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EC number: 279-365-5 | CAS number: 80010-51-1
- 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
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
It was concluded that in the absence of any evidence for general systemic toxicity or effects on reproductive performance that the no observed adverse effect level was 1000 mg/kg/day.
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 01 April 2016 to 16 Jan 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- 27 July 1995
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Commission Regulation (EC) No 440/2008
- Version / remarks:
- 30 May 2008 pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Justification for study design:
- SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:
- Premating exposure duration for parental (P0) animals: 15 days
- Basis for dose level selection: Dose levels were selected in collaboration with the sponsor based an available toxicological data, including a Rat Oral Gavage Fourteen Day Range Finding Toxicity Study performed at this Test Facility (Study Number WP89QB), where a dosage of 1000 mg/kg bw/day AI was considered to be well tolerated. Dosages of 0 (Control), 100, 300 and 1000 mg/kg bw/day AI were therefore selected for use on this study.
- Inclusion/exclusion of extension of Cohort 1B: 14 Days
- Route of administration: Oral Gavage - Specific details on test material used for the study:
- Identification: FAT 21036/G TE
Physical State/Appearance: Blue solid
Chemical Name: Sodium 1-amino-4-[[(3,5-bis[[(chloroacetyl)amino]nethyl]-2,4,6-trimethylphenyl]amino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate
Purity: 63.5 %
Batch Number: AT-0033945200
Date Received: 14 March 2016
Storage Conditions: Ambient temperature in the dark
Expiry Date: 13 April 2020 - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Han™:RccHan™:WIST strain
- Details on species / strain selection:
- The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: (P) 12 wks;
- Weight at study initiation: (P) Males: 317-357 g; Females:190-231 g
- Fasting period before study: none
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ”C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): >15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 02 June 2016 To: 25 July 2016 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled water. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services as part of this study. Results show the formulations to be stable for at least eleven days when stored at 4 °C in the dark. Formulations were therefore prepared on a weekly basis and stored at approximately 4 °C in the dark.
GROUP Adjusted for Purity Test Item as supplied
Dose level Concentration Treatment vol Concentration (mg/ml)
(mg/kg bw/day) (mg/ml) (ml/kg)
Control 0* 0* 20 0*
Low 100 5 20 7.87
Imtermediate 300 15 20 23.6
HIgh 1000 50 20 78.7
* Control animals were treated with vehicle (distilled water) alone. - Details on mating procedure:
- - M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to asday 0 of pregnancy.
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: none - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLCUV) using an external standard technique. The test item gave a chromatographic profile consisting of a profile of multiple peaks.
HPLC conditions:
HPLC: Aglient Technologies 1200
Column: Gemini 3µ C18 (100 x 4.6mm id) at 30 ”C
Mobile Phase: Eluent A : water/TBAB solution (90:10 v/v)
Eluent B: acetonitrile/TBAB solution (90:10 v/v)
TBAB solution: 5g Tetrabutylammonium bromide in 250 mL acetonitirile
Gradient: Time % B
0 0
1 0
6 100
11 100
11.5 0
15 0
Flow rate: 1 mL/min
UV: 420 nm
Injection volume: 25 μL
Retention time: 5.5 mins
Linearity
Calibration data was found to be linear over the range 0.00508 to 0.1524 mg/mL
Method accuracy and precison
Mean recovery (accuracy) and precision at 2 mg/mL = 99 % (CV=1.23 %; n=5)
Mean recovery (accuracy) and precision at 320 mg/mL = 102 % (CV=0.348 %; n=5)
LOQ (defined aslowest standard concentration) = 0.00508 mg/mL
Test item analysis
The mean concnetrations fo test item in the test formulations were within ±10 % nominal concentrations - Duration of treatment / exposure:
- 6 weeks
- Frequency of treatment:
- Daily
To reduce the viscosity of the dosing formulations a dosage volume of 20 mL/kg bw was utilized for dosing in this study. In order to reduce the potential impact of this increase in dosage volume on the animals, dosing on this study on each day was spilt over two occasions approximately four hours apart. - Details on study schedule:
- Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated twice daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iii. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
iv. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
v. The male dose groups were killed and examined macroscopically on Day 43.
vi. At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically. - Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Control group
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Remarks:
- Low dose group
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Remarks:
- Mid dose group
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Remarks:
- High dose group
- No. of animals per sex per dose:
- 12
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Guideline requirement
- Rationale for animal assignment: The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories. - Positive control:
- Not applicable
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
Individual clinical observations were performed immediately before dosing, up to thirty minutes post dosing and one hour after each dosing occasion. All observations were recorded. Additional observations may be included at the discretion of the Study Director or for the monitoring of animal health.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.
FOOD CONSUMPTION AND COMPOUND INTAKE
During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1-4). Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated during gestation and lactation.
WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes - Litter observations:
- Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)
Physical Development: All live offspring were assessed for surface righting reflex on Day 1 post partum. - Postmortem examinations (parental animals):
- Terminal Investigations
Necropsy
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Organ Weights
The epididymides and testes were removed from terminal kill adult males dissected free from fat and weighed before fixation.
Histopathology
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:
Coagulating gland Prostate
Epididymides Seminal vesicles
Ovaries Testes
Mammary gland (females only) Uterus/Cervix
Pituitary Vagina
The tissues from control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell or stage-specificity of testicular findings was noted.
Since there were indications of treatment-related changes, examination were not extended to include animals in the low and intermediate groups. - Statistics:
- Statistical Analysis
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Absolute Organ Weights, Body Weight-Relative Organ Weights
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). - Reproductive indices:
- The following parameters were calculated from the individual data during the mating period of the parental generation:
i. Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii. Fertility Indices
For each group the following were calculated:
Mating Index (%) = Number mater/number paired x 100
Pregnancy Index (%) = Number of pregnant females/number mated x 100
Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = No.females delivering live offspring/no. pregnant females x 100 - Offspring viability indices:
- Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter
ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = No. offspring alive on Day 1/No. offspring born x 100
Viability Index (%) = No. offspring alive on Day 4/No. offspring alive on Day 1 x 100
iii. Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum - Clinical signs:
- no effects observed
- Description (incidence and severity):
- There were no findings observed that indicated any adverse systemic toxicity of the test item at 100, 300 and 1000 mg/kg bw/day.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- There were two unscheduled deaths on the study, both occurring at 300 mg/kg bw/day but these deaths were considered to represent intubation error and were unrelated to treatment. Female number 65 was found dead on Day 6. Clinical signs prior to this event had been restricted to staining of the fur by the test item. At necropsy examination, the lungs were observed to be filled with a blue colored liquid and, in view of this finding, the cause of death is considered to reflect an intubation error during administration of the dosing formulation. Other necropsy findings were mainly restricted to blue discoloration of various tissues of the body (including the heart, kidneys, liver, lymph nodes, mammary gland, muscle, thymus, urinary bladder and the tissues and contents of the stomach and gastrointestinal tract).
Male number 55 was found dead on Day 18. Clinical signs prior to this event had been restricted to staining of the fur by the test item. At necropsy examination, the lungs were observed to be filled with a blue coloured liquid and, in view of this finding, the cause of death is considered to reflect an intubation error during administration of the dosing formulation. Other necropsy findings were mainly restricted to blue discoloration of various tissues of the body, including the adrenals, heart, bone marrow, kidneys, liver, lymph nodes, mammary gland, muscle, oesophagus, pancreas, seminal vesicles, thymus, urinary bladder and the tissues and contents of the stomach and gastrointestinal tract. - Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- For males at 1000 mg/kg bw/day, there was a suggestion of slightly lower mean body weight gain during the first week of treatment, compared to control. However, there was no statistical significance and, while three males showed mean body weight loss during this period, body weight gains for the remaining animals were within the concurrent control range. Subsequent mean body weight gains of males (with the exception of Days 29 to 36) were generally similar to, but slightly lower than, control, with differences failing to attain statistical significance. Overall mean body weight gain at the end of the study was also slightly lower than control but again there was no statistical significance. There was no obvious effect of treatment on body weight and body weight gain of males at 100 or 300 mg/kg bw/day. At all dosages higher mean body weight gain for males during Week 5 (Days 29 to 36) attained statistical significance when compared with control. However there was no dosage relationship and this isolated increase was considered to be incidental and unrelated to treatment. There was no obvious effect of treatment on body weight and body weight gain of females during the two week pre-pairing, gestation and early lactation phases of the study at 100, 300 or 1000 mg/kg bw/day.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- There was no obvious effect of treatment on food consumption for males during the prepairing and post-pairing phases of the study or for females during the pre-pairing, gestation and early lactation phases of the study at 100, 300 or 1000 mg/kg bw/day. There was no obvious consistent effect of treatment on food conversion efficiency although values for males at 1000 mg/kg bw/day were slightly lower than control during the first week of treatment.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- Visual inspection of water consumption throughout the study did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry 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
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Several animals were not pregnant following mating. These occurred across all Groups including controls and no findings were noted in these animals or their corresponding male partners to account for the lack of pregnancy. Animal 38 (Group 2 female) appeared to have been pregnant and was lactating. There was evidence of uterine infection but no indication of an effect due to the administration of the test item. There were no treatment-related pathological findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no treatment-related related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries. Female 65 (Group 3) was found dead early in the study. There were no findings at histopathology to account for death. Male 55 (Group 3) was also found dead without significant histopathological change.
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Reproductive function: oestrous cycle:
- not examined
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- Mating
There was no obvious effect of treatment on mating, as assessed by pre-coital interval, at 100, 300 or 1000 mg/kg bw/day. At 100 mg/kg bw/day one male/female pairing failed to mate, but this isolated occurrence, in the absence of any similar occurrence at 1000 mg/kg bw/day, was considered to be incidental and unrelated to treatment.
Fertility
There was no obvious effect of treatment on fertility, as assessed by pregnancy rate, at 100, 300 or 1000 mg/kg bw/day.
A total of 2, 1, 0 and 1 females at 0 (control), 100, 300 or 1000 mg/kg bw/day failed to achieve pregnancy. The incidence of non-pregnancy showed no dosage relationship and was considered to be unrelated to treatment.
Gestation Length
The intergroup distribution of gestation lengths did not indicate any obvious effect of treatment at 100, 300 or 1000 mg/kg bw/day.
Litter responses
At 1000 mg/kg bw/day, one female achieved pregnancy but was not observed to give birth to a litter; therefore this female either represents a total litter loss in-utero or a total litter loss post partum shortly after parturition. The female only had three implantations and it is not unusual for such small litters not to be maintained to term; in isolation this finding is considered to be unrelated to maternal treatment.
For one female at 100 mg/kg bw/day, no implantations were apparent at macroscopic necropsy but microscopic evaluation of the uterus indicated that the female had been pregnant. Again this female either represents a total litter loss in-utero or a total litter loss post partum shortly after parturition. this isolated occurrence was considered to be unrelated to treatment.
Additionally, as previously indicated, a total of 2, 1, 0 and 1 mated females failed to achieve pregnancy in the 0 (control), 100, 300 and 1000 mg/kg bw/day groups respectively. The following assessment is based on the 10, 9, 11 and 10 females that successfully maintained a litter to Day 4 of lactation.
Offspring Litter Size, Sex Ratio and Viability
There was considered to be no effect of treatment on the corpora lutea count, preimplantation loss, numbers of implantations, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to Day 4 of age at 100, 300 or 1000 mg/kg bw/day. Sex ratio for the offspring was similar in all groups and did not indicate any selective effect of maternal treatment on survival for either sex. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- other: No adverse effects seen
- Critical effects observed:
- no
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- The low incidence of clinical sign observed for the offspring did not indicate any obvious underlying effect on offspring growth and development at any of the dosages investigated.
- Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There was considered to be no obvious effect of treatment on offspring body weight on Day 1 and subsequent body weight gain to Day 4 at 100, 300 or 1000 mg/kg bw/day.
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- not examined
- Histopathological findings:
- not examined
- Other effects:
- no effects observed
- Description (incidence and severity):
- There was no obvious effect of maternal treatment on offspring surface righting performance at 100, 300 or 1000 mg/kg bw/day.
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- other: No adverse effects seen
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- no
- Lowest effective dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Treatment related:
- no
- Conclusions:
- The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity, including the survival, growth and development of the offspring, was considered to be 1000 mg/kg bw/day.
- Executive summary:
A study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 "Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).
This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
Method: The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day (incorporating a correction factor for 63.5% purity). A control group of twelve males and twelve females was dosed with vehicle alone (Distilled water) over the same treatment period. Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Surviving adult males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.
Results
Adult Responses
Mortality
There were two unscheduled deaths on the study, both occurring at 300 mg/kg bw/day but these deaths were considered to represent intubation error and were unrelated to treatment.
Clinical Observations
There were no findings observed that indicated any adverse systemic toxicity of the test item at 100, 300 and 1000 mg/kg bw/day. Fur staining by the Test Item was apparent at 1000 mg/kg bw/day and to a lesser extent at 300 mg/kg bw/day. One female at 100 mg/kg bw/day also showed similar fur staining on Day 6 of the study. Staining of the fur was not unexpected given the colored nature of the test item.
Body Weight
There was no obvious adverse effect of treatment on body weight or body weight gain for males during the pre-pairing and post-pairing phases of the study or for females during the pre-pairing, gestation and early lactation phases of the study at 100, 300 or 1000 mg/kg bw/day.
Food Consumption
There was no obvious effect of treatment on food consumption for males during the prepairing and post-pairing phases of the study or for females during the pre-pairing, gestation and early lactation phases of the study at 100, 300 or 1000 mg/kg bw/day.
There was no obvious adverse effect of treatment on food conversion efficiency for either sex during the pre-pairing phase of the study or for males during post-pairing study phase at 100, 300 or 1000 mg/kg bw/day.
Water Consumption
Visual inspection of water consumption throughout the study did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.
Reproductive Performance
Mating: There was no obvious effect of treatment on mating, as assessed by pre-coital interval, at 100, 300 or 1000 mg/kg bw/day.
Fertility: There was no obvious effect of treatment on fertility, as assessed by pregnancy rate, at 100, 300 or 1000 mg/kg bw/day.
Gestation Length: The intergroup distribution of gestation lengths did not indicate any obvious effect of treatment at 100, 300 or 1000 mg/kg bw/day.
Litter Responses
Offspring Litter Size, Sex Ratio and Viability: There was considered to be no effect of maternal treatment on the corpora lutea count, preimplantation loss, numbers of implantations, post-implantation loss, litter size and sex ratio at birth/Day 1 and Day 4 and offspring survival to Day 4 of age at 100, 300 or 1000 mg/kg bw/day.
Offspring Growth and Development
There was considered to be no obvious effect of maternal treatment on offspring body weight on Day 1 and subsequent body weight gain to Day 4 of age at 100, 300 or 1000 mg/kg bw/day. There was no obvious effect of maternal treatment on offspring surface righting performance at 100, 300 or 1000 mg/kg bw/day.
Offspring Observations
The low incidence of clinical sign observed for the offspring did not indicate any obvious underlying effect on offspring growth and development at any of the dosages investigated.
Necropsy
Offspring :Necropsy findings did not indicate any obvious effect of maternal treatment at dosages of 100, 300 or 1000 mg/kg bw/day.
Adults: Macroscopic necropsy revealed blue coloration or blue contents for various regions of the gastrointestinal tract for both sexes at 1000 mg/kg bw/day. A lower incidence of similar findings were apparent for both sexes at 100 and 300 mg/kg bw/day. This finding was consistent with the colored nature of the test item.
Organ Weights: There was no obvious effect of treatment on absolute or body weight relative testes or epididymides weights at 100, 300 or 1000 mg/kg bw/day.
Histopathology: Microscopic evaluation of male reproductive tissues at 1000 mg/kg bw/day did not indicate any obvious effect of treatment.
Conclusion
Based on the results of this study, the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity, including the survival, growth and development of the offspring, was considered to be 1000 mg/kg bw/day.
Reference
Analytical investigation: The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision. The homogeneity and stability was confirmed for test item in water formulations at nominal concentrations of 7.87 mg/mL and 78.7 mg/mL when stored refrigerated for 11 days. The mean concentratio of test item waas within 10% of nominal concentratios with the exception of analysis 2, comfirming accurate formulation.
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 1 000 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- High quality GLP-study
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Effects on developmental toxicity
Description of key information
It was concluded that in the absence of any evidence for general systemic toxicity or effects on reproductive performance/offspring development that the no observed adverse effect level was 1000 mg/kg/day.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 01 April 2016 to 16 Jan 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- Identification: FAT 21036/G TE
Physical State/Appearance: Blue solid
Chemical Name: Sodium 1-amino-4-[[(3,5-bis[[(chloroacetyl)amino]nethyl]-2,4,6-trimethylphenyl]amino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate
Purity: 63.5%
Batch Number: AT-0033945200
Date Received: 14 March 2016
Storage Conditions: Ambient temperature in the dark
Expiry Date: 13 April 2020 - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Han™:RccHan™:WIST strain
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: (P) 12 wks;
- Weight at study initiation: (P) Males: 317-357 g; Females:190-231 g
- Fasting period before study: none
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing
phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3 ”C
- Humidity (%): 50±20 %
- Air changes (per hr): >15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 02 June 2016 To: 25 July 2016 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Distilled water. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services as part of this study. Results show the formulations to be stable for at least eleven days when stored at 4 °C in the dark. Formulations were therefore prepared on a weekly basis and stored at approximately 4 °C in the dark.
GROUP Adjusted for Purity Test Item as supplied
Dose level Concentration Treatment vol Concentration (mg/ml)
(mg/kg bw/day) (mg/ml) (ml/kg)
Control 0* 0* 20 0*
Low 100 5 20 7.87
Imtermediate 300 15 20 23.6
HIgh 1000 50 20 78.7
* Control animals were treated with vehicle (distilled water) alone. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLCUV) using an external standard technique. The test item gave a chromat
ographic profile consisting of a profile of multiple peaks.
HPLC conditions:
HPLC: Aglient Technologies 1200
Column: Gemini 3μ C18 (100 x 4.6mm id) at 30”C
Mobile Phase: Eluent A : water/TBAB solution (90:10 v/v)
Eluent B: acetonitrile/TBAB solution (90:10 v/v)
TBAB solution: 5g Tetrabutylammonium bromide in 250 mL acetonitirile
Gradient: Time %B
0 0
1 0
6 100
11 100
11.5 0
15 0
Flow rate: 1 mL/min
UV: 420 nm
Injection volume: 25 μL
Retention time: 5.5 mins
Linearity
Calibration data was found to be linear over the range 0.00508 to 0.1524 mg/mL
Method accuracy and precison
Mean recovery (accuracy) and precision at 2 mg/mL = 99 % (CV=1.23 %; n=5)
Mean recovery (accuracy) and precision at 320 mg/mL = 102 % (CV=0.348 %; n=5)
LOQ (defined aslowest standard concentration) = 0.00508 mg/mL
Test item analysis
The mean concnetrations fo test item in the test formulations were within ±10%nominal concentrations - Details on mating procedure:
- - M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear] referred to as [day 0 of pregnancy]
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: none - Duration of treatment / exposure:
- 6 weeks
- Frequency of treatment:
- daily
To reduce the viscosity of the dosing formulations a dosage volume of 20 mL/kg bw was utilized for dosing in this study. In order to reduce the potential impact of this increase in dosage volume on the animals, dosing on this study on each day was spilt over two occasions approximately four hours apart. - Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Control group
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Remarks:
- Low dose group
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Remarks:
- Mid dose group
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Remarks:
- High dose group
- No. of animals per sex per dose:
- 12
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Guideline requirement
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were
then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories. - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
Individual clinical observations were performed immediately before dosing, up to thirty minutes post dosing and one hour after each dosing occasion. All observations were recorded. Additional observations may be included at the discretion of the Study Director or for the monitoring of animal health.
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During the pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.
FOOD CONSUMPTION AND COMPOUND INTAKE
During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1-4). Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated during gestation and lactation.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes
OTHER:
Estrous cyclicity (parental animals): No details
Postmortem examinations:
Terminal Investigations
Necropsy
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum. For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5 % ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted. All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Organ Weights
The epididymides and testes were removed from terminal kill adult males dissected free from fat and weighed before fixation.
Histopathology
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where stated:
Ovaries
Mammary gland
Uterus/Cervix
Pituitary
Vagina
The tissues from control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined. Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell or stage-specificity of testicular findings was noted. Since there were indications of treatment-related changes, examination were not extended to include animals in the low and intermediate groups. - Fetal examinations:
- Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded.
Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)
Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum. - Statistics:
- Statistical Analysis
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Absolute Organ Weights, Body Weight-Relative Organ Weights
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). - Indices:
- The following parameters were calculated from the individual data during the mating period of the parental generation:
i. Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii. Fertility Indices
For each group the following were calculated:
Mating Index (%) = Number mater/number paired x 100
Pregnancy Index (%) = Number of pregnant females/number mated x 100
Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii. Parturition Index
The following was calculated for each group:
Parturition Index (%) = No.females delivering live offspring/no. pregnant females x 100 - Clinical signs:
- no effects observed
- Description (incidence and severity):
- There were no findings observed that indicated any adverse systemic toxicity of the test item at 100, 300 and 1000 mg/kg bw/day.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- There were two unscheduled deaths on the study, both occurring at 300 mg/kg bw/day but these deaths were considered to represent intubation error and were unrelated to treatment. Female number 65 was found dead on Day 6. Clinical signs prior to this event had been restricted to staining of the fur by the test item. At necropsy examination, the lungs were observed to be filled with a blue colored liquid and, in view of this finding, the cause of death is considered to reflect an intubation error during administration of the dosing formulation. Other necropsy findings were mainly restricted to blue discoloration of various tissues of the body (including the heart, kidneys, liver, lymph nodes, mammary gland, muscle, thymus, urinary bladder and the tissues and contents of the stomach and gastrointestinal tract). Male number 55 was found dead on Day 18. Clinical signs prior to this event had been restricted to staining of the fur by the test item. At necropsy examination, the lungs were observed to be filled with a blue coloured liquid and, in view of this finding, the cause of death is considered to reflect an intubation error during administration of the dosing formulation. Other necropsy findings were mainly restricted to blue discoloration of various tissues of the body, including the adrenals, heart, bone marrow, kidneys, liver, lymph nodes, mammary gland, muscle, oesophagus, pancreas, seminal vesicles, thymus, urinary bladder and the tissues and contents of the stomach and gastrointestinal tract.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- For males at 1000 mg/kg bw/day, there was a suggestion of slightly lower mean body weight gain during the first week of treatment, compared to control. However, there was no statistical significance and, while three males showed mean body weight loss during this period, body weight gains for the remaining animals were within the concurrent control range. Subsequent mean body weight gains of males (with the exception of Days 29 to 36) were generally similar to, but slightly lower than, control, with differences failing to attain statistical significance. Overall mean body weight gain at the end of the study was also slightly lower than control but again there was no statistical significance. There was no obvious effect of treatment on body weight and body weight gain of males at 100 or 300 mg/kg bw/day. At all dosages higher mean body weight gain for males during Week 5 (Days 29 to 36) attained statistical significance when compared with control. However there was no dosage relationship and this isolated increase was considered to be incidental and unrelated to treatment.
There was no obvious effect of treatment on body weight and body weight gain of females during the two week pre-pairing, gestation and early lactation phases of the study at 100, 300 or 1000 mg/kg bw/day. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- There was no obvious effect of treatment on food consumption for males during the prepairing and postpairing phases of the study or for females during the pre-pairing, gestation and early lactation phases of the study at 100, 300 or 1000 mg/kg bw/day. There was no obvious consistent effect of treatment on food conversion efficiency although values for males at 1000 mg/kg bw/day were slightly lower than control during the first week of treatment.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- Visual inspection of water consumption throughout the study did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry 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):
- There was no obvious effect on absolute and body weight-relative testes and epididymidal weights at 100, 300 or 1000 mg/kg bw/day.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- At 1000 mg/kg bw/day, females showed blue coloured contents and/or blue coloration of one of more of the following: colon, caecum, stomach, duodenum, jejunum, ileum and rectum. For female number 94, the right horn of the uterus was observed to be filled with a thick yellow liquid; this female was pregnant but was not observed to give birth to a litter. At 100 and 300 mg/kg bw/day, females showed similar blue coloured contents and/or blue coloration as observed at the high dosage but generally at a lower incidence for one of more of the following: colon, caecum, stomach, duodenum, jejunum and ileum. Control female 16 showed a fluid filled left ovary and the uterus and cervix was also filled with a yellow fluid. This female failed to achieve pregnancy.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Several animals were not pregnant following mating. These occurred across all Groups including controls and no findings were noted in these animals or their corresponding male partners to account for the lack of pregnancy. Animal 38 (Group 2 female) appeared to have been pregnant and was lactating. There was evidence of uterine infection but no indication of an effect due to the administration of the test item. There were no treatment-related pathological findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no treatment-related related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries. Female 65 (Group 3) was found dead early in the study. There were no findings at histopathology to account for death. Male 55 (Group 3) was also found dead without significant histopathological change.
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Number of abortions:
- no effects observed
- Pre- and post-implantation loss:
- effects observed, non-treatment-related
- Description (incidence and severity):
- At 1000 mg/kg bw/day, one female achieved pregnancy but was not observed to give birth to a litter; therefore this female either represents a total litter loss in-utero or a total litter loss post partum shortly after parturition. The female only had three implantations and it is not unusual for such small litters not to be maintained to term; in isolation this finding is considered to be unrelated to maternal treatment. For one female at 100 mg/kg bw/day, no implantations were apparent at macroscopic necropsy but microscopic evaluation of the uterus indicated that the female had been pregnant. Again this female either represents a total litter loss in-utero or a total litter loss post partum shortly after parturition. this isolated occurrence was considered to be unrelated to treatment.
- 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
- Description (incidence and severity):
- There was considered to be no effect of treatment on the corpora lutea count, preimplantation loss, numbers of implantations, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to Day 4 of age at 100, 300 or 1000 mg/kg bw/day. Sex ratio for the offspring was similar in all groups and did not indicate any selective effect of maternal treatment on survival for either sex.
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Remarks on result:
- other: No adverse efefcts seen
- Fetal body weight changes:
- no effects observed
- 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
- Changes in postnatal survival:
- no effects observed
- External malformations:
- no effects observed
- Skeletal malformations:
- no effects observed
- Visceral malformations:
- no effects observed
- Other effects:
- no effects observed
- Description (incidence and severity):
- There was no obvious effect of maternal treatment on offspring surface righting performance at 100, 300 or 1000 mg/kg bw/day.
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- other: No adverse effects seen
- Abnormalities:
- no effects observed
- Key result
- Developmental effects observed:
- no
- Conclusions:
- The ‘No Observed Effect Level’ (NOEL) for the survival, growth and development of the offspring, was considered to be 1000 mg/kg bw/day.
- Executive summary:
The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, and provides an initial hazard assessment for effect on reproduction. The study iscompatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995). This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
Methods
The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day (incorporating a correction factor for 63.5% purity). A control group of twelve males and twelve females was dosed with vehicle alone (Distilled water) over the same treatment period. Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Surviving adult males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.
Results
Adult Responses
Mortality: There were two unscheduled deaths on the study, both occurring at 300 mg/kg bw/day but these deaths were considered to represent intubation error and were unrelated to treatment.
Clinical Observations
There were no findings observed that indicated any adverse systemic toxicity of the test item at 100, 300 and 1000 mg/kg bw/day. Fur staining by the Test Item was apparent at 1000 mg/kg bw/day and to a lesser extent at 300 mg/kg bw/day. One female at 100 mg/kg bw/day also showed similar fur staining on Day 6 of the study. Staining of the fur was not unexpected given the colored nature of the test item.
Body Weight
There was no obvious adverse effect of treatment on body weight or body weight gain for females during the pre-pairing, gestation and early lactation phases of the study at 100, 300 or 1000 mg/kg bw/day.
Food Consumption
There was no obvious effect of treatment on food consumption for females during the pre-pairing, gestation and early lactation phases of the study at 100, 300 or 1000 mg/kg bw/day. There was no obvious adverse effect of treatment on food conversion efficiency for either sex during the prepairing phase of the study or for males during post-pairing study phase at 100, 300 or 1000 mg/kg bw/day.
Water Consumption
Visual inspection of water consumption throughout the study did not indicate any obvious effect of treatment for either sex at 100, 300 or 1000 mg/kg bw/day.
Reproductive Performance
Mating
There was no obvious effect of treatment on mating, as assessed by pre-coital interval, at 100, 300 or 1000 mg/kg bw/day.
Fertility
There was no obvious effect of treatment on fertility, as assessed by pregnancy rate, at 100, 300 or 1000 mg/kg bw/day.
Gestation Length
The intergroup distribution of gestation lengths did not indicate any obvious effect of treatment at 100, 300 or 1000 mg/kg bw/day.
Litter Responses
Offspring Litter Size, Sex Ratio and Viability
There was considered to be no effect of maternal treatment on the corpora lutea count, preimplantation loss, numbers of implantations, post-implantation loss, litter size and sex ratio at birth/Day 1 and Day 4 and offspring survival to Day 4 of age at 100, 300 or 1000 mg/kg bw/day.
Offspring Growth and Development
There was considered to be no obvious effect of maternal treatment on offspring body weight on Day 1 and subsequent body weight gain to Day 4 of age at 100, 300 or 1000 mg/kg bw/day. There was no obvious effect of maternal treatment on offspring surface righting performance at 100, 300 or 1000 mg/kg bw/day.
Offspring Observations
The low incidence of clinical sign observed for the offspring did not indicate any obvious underlying effect on offspring growth and development at any of the dosages investigated.
Pathology
Necropsy Offspring : Necropsy findings did not indicate any obvious effect of maternal treatment at dosages of 100, 300 or 1000 mg/kg bw/day. Necropsy Adults: Macroscopic necropsy revealed blue coloration or blue contents for various regions of the gastrointestinal tract for both sexes at 1000 mg/kg bw/day. A lower incidence of similar findings were apparent for both sexes at 100 and 300 mg/kg bw/day. This finding was consistent with the colored nature of the test item.
Organ Weights
There was no obvious effect of treatment on absolute organ weigts at 100, 300 or 1000 mg/kg bw/day.
Histopathology
Microscopic evaluation of male reproductive tissues at 1000 mg/kg bw/day did not indicate any obvious effect of treatment.
Conclusion
Based on the results of this study, the ‘No Observed Effect Level’ (NOEL) for reproductive toxicity, including the survival, growth and development of the offspring, was considered to be 1000 mg/kg bw/day.
Reference
Analytical investigation: The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.
The homogeneity and stability was confirmed for test item in water formulations at nominal concentrations of 7.87 mg/mL and 78.7 mg/mL when stored refrigerated for 11 days.
The mean concentratio of test item waas within 10% of nominal concentratios with the exception of analysis 2, comfirming accurate formulation.
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no study available
- Dose descriptor:
- NOAEL
- 1 000 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- High quality GLP-study
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Additional information
The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, and provides an initial hazard assessment for effect on reproduction. The study is compatible with the requirements of the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995). This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). Treatment with FAT 21036/G TE at dosages of up to 1000 mg/kg bw/day was generally well tolerated by both sexes. Clinical signs were generally restricted to fur staining from the test item mainly for animals at 300 mg/kg bw/day and, to a greater extent, 1000 mg/kg bw/day. Isolated instance of noisy respiration were observed for one male and two females at 1000 mg/kg bw/day, but this was considered to reflect occasional difficulties with dosing individual animals rather than any underlying toxicological effect. At 1000 mg/kg bw/day, lower body weight gain was apparent, although these lower gains failed to attain statistical significance and there was no similar effect apparent for females at this dosage. At the level observed, this lower body weight gain was considered to be insufficient to represent an adverse effect of treatment. At 300 mg/kg bw/day, there were two unscheduled deaths (one male and one female) but these mortalities were considered to represent intubation error and were therefore unrelated to treatment. There was no effect of treatment on reproduction. One pregnant female at 100 mg/kg bw/day and another at 1000 mg/kg bw/day were not observed to give birth to a litter however, these isolated occurrences, in the absence of any supporting increase in post-implantation loss or offspring mortality in remaining litters, were considered to be incidental and unrelated to maternal treatment. There was no effect on maternal treatment on litter data or initial offspring body weight and subsequent survival, growth and development. Macroscopic necropsy revealed blue coloured coloration/contents for various regions of the gastrointestinal tract for both sexes at 1000 mg/kg bw/day. A lower incidence of similar findings were also apparent for both sexes at 100 and 300 mg/kg bw/day. These findings were consistent with the colored nature of the test item and the route of administration; reproductive organs/tissues appeared to be unaffected. At 1000 mg/kg bw/day, one male showed small testes and epididymides and another showed a small left seminal vesicle but their respective mating partners both achieved pregnancy. As there was no obvious effects on mean testes and epididymal weights or any evidence of microscopic change for reproductive tissues for either sex these isolated findings were considered incidental and unrelated to treatment. Overall, the No Observed Effect Level (NOEL) for reproductive toxicity, including the survival, growth and development of the offspring was considered to be 1000 mg/kg bw/day.
Justification for classification or non-classification
Acid Blue 225 does not warrant classification for reproductive toxicity as per the criteria of Regulation (EC) No. 1272/2008.
Additional information
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