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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 4 December 2015 to 29 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to the OECD TG 414 (2001 version) without deviation.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
Principles of method if other than guideline:
One group of 22 females received Macrolex Blau 3R at the limit dose of 1000 mg/kg bw/day by daily oral gavage administration at a dose volume of 10 mL/kg bw/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 (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
1,4-bis[(2-ethyl-6-methylphenyl)amino]anthraquinone
EC Number:
255-460-7
EC Name:
1,4-bis[(2-ethyl-6-methylphenyl)amino]anthraquinone
Cas Number:
41611-76-1
Molecular formula:
C32H30N2O2
IUPAC Name:
1,4-bis[(2-ethyl-6-methylphenyl)amino]-9,10-dihydroanthracene-9,10-dione
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 %
Expiry date: 16 January 2017
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at ambient temperature in the dark
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The homogeneity and stability of formulations during storage were confirmed as part of a separate study (Sponsor Study No. 7L447). In that GLP-compliant study, the stability and homogeneity of the test material in the vehicle at concentrations of 0.4 and 100 mg/mL were confirmed as eight days under refrigerated storage conditions
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: n/a
- Reactivity of the test material with the incubation material used (e.g. plastic ware): n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): The required amount of test substance was ground in a mortar using a pestle and mixed with some vehicle to form apaste. Furtheramountsofvehicleweregraduallyadded and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogeniser.
- Preliminary purification step (if any): not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material) not applicable

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited
- Age at study initiation: approximately 70 days of age
- Weight at study initiation: 232 to 279 g
- Fasting period before study: none
- Housing: up to four animals during acclimatisation; individually during gestation. Solid (polycarbonate) bottom cages, containing softwood based bark-are fibre bedding which was changes at appropriate intervals each week.
- Diet (e.g. ad libitum): SDS VRF1 Certified pellet diet ad libitum. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): potable water from the public supply ad libitum.
- Acclimation period: 5 days before commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): n/a (filtered fresh air which was passed to atmosphere and not recirculated)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 9 December 2015 From: To: 8 January 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% CMC-Na solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The required amount of test substance was ground in a mortar using a pestle and mixed with some vehicle to form apaste. Furtheramountsofvehicleweregraduallyadded and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogeniser.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly

VEHICLE
- Justification for use and choice of vehicle (if other than water): the same vehicle was used for the 28-day study
- Concentration in vehicle: 100 mg/mL
- Amount of vehicle (if gavage): volume dose = 10 mL/kg bw
- Lot/batch no. (if required): n/a
- Purity: n/a
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The samples were analysed in accordance with the validated Envigo Analytical Procedure FIA/M118/15.
The analytical method involved dispersion in water (5 mL) followed by dissolution in acetonitrile. Extracted samples were diluted in acetonitrile/water 70/30 v/v followed by reverse phase high performance liquid chromatographic analysis with ultra violet detection at 254 nm. Sample concentrations were determined with reference to external standards prepared in the concentration range 1 |ig/mL to 5 µg/mL.
For Week 1 and the Last week oftreatment, freshly prepared test formulations were sampled (4 x 1 mL, 2 x 3 mL for controls, accurately weighed) by Pharmacy personnel and submitted for analysis. Duplicate samples were analysed in accordance with the analytical procedure, and the samples were retained for contingency. These were discarded following acceptable results.
The mean concentrations of Macrolex Blau 3R in test formulations analysed for the study were within 1% of nominal concentrations, confirming accurate formulation. The precision of individual results from the mean value was within 3%, confirming the precision of analysis. Procedural recovery results confirmed the continued accuracy of the method and were not used to correct the final reported values.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Verification of same strain and source of both sexes: yes, 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.
- Proof of pregnancy: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm referred to as day 0 of pregnancy. Only females showing at least two copulation plugs were allocated.
- Any other deviations from standard protocol:
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
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels for this main embryo-fetal study in the rat were based on the results of a preliminary embryo-fetal rat toxicity study performed in Sprague Dawley rats (Envigo Study No. TMR0140). In that study there were no toxicologically relevant observed effects at 1000 mg/kg bw/day upon the dams or on embryo-fetal survival or growth. Based upon this absence of effects in the preliminary study, and the known low solubility and very low bioavailability of the test item, it was considered that the limit test design was appropriate for this test item, investigating a single high dose level of 1000 mg/kg bw/day.
- Rationale for animal assignment (if not random): To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups.
Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.
- Fasting period before blood sampling for (rat) dam thyroid hormones: not applicable
- Time of day for (rat) dam blood sampling: not applicable
- Other: n/a

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: n Days 0, 5, 12, 18 and 20 after mating

BODY WEIGHT: Yes
- Time schedule for examinations: on Days 0, 3, and 6-20 after mating

FOOD CONSUMPTION : Yes
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 20 after mating
- Organs examined: All adult animals were subject to a detailed necropsy. Afterareviewofthehistoryofeach animal, a full macroscopic examination of the tissues was performed. All external features andorificeswereexaminedvisually. Anyabnormalityintheappearanceorsizeofanyorgan and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. Particular attention was paid to organ colour, taking into account that the test material is blue, with appropriate data recording.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (including cervix and ovaries):
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Blood sampling:
- Plasma: No
- Serum: No
Fetal examinations:
- External examinations: Yes: (all per litter)
- Soft tissue examinations: Yes: [ half per litter]
- Skeletal examinations: Yes: [ half per litter]
- Head examinations: Yes: [ half per litter]
- Anogenital distance of all live rodent pups: no
Statistics:
The following data types were analysed at each timepoint separately:
Body weight, using absolute values and gains over appropriate study periods Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
Litter size and survival indices
Fetal, placental and litter weight
(further details in the field "Any other information materials and methods incl. tables")
Indices:
Pre-implantation loss (%) = (Number of corpora lutea - Number of implantations) / Number of corpora lutea x 100.
Post-implantation loss (%) = (Number of implantations - Number of live fetuses) / Number of implantations x 100.
Historical control data:
Control Incidence Major / Minor Skeletal / Minor Visceral abnormalities - Crl:CD(SD)Rat - from 8 studies are included. (Cf. attachement to the ESR)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day a total of 17 females were recorded with the clinical sign of blue staining of the tail. This was considered to be related to the blue coloured test material being excreted in the faeces. Two animals were recorded with abnormally coloured staining of the head and muzzle, where a further qualifier of colour was omitted in error, however these were recorded on Day 0 after mating, and therefore could not have been related to the test material. In addition four treated females (numbers 31,32, 33 and 34) were recorded with abnormal colouration of the tail on Day 20 after mating; however, no further qualifier of colour was recorded in error. Three out of the four of these females were confirmed as having a blue tail at necropsy on Day 20 after mating.
There were no other consistent clinical signs, nor any dosing signs, observed that were considered to be related to treatment at the limit dose of 1000 mg/kg bw/day.
Mortality:
no mortality observed
Description (incidence):
There were no premature decedents.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean overall bodyweight gain during gestation was similar to controls and unaffected by treatment at 1000 mg/kg/day.
There were a number of incidences of slight but statistically significant differences in mean absolute body weight at 1000 mg/kg/day when compared to the Controls (approximately 3% on Days 6, 10, 11, 12, 16, 17, 18 of gestation), however, this was principally due to slightly lower gain experienced during Days 3 to 6 of gestation, prior to the commencement of treatment, such that mean bodyweight at the start of treatment was statistically significantly lower than Controls for females receiving 1000 mg/kg/day. After the commencement of treatment, mean body weight gain during Days 6-20 of gestation for treated females was not significantly different from Controls (Controls 113g; dose group 111g).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean overall values of food consumption during Days 6-19 of gestation for females receiving 1000 mg/kg bw/day were no different from Controls and were unaffected by treatment.
Mean food consumption for treated females immediately prior to the commencement of treatment (during Days 3-5 of gestation) was marginally lower than Controls, attaining statistical significance, however, subsequent food consumption for the remainder of gestation was unaffected by treatment.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Mean gravid uterine weight was similar to Controls and unaffected by treatment and when mean values of body weight and body weight gain were adjusted for the contribution of the gravid uterus, the maternal portion of mean body weight gain during Days 6-20 of gestation was similar to Controls and unaffected by treatment at 1000 mg/kg bw/day.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Several females receiving 1000 mg/kg bw/day showed blue colouration of gastro-intestinal content.
Three females at 1000 mg/kg bw/day showed blue colouration of the rectal tissue, 5 females showed blue coloration of the 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.
There were no other blue coloured organs observed in any female, and no further maternal findings observed at macroscopic examination that were considered to relate to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
One Control female was found to be non-pregnant. All treated females were pregnant with a live litter on Day 20 of gestation.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
Embryo-fetal survival was clearly unaffected by treatment at 1000 mg/kg bw/day with mean numbers of implantations, resorptions, live young and percentages of sex ratio and pre and post-implantation loss being similar to Control values.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
changes in number of pregnant
changes in pregnancy duration
clinical biochemistry
clinical signs
dead fetuses
early or late resorptions
effects on pregnancy duration
gross pathology
haematology
histopathology: non-neoplastic
maternal abnormalities
mortality
necropsy findings
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean male, female and overall fetal weights in litters of females receiving 1000 mg/kg bw/day were lower than Controls, and although statistical significance was attained, the difference was marginal (approximately 0.2g or 5-6% lighter than Controls) and considered not adverse since embryo-fetal survival and development were clearly unaffected by treatment.
Mean placental weights at 1000 mg/kg/day were similar to Controls and unaffected by treatment.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
Fetal development was clearly unaffected by treatment at the limit dose of 1000 mg/kg bwday.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants showed no relationship to treatment.
At 1000 mg/kg bw/day there was a slightly increased incidence of delayed/ incomplete ossification/unossified thoracic vertebrae compared to concurrent control, however, this was within the Historical Control range.
Visceral malformations:
no effects observed
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

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--Adverse-Effect-Level (NOAEL) of Macrolex Blau 3R for maternal toxicity was the limit dose of 1000 mg/kg bw/day.
For embryo-fetal development, the limit dose of 1000 mg/kg bw/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. The study was conducted according to the OECD TG 414 and in compliance with GLP.


One group of 22 females received Macrolex Blau 3R suspended in 0.5% CMC-Na solution at the limit dose of 1000 mg/kg bw/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, 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 bw/day was well tolerated and elicited no deaths and no toxicity related changes in clinical condition of the adult females.


At 1000 mg/kg bw/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 bw/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 bw/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 bw/day.