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EC number: 204-155-7 | CAS number: 116-75-6
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Toxicity to reproduction
Administrative data
- Endpoint:
- screening for reproductive / developmental toxicity
- Remarks:
- based on test type (migrated information)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: well performed GLP compliant OECD guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 012
- Report date:
- 2013
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
Test material
- Reference substance name:
- 1,4-bis(mesitylamino)anthraquinone
- EC Number:
- 204-155-7
- EC Name:
- 1,4-bis(mesitylamino)anthraquinone
- Cas Number:
- 116-75-6
- Molecular formula:
- C32H30N2O2
- IUPAC Name:
- 1,4-bis(mesitylamino)anthraquinone
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source:Charles River Italy S.p.A., Calco (Lecco), Italy
- Age at study initiation: 8-9 weeks
- Weight at study initiation: (P) Males: 317-322g; Females: 210-216 g;
- Housing:
From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulphone solid bottomed cages measuring approximately 59.5x38x20 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During the mating period, animals were housed on the basis of one male to one female in clear polycarbonate cages measuring approxiamtely 43x27x18 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
The males were re-caged after mating as they were before mating.
After mating, the females were transferred to individual clear polycarbonate solid bottomed cages measuring approxiamtely 43x27x18 cm
- Acclimation period: 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C):20-24
- Humidity (%):40-70
- Air changes (per hr):15-20
- Photoperiod (hrs dark / hrs light):12/12
IN-LIFE DATES: From: To:05 July 2012-05 September 2012
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- The required amount of the test item was suspended in the vehicle (corn oil), brought to the final volume appropriate for each concentration (concentrations of 20, 60 and 200 mg/mL) and kept under magnetic stirrer at room temperature prior to use and until the time of dosing of the last animal.
Concentrations were calculated and expressed in terms of test item as supplied. - Details on mating procedure:
- Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
The female was paired with the same male until positive identification occurred. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable (content check and homogeneity) and the stability of formulation was satisfactory. Results of the analyses were within the limits of acceptance.
The stability was found to be 24 hours at room temperature in the concentration range of 20 to 200 mg/mL.
Samples of the formulations prepared on Day 1 and on Week 4 of the study were also analysed to check the concentration and homogeneity. Results of the analyses were within the limits of acceptance.
Chemical analyses were carried out by the Analytical Chemistry Department at RTC, using a validated spectrophotometric detection in the range from 1.0 to 250 mg/mL.
The software used for this activity was Empower Probuild No. 2154. - Duration of treatment / exposure:
- Males were dosed for 32 days (including 2 weeks prior to pairing during the pre pairing period and approximately 2 weeks post pairing). Females were dosed 2 weeks before pairing, during the pairing period, gestation period and up to Day 3 post partum.
- Frequency of treatment:
- Males were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before necropsy.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Females were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum or the day before sacrifice. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant. - Details on study schedule:
- Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 32 days.
Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum or until the day before necropsy.
The following investigations were performed on parental animals of all groups: body weight, clinical signs, food consumption, oestrous cycle, mating performance, litter data, macroscopic observations, organ weights and histopathological examination of abnormalities. Histopathological examination of testes, epididymides and ovaries was performed only on control and high dose groups.
Doses / concentrations
- Remarks:
- Doses / Concentrations:
Doses: 0, 100, 300, 1000 mg/kg bw
Basis:
actual ingested
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- Dose selection rationale: Dose levels had been selected in consultation with the Sponsor based on information from a previous non GLP compliant dose range finding study.
- Positive control:
- no
Examinations
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule:Daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Daily
BODY WEIGHT: Yes
Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.
FOOD CONSUMPTION: Yes
Food consumption was recorded at weekly intervals whenever possible, for each cage of rats from allocation to pairing. For female animals, food consumption was also recorded on Days 7, 14 and 20 post coitum, starting from Day 0 post coitum and on Day 4 post partum (starting from Day 1 post partum). - Oestrous cyclicity (parental animals):
- Vaginal smears were taken daily in the morning from the first day of treatment and up to the end of the mating period, until a positive identification of mating is made. The vaginal smear data were examined to determine the following:
a) anomalies of the oestrous cycle;
b) 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
The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed in control and high dose males. - Litter observations:
- A parturition check was performed from Day 20 post coitum. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring are first detected in the cage was considered Day 0 post partum.
As soon as possible, after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
Live pups were individually weighed on Days 1 and 4 post partum.
Pups dying during the lactation period were weighed before the despatch to necropsy.
Observations were performed once daily for all litters. - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All animals after 32 days ofdosing
- Maternal animals: all live females with pups were killed on Day 4 post partum with pups; non rpegnant female after day 25 of post coitum
GROSS NECROPSY
The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.
Females:
All females were examined also for the following:
a) number of visible implantation sites;
b) number of corpora lutea (pregnant animals).
Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.
Organ weights
From all animals completing the scheduled test period, the organs indicated below were dissected free of fat and weighed. The ratios of organ weight to terminal body weight were calculated for each animal.
Tissues fixed and preserved
Samples of all the tissues listed in section 4.4.6 were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in modified Davidson's fluid) from all animals.
Histopathological examination and staging of spermatogenic cycle
The tissues required for histopathological examination are listed below. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre
thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous
epithelium (staging of spermatogenic cycle) was performed in control and high dose males.
In the first instance the examination was restricted as detailed below:
a) Tissues specified in section 4.4.6 from all animals in the control and high dose group killed at term.
b) Tissues specified in section 4.4.6 from all animals dying during the treatment period.
c) All abnormalities in all groups. - Postmortem examinations (offspring):
- All pups found dead in the cage were examined for external and internal abnormalities.
All live pups were killed on Day 4 post partum and examined for external abnormalities and sex confirmation by gonadal inspection. - Statistics:
- Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p<0.05. - Reproductive indices:
- Group mean values were calculated for all parameters. Data from females not pregnant were excluded from group mean calculations as considered appropriate by the Study Director.
The following reproductive indices were calculated:
Males
Copulatory Index (%) = (no. of animals mated x 100) / (no. of animals paired)
Fertility Index (%) = (no. of males which induced pregnancy x 100) / (no. of males paired)
Females
Copulatory Index (%) = (no. of animals mated x 100) / (no. of animals paired)
Fertility Index (%) = (no. of pregnant females x 100) / (no. of females paired)
Males and females
Copulatory Interval = Mean number of days between pairing and mating
Females
Pre-birth loss was calculated as a percentage from the formula:
(((No. of visible implantations) - (total litter size at birth)) x 100) / (No. of visible implantations)
Pup loss at birth was calculated as a percentage from the formula:
(((Total litter size) - (live litter size)) x 100) / (Total litter size)
Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:
(((Total litter size at birth) - (live litter size at Day 4)) x 100) / (Total litter size at birth)
Sex ratios were calculated at birth and on Day 4 post partum and presented as the percentage of males per litter. - Offspring viability indices:
- Pup loss at birth
Cumulative pup loss on Day 4 post partum
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
- Other effects:
- not examined
Reproductive function / performance (P0)
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
Details on results (P0)
One control female and one low dose female were found dead. On the basis of the macroscopic and microscopic observations, the cause of deaths is attributable to a mis-dosing.
One low dose female was not pregnant and one mid-dose female showed unilateral implantation. The number of females with live pups on Day 4 post partum was 9 in the control group, 8 in the low dose group and 10 in each of the mid- and high dose groups.
Clinical signs:
As consequence of the colour of the test item blue staining of the bedding material and tail was noted in the animals of mid- and high dose groups.
Body weight and body weight gain:
No signs of toxicological significance were seen in body weight or body weight gain during the study in treated animals compared to controls.
Food consumption:
No effects on food consumption were seen.
Oestrus cycle, mating performance and reproductive parameters:
No treatment-related anomalies were noted in the oestrus cycle of treated females when compared to controls. Pre-coital interval, copulation plugs, copulatory and fertility index were similar between treated and control animals.
Implantation, pre-birth loss data and gestation length:
No differences were observed in treated and control groups for these parameters.
Litter data and sex ratio:
Litter data and sex ratios at birth and on Day 4 post partum were unaffected by treatment.
Clinical signs of pups:
Pre-weaning clinical signs did not show treatment-related effects.
Necropsy findings in pups:
Necropsy findings in decedent pups and pups sacrificed on Day 4 post partum did not reveal any treatment-related effect.
Terminal body weight and organ weights:
Terminal body weight and organ weights were unaffected by treatment in both sexes.
Macroscopic and microscopic observations including examination of spermatogenic cycle:
The lesions detected in some animals were also noted in untreated laboratory rats; they were therefore considered to be incidental and/or spontaneous in origin.
No treatment-related changes were seen in testis, epididymides (including spermatogenic cycle) and ovaries evaluated in high dose males or females receiving the test substance.
Conclusions:
On the basis of the above mentioned results, the dosage of 1000 mg/kg/day of the test item was established as the NOAEL (No Observed Adverse Effect Level) for animals of both sexes.
Effect levels (P0)
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects up to the highest dose tested
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:
- NOAEL
- Generation:
- F1
- Effect level:
- 1 000 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects up to the highest dose tested
Overall reproductive toxicity
- Reproductive effects observed:
- not specified
Applicant's summary and conclusion
- Conclusions:
- Clinical signs, body weight and food consumption were unaffected by treatment in both sexes.
Reproductive parameters such as fertility index, pre-coital interval and copulatory index did not show significant differences in treated groups compared to controls.
Gestation length, litter data, sex ratios and implantation losses (pre and post) were unaffected by treatment.
At macroscopic and microscopic examination, no treatment related lesions were seen.
On the basis of the results obtained in this OECD Reproduction / Developmental Toxicity Screening Test, the maximum dosage of 1000 mg/kg/day of the test item was established as the NOAEL (No Observed Adverse Effect Level) for parental animals of both sexes (general toxicity and reproduction) and for the offspring (developmental toxicitxy).
- Executive summary:
Study design
The toxic effects on rats after repeated dosing, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition, development of the concepts and early lactation were investigated.
The dosage groups were as follows:
Group Number
Treatment (mg/kg/day)
Number of animals
1
0
10M+10F
2
100
10M+10F
3
300
10M+10F
4
1000
10M+10F
M = Males; F = Females
The test item was administered by oral gavage at a dose volume of 5 mL/kg.
Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 32 days. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum or until the day before necropsy. The following investigations were performed on parental animals of all groups: body weight, clinical signs, food consumption, oestrous cycle, mating performance, litter data, macroscopic observations, organ weights and histopathological examination of abnormalities. Histopathological examination of testes, epididymides and ovaries was performed only on control and high dose groups.
Mortality and fate of females
One control female and one low dose female were found dead. On the basis of the macroscopic and microscopic observations, the cause of deaths is attributable to a misdosing. One low dose female was not pregnant and one mid-dose female showed unilateral implantation. The number of females with live pups on Day 4 post partum was 9 in the control group, 8 in the low dose group and 10 in each of the mid- and high dose groups.
Clinical signs
As consequence of the colour of the test item blue staining of the bedding material and tail was noted in the animals of mid- and high dose groups.
Body weight and body weight gain
No signs of toxicological significance were seen in body weight or body weight gain during the study in treated animals compared to controls.
Food consumption
No effects on food consumption were seen.
Oestrus cycle, mating performance and reproductive parameters
No treatment-related anomalies were noted in the oestrus cycle of treated females when compared to controls. Pre-coital interval, copulation plugs, copulatory and fertility index were similar between treated and control animals.
Implantation, pre-birth loss data and gestation length
No differences were observed in treated and control groups for these parameters.
Litter data and sex ratio
Litter data and sex ratios at birth and on Day 4 post partum were unaffected by treatment.
Clinical signs of pups
Pre-weaning clinical signs did not show treatment-related effects.
Necropsy findings in pups
Necropsy findings in decedent pups and pups sacrificed on Day 4 post partum did not reveal any treatment-related effect.
Terminal body weight and organ weights
Terminal body weight and organ weights were unaffected by treatment in both sexes.
Macroscopic and microscopic observations including examination of spermatogenic cycle
The lesions detected in some animals were also noted in untreated laboratory rats; they were therefore considered to be incidental and/or spontaneous in origin. No treatment-related changes were seen in testis, epididymides (including spermatogenic cycle) and ovaries evaluated in high dose males or females receiving the test item.
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