rats delivered live pups. The gestation index was 100% in all groups. One dead pup was observed at birth in Groups 2 and 3. The incidence of dams with dead pups at birth was 0%, 13%, 13% and 0% in Group 1, 2, 3 and 4, respectively. On P4, a decreased number of live pups was observed in all groups. The 4-day survival index was 99.3%, 96.7%, 96.6% and 98.1% in Groups 1 to 4, respectively. There was no difference in litter size between control and substance-treated groups. No external abnormally pups were observed at birth in the control and treated groups. At birth, 44.6% to 51.9% of pups were male and on P4, 47.0% to 56.5% pups were male. The gender difference at birth and on P4 was a consequence of difficult sex determination at birth. The litter weights in all groups increased from P0 to P4.


Male Gross Examination, Organ Weight and Histopathology

There were no statistically significant differences in testis and epididymis weights between control and treated groups. No macro- or microscopical changes were observed in any males.


Females Gross, Uterus Examination and Histopathology

There were no statistically significant differences in the number of corpora lutea and implantations between control and treated groups. Pre-implantation and pre-natal losses were observed in all groups including the controls. Decreased size of thymus was observed in one animal of Group 2 and two animals of Group 4. No histopathological abnormalities were noted.

Applicant's summary and conclusion

Based on the results of the present study, the NOAEL was considered to be 1000 mg/kg body weight/day for males, females and offspring.
Executive summary:

In a reproduction/developmental toxicity screening test (OECD 421) the test item was adminstered orally to 10 male and female rats in corn oil by gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. Male rats were trared for 28 days and female rats for 40 to 53 days, depending on the time of copulation and gestation.The following parameters were monitored: mortality and clinical observations in adults and pups, adult mating behaviour, body weight in adults and pups, adult food consumption, adult male sexual organ weights, adult and pups macroscopic observations at necropsy, and adult microscopic examinations of collecteted tissues.

The dose formulations were homogeneous and target concentrations were confirmed. Test article was not detected in the control group. No death, abortion or prematurely delivery were observed. Dose-related clinical observations were test article-like colored feces, test article-like colored hair stained and salivation in treated males and/or females. The test article treatment did not cause adverse effects on body weight and food intake in premating, gestation and lactation phases. There were no test article-related effects in mating index of both gender, and fertility index, pre-coital interval and pregnancy day in the females. There were no treatment related effects on pups viability, body weight, sex and gross external examinations. Macroscopic and microscopic evaluations did not reveal treatment-related effects in male and female fertility or reproductive organs.

The NOAEL in parental animals and in offspring was determined tob e 1000 mg/kg bw/day.