Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
One group of 22 females received Macrolex Blau 3R at the limit dose of 1000 mg/kg/day by daily oral gavage administration at a dose volume of 10 mL/kg/day from Day 6 to 19 after mating. A similarly constituted control group received the vehicle, 0.5% CMC-Na solution, according to the same dose volume and regimen. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.
Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating with the weight of the gravid uterus recorded. All fetuses were weighed, subjected to an external macroscopic examination and then subsequently to detailed internal visceral or skeletal examination.
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Test substance: Macrolex Blau 3R
Test substance identity
(including alternative names): Macrolex Blau 3R, Reinblau RLW, Macrolex Blue 3R
CAS name: 1,4-Bis[(2-ethyl-6-methylphenyl)amino]-9,10-anthracenedione
CAS number: 41611-76-1
Appearance: Blue powder
Purity: 99.6 %

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% CMC-Na solution
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
Mating
Male/female ratio: 1:1 with identified stock males.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
Day 0 of gestation: When positive evidence of mating was detected.
A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
Duration of treatment / exposure:
Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.
Frequency of treatment:
Once daily
Duration of test:
21 days
Doses / concentrations
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
1000 mg/kg/day: 22 females
0 mg/kg/day: 22 females (control group)
Control animals:
yes, concurrent vehicle

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

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

Results (fetuses)

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

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Treatment of pregnant female Sprague Dawley rats with Macrolex Blau 3R daily from Day 6 to Day 19 after mating, inclusive, at the limit dose of 1000 mg/kg/day was well tolerated and elicited no deaths and no toxicity related changes in clinical condition of the adult females.

At 1000 mg/kg/day a total of 17 females were recorded with the clinical sign of blue staining of the tail. At necropsy several females receiving 1000 mg/kg/day showed blue colouration of gastro-intestinal contents, three females showed blue colouration of rectal tissue, 5 females were recorded with blue coloration of stomach tissue and 17 females were confirmed as having blue colouration of the tail. The blue staining observed was considered discolouration caused by the test material itself (a blue dye) and is considered not to represent toxicity.

Body weight gain and food consumption during gestation were unaffected by treatment and there were no other maternal findings observed at macroscopic examination that were considered to relate to treatment.

There was no effect of treatment at 1000 mg/kg/day upon embryo-fetal survival or placental weights, and fetal development was also unaffected by treatment at this limit dose, with the incidence of major and minor abnormalities and skeletal variants showing no relationship to treatment.

Mean male, female and overall fetal weights at 1000 mg/kg/day were lower than Controls, but the difference was considered marginal (approx. 5-6%) and was clearly not adverse since embryo fetal survival and development were unaffected by treatment.

Based on the results of this embryo-fetal developmental toxicity study in rats, it was concluded that the No-Observed-Effect-Level (NOEL) of Macrolex Blau 3R for maternal toxicity was the limit dose of 1000 mg/kg/day.

For embryo-fetal development, the limit dose of 1000 mg/kg/day was considered to be the No-Observed-Adverse-Effect-Level (NOAEL), when administered during the organogenesis and fetal growth phases of gestation in the rat, as there was a marginal reduction in fetal weight which was considered not adverse due to there being no effect of treatment upon embryo-fetal survival or development.

Applicant's summary and conclusion

Conclusions:
Based on the results of this embryo-fetal developmental toxicity study in rats, it was concluded that the No-Observed-Effect-Level (NOEL) of Macrolex Blau 3R for maternal toxicity was the limit dose of 1000 mg/kg/day.
For embryo-fetal development, the limit dose of 1000 mg/kg/day was considered to be the No-Observed-Adverse-Effect-Level (NOAEL), when administered during the organogenesis and fetal growth phases of gestation in the rat, as there was a marginal reduction in fetal weight which was considered not adverse due to there being no effect of treatment upon embryo-fetal survival or development.
Executive summary:

The objective of this study was to assess the influence of Macrolex Blau 3R, a blue powder, on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Sprague Dawley CD rat.

One group of 22 females received Macrolex Blau 3R at the limit dose of 1000 mg/kg/day by daily oral gavage administration at a dose volume of 10 mL/kg/day from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, 0.5% CMC-Na solution, according to the same dose volume and regimen. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating with the weight of the gravid uterus recorded. All fetuses were weighed, subjected to an external macroscopic examination and then subsequently to detailed internal visceral or skeletal examination.

Treatment of pregnant female Sprague Dawley rats with Macrolex Blau 3R daily from Day 6 to Day 19 after mating, inclusive, at the limit dose of 1000 mg/kg/day was well tolerated and elicited no deaths and no toxicity related changes in clinical condition of the adult females.

At 1000 mg/kg/day a total of 17 females were recorded with the clinical sign of blue staining of the tail. At necropsy several females receiving 1000 mg/kg/day showed blue colouration of gastro-intestinal contents, three females showed blue colouration of rectal tissue, 5 females were recorded with blue coloration of stomach tissue and 17 females were confirmed as having blue colouration of the tail. The blue staining observed was considered discolouration caused by the test material itself (a blue dye) and is considered not to represent toxicity.

Body weight gain and food consumption during gestation were unaffected by treatment and there were no other maternal findings observed at macroscopic examination that were considered to relate to treatment.

There was no effect of treatment at 1000 mg/kg/day upon embryo-fetal survival or placental weights, and fetal development was also unaffected by treatment at this limit dose, with the incidence of major and minor abnormalities and skeletal variants showing no relationship to treatment.

Mean male, female and overall fetal weights at 1000 mg/kg/day were lower than Controls, but the difference was considered marginal (approx. 5-6%) and was clearly not adverse since embryo fetal survival and development were unaffected by treatment.

Based on the results of this embryo-fetal developmental toxicity study in rats, it was concluded that the No-Observed-Adverse- Effect-Level (NOAEL) of Macrolex Blau 3R for maternal toxicity and embryo-fetal development was the limit dose of 1000 mg/kg/day.