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Reaction mass of tetrasodium 5-{[4-chloro-6-(4-{[2-(sulfonatooxy)ethyl]sulfonyl}anilino)-1,3,5-triazin-2-yl]amino}-4-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl) diazenyl]naphthalene-2,7-disulfonate and trisodium 5-({4-chloro-6-[4-(vinylsulfonyl) anilino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-2,7-disulfonate
EC number: 701-360-8 | CAS number: -
- 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
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- 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
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- Toxicological Summary
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- Acute Toxicity
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- Repeated dose toxicity
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- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
No adverse effects on reproduction in rats, related to test substance toxicity, were seen
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 28 April 2011 to 23 June 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Species and strain: Wistar RJHan:WI rats
Source: Laboratoire Elevage Janvier, B.P. 4105, Route des Chênes Secs, 53940 Le Genest-St-Isle CEDEX France
Hygienic level: SPF at arrival; standard laboratory conditions during the study
Justification of species/strain: The rat is regarded as suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility.
Number of animals: Main groups: 48 male, 48 female rats, 12 animals/sex/group, 4 groups; a sufficient number of at least 8 pregnant females/group was achieved.
Recovery groups: 10 male, 10 female rats, 5 animals/sex/group, 2 groups, Control and High dose
Positive Control MNT group: 12 male and 12 female rats, 1 group
At the completion of the study, the spare animals were returned to CiToxLAB Hungary Ltd. spare colony, as their use was not required (no replacements with spare animals were performed)
Age of animals: Young adult rats, approximately 10-11 weeks old at starting and 12-13 weeks at mating. The age range within the study was kept to the minimum practicable.
Body weight range: Males: 366 g – 407 g, Females: 219 g - 249 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment
Acclimation period: At least 7 days (5 days from animal arrival to pre-treatment ophthalmoscopy examination, 7 days from animal arrival to onset of treatment)
Animal health: Only healthy animals were used for the test, as certified by the veterinarian. Females were nulliparous and non-pregnant.
Room number: 524, 505
Cage type: Type II and/or III polypropylene/polycarbonate
Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). Details of bedding quality are reported.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.9 – 24.2°C
Relative humidity: 33 - 69%
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 5 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).
The temperature and humidity were measured twice daily; no deviations from the target ranges were noted during the study.
Food and water supply: Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum, and tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum. Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary).
The food and water are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Animal identification: Each parental/adult animal (P Generation) was identified by a number unique within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at CiToxLAB Hungary Ltd.
This number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group, as indicated in the Experimental design section. The boxes were arranged in such a way that possible effects due to cage placement were minimized and were identified by cards showing the study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery. The new-borns (Offspring, F1 Generation) were identified by cutting off digit-tips up to one day after birth.
Randomization: All parental/adult (P) male and female animals were sorted according to body weight by computer and divided to weight ranges. An equal number of animals from each weight group was randomly assigned to each dose group to ensure that test animals were as nearly as practicable of a uniform weight. The grouping was controlled by SPSS/PC software according to the actual body weight, verifying the homogeneity/variability between/within the groups and cages. Males and females were randomized separately. - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- Vehicle
Name: Distilled, sterile water for injection, PhEUR
Lot No.: 8490910, 0110111
Manufacturer: TEVA Pharmaceutical Corporation
Expiry Date: September 2013, January 2014 respectively
Storage: Room temperature - Details on mating procedure:
- Main and Positive Control MNT animals: Mating began after the animals have attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male of the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred, for up to 6 days. A vaginal smear were prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were caged individually. Mating pairs were clearly identified in the data, mating of siblings was avoided.
Recovery animals: Recovery animals were not mated and, consequently, were not used for the assessment of reproduction/developmental toxicity. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The samples were evaluated by UV-HPLC method
- Duration of treatment / exposure:
- Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period).
Females were dosed for 14 days pre-mating, for up to 5 days mating period, through gestation and up to the day of necropsy (at least 4 days post-partum dosing). - Frequency of treatment:
- Test item or Control (water)-treated Groups 1-4 Main animals were administered the dosing solutions daily on a 7 days/week basis.
- Details on study schedule:
- Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination, as no additional mating was considered required.
Females were dosed for 14 days pre-mating, for up to 5 days mating period, through gestation and up to the day of necropsy (at least 4 days post-partum dosing). The day of birth (viz. when parturition was complete) was defined as Day 0 post-partum. Females showing no-evidence of copulation were sacrificed as practical, 26-27 days after the end of the mating period. - Dose / conc.:
- 62.5 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 250 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- Number of animals: Main groups: 48 male, 48 female rats, 12 animals/sex/group, 4 groups; a sufficient number of at least 8 pregnant females/group was achieved.
Recovery groups: 10 male, 10 female rats, 5 animals/sex/group, 2 groups, Control and High dose - Control animals:
- yes, concurrent vehicle
- Details on study design:
- Rationale for dose selection and route of administration: The dose levels and the vehicle were selected by the Sponsor in consultation with the Study Director based on available data, formulation and analytical trials and information from previous experimental work, including the results of an acute oral toxicity study (CiToxLAB Hungary Ltd. study code 11/008-001P) and a repeated dose range finding study in the rat (CiToxLAB Hungary Ltd. study code 11/008-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. The oral route was selected as it is a possible route of exposure to the test item in humans.
- Parental animals: Observations and examinations:
- Clinical observations and functional observation battery (FOB)
All animals: Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and end of the working day. General clinical observations were performed daily, after treatment at approximately the same time with minor variations, or in the afternoon (pm) as practical during the working day, as no peak period of effects was noted after dosing during the first days of treatment. During Recovery period, the animals were similarly observed daily as practical.
All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including onset, degree and duration of signs as applicable.
More detailed examinations were made once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning (am) or before treatment. These observations were made outside the home cage in a standard arena, at similar times as practical. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. No such clinical signs were observed during the study.
Main animals, 5 males and 5 females/group, “subgroup A”:
Assessment of any potential test item related neurotoxicity was performed during the last exposure week (males, on Day 26 am, females, on PPD 3 am). In order to avoid hyperthermia of pups, dams were removed from the pups for not more than approximately 30-40 minutes. Selected animal were subjected to the functional observation battery, including qualitative assessment of the grip strength, and to measurements of the landing foot splay and fore/hind grip strength.
To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rats were dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured.
Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support; results are tabulated with individual and mean data.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed. Parameters such as, but not limited to body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated.
Recovery animals: Neurotoxicity evaluation was similarly conducted in the Recovery animals towards the end of the Recovery period, on Day 54, for necropsy on Day 56.
Body weight measurement : All adult Main and Recovery animals were weighed with accuracy of 1 g for randomization purposes, then on Day 0, afterwards at least weekly and at termination. Parent females were weighed on gestation Days GD0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition) and PPD4 (before termination). Body weights of the female animals were additionally weighed on gestational Days GD10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.
Food consumption measurement: Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly.
Ophthalmology: The fundus of eyes of all animals was examined before treatment. Five male and 5 female Control and High dose animals (“subgroup C”) randomly selected from groups 1 and 4, during the last week of treatment prior to necropsy (males, Day 26 pm, females, PPD 3 pm). Mydriasis was produced after instillation of a mydriatic agent (eye drops "Mydrum") into the conjunctival sac. The examination was performed using a Gowlland ophthalmoscope. As no ophthalmoscopic alterations were found, no additional examination was performed in other animals.
Observation of the delivery process, offspring and nursing instinct: Main Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All observations were recorded as applicable. No evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy. Dams were observed to record whether they form a nest from the bedding material and cover their new-borns or not. The efficiency of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded as applicable.
Observations are reported individually for each adult animal. In addition to the observations on parent animals, the pups (offspring) were monitored for any behavioural changes.
CLINICAL PATHOLOGY
All animals selected for blood sampling were fasted (overnight period of food deprivation).
For terminal blood sampling of Recovery animals, 3 samples were taken from each animal: one for haematology (1.2 mL blood, in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (1.4 mL blood for APTT and PT measurements, in tubes with sodium citrate as anticoagulant) and one to obtain serum (approximately 1 mL blood as practical, in tubes with no anticoagulant) for clinical chemistry.
For Day 14 blood sampling of Main animals selected (subgroup B), 2 samples were taken from each scheduled animal: one for haematology (1.2 mL blood, in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood) and one to obtain serum (approximately 1 mL blood as practical, in tubes with no anticoagulant) for clinical chemistry.
For terminal blood sampling of Main animals selected (subgroup B), one sample for blood clotting times (1.4 mL blood for APTT and PT measurements, in tubes with sodium citrate as anticoagulant) were taken from scheduled animals.
For urine collection, the selected animals (Main subgroup B and Recovery) were placed in metabolic cages for approximately 16 hours and food and water deprived, then water were provided at libitum for at least approximately 2 hours prior to necropsy and organ weight measurements.
Main animals, 5 males and 5 females/group, “subgroup B”:
Laboratory examinations for haematology and clinical chemistry evaluation were conducted at the end of pre-mating period, on blood samples collected from the sublingual vein, prior to the start of mating on Day 14 from 5 animals/sex/group randomly selected (“subgroup B”).
Coagulation evaluation (APTT and PT) was performed at the completion of the treatment, on blood samples collected by cardiac puncture from subgroup B animals under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
Urine sampling (approximately 16 hours sampling period) was performed prior to necropsy from the same subgroup B (urinalysis on Day 28-males, PND 5-females).
Recovery animals: Haematology, coagulation and clinical chemistry investigations were conducted at the completion of the Recovery period, 14 days after the first scheduled euthanasia of Main dams. Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
Urine sampling (approximately 16 hours sampling period) was performed from the Recovery animals prior to necropsy (urinalysis on the day of necropsy, conducted 14 days after the first scheduled euthanasia of the Main dams). - Oestrous cyclicity (parental animals):
- A vaginal smear were prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines).
- Sperm parameters (parental animals):
- For the adult animals, detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups (Main and Recovery) and all macroscopic findings (abnormalities) from all animals. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
- Litter observations:
- Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities.
In addition to the observations on parent animals, the pups (offspring) were monitored for any behavioural changes. Live pups were counted, sexed, weighed individually within 24 hours of parturition (ex. Day 0 or 1 post-partum, PND0 or 1) and on PND4, with accuracy of 0.01g. All the litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination in order to identify the possible cause of death. All observed abnormalities were recorded. - Postmortem examinations (parental animals):
- Pathology
Gross necropsy was performed on all animals. Terminally, after completion of the treatment or Recovery periods as applicable, animals were sacrificed under pentobarbital anaesthesia followed by exsanguination. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.
Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded in the Main females as applicable.
At the time of termination, body weight and weight of the following organs of all parental animals were determined:
- With a precision of 0.01 g: uterus (with and without cervix), vagina, testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands, brain
- With a precision of 0.001 g: ovaries, pituitary
The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerve were retained in modified Davidson’s fixative. Testes and epididymides were preserved in Bouin’s solution, all other organs in 10% buffered formalin solution.
From subgroup B Main animals and Recovery animals, the following organs were weighed in addition to the ones previously mentioned:
- With a precision of 0.01 g: heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals.
For all organs, paired organs were weighed individually. Individual and/or paired absolute organ weight are reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) were calculated and reported.
Histopathological examination was performed on the selected list of specified tissues from the Control and High Dose Main and Recovery rats and all macroscopic lesions from all toxicology animals. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Additionally selected organs/tissues for 5 animals/sex/group (identified as subgroup B Main) were microscopically evaluated from the Control and High dose Main animals, and kidneys and stomach samples from the Subgroup B Low and Mid Dose animals were examined, due to the test item-related microscopic findings noted at the first instance in the animals examined.
The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. - Postmortem examinations (offspring):
- Pups euthanized at PND4 were carefully examined at least externally for gross abnormalities. Any pups showing abnormalities in structure or behaviour, including the pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination, in order to identify the probable cause of death if possible.
- Statistics:
- Data were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., and then tabulated using the Microsoft Office Word and/or Excel, as appropriate. Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations.
The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible. - Reproductive indices:
- Formulae for Calculation of Mating and Fertility Indices are included in pdf attached under background information.
- Offspring viability indices:
- Formulae for Calculation of Pups’ Mortality and Sex Ratio Indices are included in pdf attached under background information.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- See below for details
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Effect on body weight - see below
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Effect on body weight - see below
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- See below for details
- Other effects:
- not examined
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- effects observed, treatment-related
- Description (incidence and severity):
- See below for details
- Dose descriptor:
- NOAEL
- Remarks:
- (reproductive toxicity)
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: No effects seen at highest dose tested (1000 mg/kg bw/day).
- Dose descriptor:
- NOAEL
- Remarks:
- (reproductive toxicity)
- Effect level:
- 250 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: Due to the higher intrauterine, postnatal and total foetal mortality at 1000 mg/kg bw/day
- Critical effects observed:
- no
- Clinical signs:
- no effects observed
- Mortality / viability:
- mortality observed, treatment-related
- Description (incidence and severity):
- See below for details
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- See below for details
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- not examined
- Dose descriptor:
- NOAEL
- Remarks:
- (teratogenicity)
- Generation:
- F1
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No teratogenic effects at highest dose tested (1000 mg/kg bw/day)
- Dose descriptor:
- NOAEL
- Remarks:
- (developmental effects)
- Generation:
- F1
- Effect level:
- 250 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: Slight effect on pups survival and birth weight at 1000 mg/kg bw/day.
- Critical effects observed:
- no
- Reproductive effects observed:
- no
- Conclusions:
- The no observed adverse effect level (NOAEL) for the test substance for parental/adult systemic toxicity is considered to be 62.5 mg/kg bw/day. For reproductive toxicity, the no observed effect level (NOEL) for parental/adult effects is considered to be 1000 mg/kg bw/day for the males and 250 mg/kg bw/day for the females, due to the higher intrauterine, postnatal and total foetal mortality at 1000 mg/kg bw/day, correlated with a NOEL of 250 mg/kg bw/day for F1 offspring, based on a possible treatment-related slight effect on pups survival and weight at 1000 mg/kg bw/day. No structural adverse effects were observed in the surviving pups at up to and including 1000 mg/kg bw/day, under the conditions of this study.
- Executive summary:
This study has been performed in accordance with the Study Plan, OECD Guidelines for Testing of Chemicals No. 422 (22nd March 1996) and the Principles of Good Laboratory Practice.
The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat study was to obtain information on the possible toxic effects of the test item following repeated daily administration by oral gavage to Wistar rats. Reversibility of any treatment-related changes was evaluated following a 14-day Recovery period. The study also comprised a reproductive/developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Day 4 post-partum.
In the Main Groups, male and female Wistar rats were treated for 2 weeks pre-mating, then during the mating/postmating period, males for 28 days and females throughout gestation period and up to and including postpartum/lactation Day PPD5. Additional 5 male and 5 female rats from the Control and High dose Recovery Groups 1 and 4 scheduled for follow-up observations were not mated, but treated up to the first scheduled euthanasia of the Main dams (Day 41), then kept at least for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects, and subjected to necropsy with macroscopic examination on Day 56.
Parameters measured during the study included signs of morbidity and mortality twice daily, daily or detailed weekly observation of clinical signs, neurological and ophthalmoscopic assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. In addition, the reproductive performance and indices, pregnancy, parturition and postpartum/lactation period were monitored in the adult Main animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND4. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals.
For the adult animals, detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups (Main and Recovery), all macroscopic findings (abnormalities) from all animals and kidney and stomach samples from the Mid and Low dose Subgroup B Main groups, based on the results observed at the initial evaluation of the High dose group. In addition, bone marrow smears were prepared at necropsy and evaluation of induction of micronuclei in bone marrow erythrocytes of treated and Control animals was conducted from the Control and High dose Main animals.
Analysis of formulations (concentration, homogeneity) and assessment of test item stability in this vehicle in the conditions employed on the study was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Stability tests (CiToxLAB Hungary Ltd. study code 11/008-316AN and additional evaluation during the current study) at concentrations from approximately 1 to 100 mg/mL in ultrapure water indicated 1 day stability at room temperature and up to 7 days, while stored refrigerated at 2-8ºC in the dark, when the recovery range was 91%-105%, which lies within the acceptance range of 100 ± 10%.
Concentration and homogeneity of formulations were evaluated by UV-HPLC method on duplicate samples collected from the top, middle and bottom of test item solutions, and one sample from the control taken and analysed fresh on 3 occasions during the study. The measured concentrations varied between 95% and 107% of the nominal concentrations (6.25, 25 and 100 mg/mL). No test item was detected in the Control solution samples. These results were considered suitable for the study purposes.
No mortality or systemic adverse effects occurred during the study. During the treatment period, slight to moderate dark pink discoloration of the skin was noted in the High dose animals after Day 12 of treatment, until the completion of the study up to Day 55 during the Recovery period. Furthermore, dark red discoloration of the faeces was noted at 1000 mg/kg bw/day from Day 1, and of the urine, from Day 2 onwards. The discoloration of the faeces persisted in the 1000 mg/kg bw/day Recovery animals for up to 3 days after the last dose administration (up to Day 44) and of the urine, until completion of the recovery phase and termination. These changes were ascribed to elimination of the test substance or its metabolites through faeces or urine and an expected staining effect.
There were no test item-related differences to Control in the mean daily food consumption. No test item related effects, or changes considered toxicologically significant were noted in the haematology parameters evaluated in the Main animals at up to and including 250 mg/kg bw/day dose levels. At 1000 mg/kg bw/day, WBC increases considered possibly related to the test substance administration were noted in the Main animals evaluated on Day 14, up to 45% and 50% higher than control, p<0.01, in the males and females, respectively. The mean values remained 92% higher than control, p<0.01, in the Recovery males evaluated on Day 56, to become comparable with the physiological values in the Recovery females.
In the Main animals evaluated at the completion on Day 14, an apparent total bilirubin T‑BIL increase was noted in the 1000 mg/kg bw/day Main High dose males and females (174% and 166%, p<0.05 and 0.01, respectively). The bile acids were also higher than controls in both males and females at 250 and 1000 mg/kg bw/day, attaining statistical significance in the Mid and Main High dose males (43%, p<0.05 and 105% higher than control, p<0.01, at 250 and 1000 mg/kg bw/day) and in the Main High dose females (180%, p<0.01). After a 14-day recovery period, the T-BIL remained up to 36.2% higher than control in the High dose Recovery males and females. This was considered to be related to a possible spectral interference with the analytical method caused by the discolouration of the serum by the test item and not to reflect an adverse effect on the liver function.
In addition, lower albumin/globulin ratio, up to -20%, and lower ALT, up to -23%, were recorded in the Main High dose animals, effects considered potentially related to test item administration at 1000 mg/kg bw/day dose level; the ALT mean value remained up to -53% lower than control in the Recovery animals, however, without attaining statistical significance.
At urinalysis, pink urine was noted in all animals evaluated from the Main Mid dose 250 mg/kg bw/day group (5/5 males and 5/5 females, subgroup B) and red urine, in all the High dose 1000 mg/kg bw/day animals examined (5/5 males and 5/5 females, subgroup B Main and in 5/5 male and 5/5 female Recovery animals). These changes were ascribed to elimination of the test substance or its metabolites through urine (urine collection in metabolic cages) and an expected staining effect.
There were no statistically significant differences between the Control and test item-treated groups with regard to reproductive ability or in the mating or gestation indices, or effects considered adverse or toxicologically significant in correlation with the test substance administration. In all groups, the mating indices were 100%, the fertility indices, 83%, 100%, 92% and 92% due to 2, 0, 1 and 1/12 non-pregnant females, respectively, in Groups 1, 2, 3 and 4, and the gestation indices, 100%, with the exception of the High dose group in which the gestation index was incidentally 91%, due to 1/11 pregnant, but not delivered females in this group, values which are comparable with concurrent control data in Wistar rats.
Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 6 days of pairing (cohabitation).
No test item effect on the duration of pregnancy or abnormalities in the gestation outcome ascribed to the treatment were observed. The mean duration of pregnancy was similar in the control and test item treated groups and varied from 22.00 days (22 days) in the Controls, 22.17 days (22 to 23 days) in the 62.5 mg/kg bw/day Low dose group, 22.45 days (p<0.05, 22 to 23 days) in the 250 mg/kg bw/day Mid dose group, to 22.09 days (22 to 23 days) in the 1000 mg/kg bw/day High dose group. These values were comparable with the contemporaneous historical control data (gestation length: 22.40 ± 0.53 days, range: 22 to 23 days, n = 22) and the minor differences to control were not regarded as toxicologically relevant, although the mean duration of pregnancy attained statistical significance in the 250 mg/kg bw/day Mid dose group only. All the parturitions were normal.
The number of corpora lutea and implantation sites were comparable in the treated groups up to and including 1000 mg/kg bw/day with the mean value recorded in the Control group.
There were no statistically significant differences or effects that could be ascribed to treatment on the pre/post-implantation, post-natal or total mortality values (%) at up to and including 250 mg/kg bw/day.
At 1000 mg/kg bw/day, the intrauterine mortality, post-natal mortality and subsequently the total mortality were higher than control. However, only the post-natal mortality attained statistical significance (intrauterine mortality: 28.19% vs. 11.44%, i.e. 146.4% higher than control; post-natal mortality: 16.34%, p<0.05, vs. 0.71%; total mortality: 33.65% vs. 12.03%, i.e. 179.6% higher than control in the High dose vs. the control group). These effects were considered to be possibly related to the test substance administration at the High dose level, although a role of the individual variability cannot be excluded (females 4501, 4503 and 4506).
The test substance administered to parental generation under the conditions of this study did not lead to any adverse effects considered related to treatment or toxicologically significant in the F1 generation at up to and including 250 mg/kg bw/day dose level.
At 1000 mg/kg bw/day, the mean number of viable pups was -26.1% lower than control on PND0 and -30.4% lower on PND4, attaining statistical significance when evaluated for the total number of pups. These results correlated with the higher post-natal and total mortality observed in the High dose dams . Survival index was -18.3% lower than control in the High dose group on PND0 and -26.2% on PND4, although without attaining statistical significance.
The pups found dead and cannibalised were counted and sex determined if possible, but not further examined macroscopically. No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. Findings such as cold to touch, occasional cannibalization or lack of suckling were noted with low incidence, within the physiological range expected in the population of Wistar rats and considered without toxicological significance, or to reflect a test item or adverse effect. There were no adverse effects, or effects considered related to test item administration on the sex ratios, and no statistical or toxicological significant differences to control were noted.
There were no effects considered adverse on the offspring weight or weight gain at up to and including 250 mg/kg bw/day.
At 1000 mg/kg bw/day, when evaluated per litter basis, there were no statistically significant differences to control, however, the mean litter weights were lower than control on both PND0 and PND4 (on PND0,11 litters , 65.7 g, vs. 10 litters, 90.1 g, thus -27.1% lower than controls mean litter weight in the High dose group; on PND4, 9 litters, 112.9 g, vs. 10 litters, 140.1 g, thus -19.4% lower than controls mean litter weight in the High dose group). When evaluated for all pups, the mean body weights were statistically significant, slightly lower up to -7% than control: on PND0, 6.39 g vs. 6.88 g, and on PND4, 10.36 g vs. 11.03 g. As the differences were minor and the dose response was not clear, a test item related adverse effect on the offspring development following administration of 1000 mg/kg bw/day to the parental generation in the conditions of this study could not be ascertained, although it cannot be excluded.
At pathology evaluation, pink/red discoloration of numerous organs/tissues was observed at necropsy in the High dose group. In the Mid dose group, discoloration was only observed in trachea, stomach and kidneys in single animals. Histopathologically, treatment-related changes were observed in the kidney and stomach of High and Mid dose rats. The severity and/or incidence of these changes indicated a relationship to dose. Degeneration/regeneration of the cortical tubules in the kidneys were present in High dose rats and Mid dose males. Degeneration/regeneration of epithelial cells of the glandular mucosa in the stomach accompanied by a minimal to mild inflammatory influx in the submucosa,were recorded in rats at dose levels of 250 and 1000 mg/kg bw. There was clear dose responded decrease in the severity when compared High Dose (1000 mg/kg bw/day) and Mid Dose (250 mg/kg bw/day) animals. A minimal epithelial degeneration/regeneration without influx of inflammatory cells was observed in two Low Dose males.
After a 14 day of recovery period, a residual effect of treatment was microscopically seen in the kidneys and stomach in the High Dose groups. In the kidneys, decreased severity of the test item-related microscopic alterations from mild to minimal was more evident in the males compared to the females. Cessation of treatment for 14 days resulted in almost completed regression of the test item-related lesions in the stomach. The decrease of the severity from mild to minimal was observed in all High Dose animals. Also, mitotic index has been decreased and inflammatory influx as well as formation of the submucosal lymphoid follicles, were no more observed.
Twenty three pups were found dead, intact and were subjected to necropsy between Days 0-3 post partum (4, 4, 5 and 10 pups from the Control, Low, Mid and High Dose groups). Negative floating test was observed in 3/4, 3/4, 4/5 and 7/10 animals from the Control, Low, Mid and High Dose groups. Positive floating test was observed in 1/5 and 2/10 Mid and High dose of the pups found dead on PND0 (no floating test was required on the remaining pups which died between PND1-3). White liquid/creamy material in the stomach was present in 1/4, 1/4 and 1/10 pups from Control, Low and High Dose. The stomach was empty in 1/5 and 2/10 offsprings from the Mid and High Dose groups. A cause of death of these pups could not be determined.
In the Main animals, there were no toxicologically relevant changes in the organ weight values, or effects considered adverse or test item related at up to and including 250 mg/kg bw/day.
At 1000 mg/kg bw/day, the absolute kidneys weight was 14% higher than control in the High dose Main males, without attaining statistical significance. However, the relative kidney weight was 20% higher than control, p<0.01, when adjusted for the body weight, and 15% higher, p<0.05, when adjusted for the brain weight. No similar effects were observed in the High dose Main females, however, kidneys weights were higher than control in both male and female Recovery animals. In view of the microscopic findings recorded, these kidneys weight variations were considered to reflect a possible test substance-effect at 1000 mg/kg bw/day administered under the conditions of this study.
In conclusion, the only effects noted were unspecific toxic effects resulting inhigher intrauterine, postnatal and total foetal mortality at 1000 mg/kg bw/day. In this study, for reproductive toxicity, the no observed effect level (NOEL) for parental/adult effects is considered to be 1000 mg/kg bw/day for the males and 250 mg/kg bw/day for the females, due to the higher intrauterine, postnatal and total foetal mortality at 1000 mg/kg bw/day, correlated with a NOEL of 250 mg/kg bw/day for F1 offspring, based on a slight secondary effect on pups survival and birth weight at 1000 mg/kg bw/day. No structural adverse effects were observed in the surviving pups at up to and including 1000 mg/kg bw/day, under the conditions of this study.The relation to test substance administration is questionable and this effect is not considered to be an adverse effect related to substance toxicity, hence, the NOAEL was considered to be 1000 mg/kg bw/day for male and female rats and their offspring.
- Endpoint:
- one-generation reproductive toxicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 23 Jul 2001 to 12 Dec 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.34 (One-Generation Reproduction Toxicity Test)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstrasse 27, 33178 Borchen, Germany
- Age at study initiation: approximately 6 weeks
- Housing: single
- Diet (ad libitum): sniff R/M-Z (V1324)
- Water (ad libitum): tap
- Acclimation period: at least five days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 16-20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 31 July 2001 To: 12 December 2001 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved daily in deionized water in concentrations of 12.5 mg/mL, 50 mg/mL and 200 mg/mL.
VEHICLE: deionized water
- Concentration in vehicle: 12.5 mg/mL, 50 mg/mL and 200 mg/mL
- Amount of vehicle: 5 mL/kg body weight - Details on mating procedure:
- - M/F ratio per cage: 1:1 (1:2-mating was performed in three high dose females because of mortality in males)
- Length of cohabitation: three weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was individually caged - Analytical verification of doses or concentrations:
- not specified
- Details on analytical verification of doses or concentrations:
- -
- Duration of treatment / exposure:
- Males : 10 weeks pre-mating, treatment continued during mating (ca. 3 weeks)
Females : 4 weeks pre-mating, treatment continued during mating (ca. 3 weeks) and during lactation until day 21 post partum - Frequency of treatment:
- daily
- Details on study schedule:
- NA
- Remarks:
- Doses / Concentrations:
12.5 mg/mL, 50 mg/mL and 200 mg/mL
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
62.5, 250 and 1000 mg/kg body weight per day
Basis: - No. of animals per sex per dose:
- 28
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: The dose rationale was based on a subacute 28-day ora toxicity study with the test compound in rats, which did not show any adverse findings up to and including the limit dose of 1000 mg/kg body weight. Accordingly, dose levels of 0, 62.5, 250 and 1000 mg/kg body weight per day were selected for the present study.
- Positive control:
- NA
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: Yes
- Time schedule for examinations: once weekly in both sexes during the pre-mating period
in females on day 0, 7, 14 and 21 during gestation and on day 0, 4, 7, 14 and 21 of lactation period.
FOOD CONSUMPTION: Food consumption was recorded together with the body weights (except the mating period for both genders, and except on day 4 of lactation for the females).
OTHER:
- Clinical Chemistry: 10 male and 10 female animals per group at scheduled sacrifice - Oestrous cyclicity (parental animals):
- daily during mating period
- Sperm parameters (parental animals):
- Parameters examined in all P male parental generations: testis weight, epididymis weight, prostate weight, seminal vesicles weight
histopathology of testis, epididymis, prostate, seminal vesicles - Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, viability, physical or behavioural abnormalities
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead. - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals were killed in the third week of the mating period
- Maternal animals: All surviving animals were killed on day 22 (or until day 24, after weekends), after birth. Animals with necropsy date on weekend were killed the next weekday
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. All abnormal findings with special attention paid to the organs of the reproductive system were recorded
HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues or organs (or pieces of them) were preserved in Bouin's solution (testes) and formaldehyde solution and processed for histopathological investigations: Epididymides, Kidneys, Liver, Ovaries with oviducts, Pituitary, Prostate, Seminal vesicle, Testes, Uterus, Vagina, all other gross lesions.
Histopathological examinations were carried out of the control and high dose animals on these organs, as well as on on heart, spleen, lung, pancreas and gastro-intestinal tract from those animals with macroscopically visible changes, i.e., blueish colored pigmentation storage of the test compound.
The following organs were weighed: Epidymides, Kidneys, Liver, Ovaries, Pituitary, Prostate, Seminal vesicle, Testes, Uterus
OTHER: In order to investigate the cause of the dental findings in the late treatment period of the high dose animals, in total five affected incisors of the high dose males and five incisors of the control animals were analyzed for calcium and phosphorous content (two high dose and two control animals, data not presented, filed in the raw data). Secondly they were extended to fluoride, calcium and phosphorous content on the remaining 3 high dose incisors and control incisors. - Postmortem examinations (offspring):
- SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 4 days of age.
- Dead or moribund pups and pups killed at day 4 were examinated for defects.
- All surviving F1-animals were killed on day 22 (or until day 24, after weekends), after birth. Animals with necropsy date on weekend were killed the next weekday
- Statistics:
- All Parameters: The assumption of a monotonic dose-response relationship for all parameters justifies the restriction of the significance level to 5 percent (per parameter and sex), using the method of: HOTHORN L, LEHMACHER W.: A Simple Testing Procedure "Control versus k Treatments" for One-sided Ordered Alternatives, with Application in Toxicology, Biom. J. 33, 179-189, Akademie Verlag
Bodyweights: The changes of parameter values compared to the treatment-free baseline values are analyzed with the t-Test:
HARTUNG J., ELPERT B., KLÖSENER K. H., Lehr- und Handbuch der angewandten
Statistik (1989), R. Oldenbourg Verlag, München
Clinical Pathology Data: Wilcoxon's Test: HOLLANDER M., WOLFE, D. A:, Nonparametric statistical methods
Organ weights (absolute): t-Test
Organ weights (relative to bodyweight): Wilcoxon's Test - Reproductive indices:
- Copulatory index (%): Number of sperm positive females x 100 / Number of mated females
Fertility index - Males (%): Number of fertile males x 100 / Number of mated males
Fertility index - Females (%): Number of pregnant females x 100 / Number of mated females
Gestation index (%): Number of females with viable pups x 100 / Number of pregnant females
Sex ratio: (Number of pups examined - Number of males (females)) x 100 / Number of pups examined - Offspring viability indices:
- Intra uterine mortality: (Number of implantations - Number of newborns) x 100 / Number of implantations
Total mortality: (Number of implantations - Number of viable pups) x 100 / Number of newborns
Viability index (%): Number of viable pups on day 4 (7, 14, 21) x 100 / Number of viable pups on day 0 (4, 7, 14)
Lactation index (%): Number of viable pups on day 21 x 100 / Number of viable pups on day 0 of lactation
Weaning index (%) - Clinical signs:
- effects observed, treatment-related
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- effects observed, treatment-related
- Dose descriptor:
- NOAEL
- Remarks:
- General health
- Effect level:
- 62.5 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- other: broken-off incisors (fluorose) from 0.3% fluor impurity
- Dose descriptor:
- NOAEL
- Remarks:
- Reproductive performance
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- other: All effects observed were due to broken-off incisors resulting in lower food consumption and a lower pregnancy index. This effect was due to fluorosis of the rats' teeth caused by the 0.3% fluor impurity
- Critical effects observed:
- no
- Clinical signs:
- no effects observed
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Sexual maturation:
- not specified
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- not examined
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- other: effects observed on body weight of high-dose pups were due to broken-off incisors in dams resulting in lower food consumption. This effect was due to fluorosis of the rats' teeth caused by the 0.3% fluor impurity
- Reproductive effects observed:
- no
- Conclusions:
- Daily oral administration of the test substance to rats during the premating, mating, gestation and lactation period at dose levels of 62.5 or 250 mg/kg body weight did not affect food consumption, body weight development, male or female mating/reproductive performance, fertility, gestation length as well as development of their progenity.
Daily oral administrations of 1000 mg/kg body weight (high-dose group) were well tolerated in rats within the first 5 weeks of treatment, but thereafter, from week 6 onwards, caused mortality due to dental lesions with subsequent disability of food uptake and starvation (clinical picture of dental fluorosis). This finding was time-dependent, with a threshold dose of 250 mg/kg body weight for males, and could be related to the fluoride impurity (0.3%) of this batch tested.
Although there was marked pigment storage of the test compound in several organs, there was no clear functional or histopathological correlate that could be related to compound-induced systemic toxicity and/or specific reproductive toxicity. Impairment of reproduction and fertility at high dose parental animals was primarily the result of severe dental problems.
In the presence of severe dental problems at 1000 mg/kg bw and threshold dose of 250 mg/kg bw for this finding, there was no evidence of selective reproductive toxicity in rats. - Executive summary:
The present study was conducted in order to determine the effects of the test substance on reproduction when administered orally by gavage to male and female Sprague Dawley rats during pre-mating, mating, gestation and lactation.
Groups of 28 male and 28 (27 in the high-dose group) female Spraque Dawley rats received the test substance orally once daily at dose levels of 0, 62.5, 250 or 1000 mg/kg body weight for a period of 10 weeks (males) and 4 weeks (females), prior to mating. Dosing of males was continued during the whole mating period until sacrifice (approx. week 11 - 13 of the study). Treatment of mated females was continued until day 21 after littering. The dosing volume was 5 mL/kg, corresponding to concentrations of 0, 12.5, 50 and 200 mg/mL. At start of the study, the animals were 5-9 weeks of age with mean body weights of 240 g for males, and 206 g for females.
Behavior and state of health were observed daily in all groups. Body weight development and food consumption were recorded throughout the study in females, and during pre-mating period in males. After the mating period the males were killed and necropsied. The dams were allowed to litter and rear their progeny to the stage of weaning. Growth, development and behavior of the
progeny were assessed during lactation. The dams as well as surviving pups were killed on day 22-24 post partum. Animals scheduled for necropsy on weekend were killed the next weekday.
At the time of sacrifice or death during the study the animals of the P generation were examined for macroscopically visible abnormalities. The main organs were weighed and the organ to body weight ratios calculated. Special attention was paid to the organs of the reproductive system. Histopathology of listed organs was performed in case of macroscopic visible changes. Moreover,
dental mineral analyses (fluoride, calcium and phosphorus) were performed externally. In addition, clinical chemistry investigations, in particular for serum electrolytes, were performed in 10 animals per sex and group as amended to the protocol.
Body weights, food consumption, clinical chemistry data, absolute and relative organ weights and litter parameters were analyzed with the aid of a statistical program to show differences compared to the controls.
RESULTS
High-dose group (1000 mg/kg body weight): There were 7 males and five females that were found dead or killed on humane grounds due to starvation and bad general health condition as a cause of broken off incisors and subsequent disability of food uptake. In addition, one female was killed with dead pups at birth, another one with live pups was killed on lactation day 6 due to
inability to suckle them properly. Teeth trimming were carried out to insure food uptake during mating procedures for as many animals concerned as possible. Mean food consumption and body weight development was decreased during pre-mating (males) and during the lactation period (surviving females). Mean gestation length, (ca. 23.0 days), was not affected. Because of these
unscheduled deaths the number of pregnancies was markedly reduced (12 cf./22 of control). The absolute number of females at term with live pups was reduced (11 cf./21 of control), with lower absolute number of implantations. One dam had dead pups only. However, relative numbers of live pups, the mean number of implatations and birth index, was not adversely affected when
related to the number of females at term with live pups. During early lactation, 4/11 females had to be killed on humane grounds, as they were not able to rear their healthy offspring due to starvation. The remaining 7 females reared their healthy offpring up to the end of the lactation period, however, mean pup body weight gains were significantly decreased from day 14 post partum up to the end of the study. Mean viability index, weaning index, survival rate at day 21 was not affected. There was 1 unreared litter recorded for this group. The pups did not show any macroscopically visible abnormalities.
Apart from significantly decreased total bilirubin levels, clinical pathology was unobstrusive, also with regard to serum electrolytes. Anatomic pathology revealed severe dental lesions (broken off, deformed and white discolored incisors), which were confirmed to contain a 3-fold concentration of fluoride. Fluoride (0.3%) was identified as an impurity of the test compound batch, tested in
this study. Massive bluish discolorations of the whole carcasse and in several inner organs were also detected at necropsy. Microscopy confirmed intratubular pigment storage in the kidneys, increased number of necrotic/apoptotic cells in the liver as a histopathological correlate of clinical starvation, and mixed cellular infiltrations in the submucosal area of the stomach, probably as a
result of irritating effects of the test compound. There were no selective changes in sexual organs that could be related to selective reproductive toxicity in these dose group animals, nor were there any correlates of target organ toxicity.
Mid-dose group (250 mg/kg body weight): There were no premature deaths. No compound related clinical findings were recorded for the females. Four males had broken-off incisors during the late treatment period (weeks 6 -12). However, food consumption, body weight development, mating and reproductive performance, fertility, mean gestation length, rearing and development of their
offspring remained unaffected by administration of the test compound. Clinical chemistry, as well as anatomic pathology (necropsy, organ weights, histopathology) in particular of the sexual organs were generally unobstrusive, apart from pigment storage (dark brownish/or bluish discolorations) in the kidneys.
Low-dose group (62.5 mg/kg body weight): There were no premature deaths. No compound-related clinical signs were recorded in the P-generation male and female animals. Food consumption, body weight development, mating and reproductive performance, fertility, mean gestation length, rearing and development of their offspring remained unaffected by administration of the test compound. Clinical Chemistry, as well as anatomic pathology (necropsy, organ weights, histopathology) in particular of the sexual organs were unobstrusive.
Referenceopen allclose all
Clinical observation: There were no clinical signs at up to and including 250 mg/kg bw/day or in the Control animals administered 10 mL/kg vehicle (distilled, sterile water for injection).
No systemic adverse clinical signs were noted following administration of the test substance daily by oral gavage at 1000 mg/kg bw/day dose level. During the treatment period, dark red discoloration of the faeces was noted in the High dose Main animals administered 1000 mg/kg bw/day on Day 1, and of the urine, on Day 2, where the treatment started on Day 0.
The discoloration of the faeces persisted in the 1000 mg/kg bw/Day Recovery animals for up to 3 days after the last dose administration (up to Day 44) and of the urine, until completion of the recovery phase and termination. Slight to moderate dark pink discoloration of the skin was also noted in the High dose animals after Day 12 of treatment, until the completion of the study up to Day 55 during the Recovery period.
These changes were ascribed to elimination of the test substance or its metabolites through faeces or urine and an expected staining effect.
Neurological assessment: There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli, grip strength or motor activity in the control or treated groups, at the evaluation performed towards the end of the treatment or Recovery periods.
Increased vocalization was observed on occasion in the animals throughout all the dose groups when subjected to the modified Irwin test (functional observation battery). However, no treatment-related differences to the Control, or dose, or gender related response, were noted, and this sign was considered to be without toxicological significance and within the normal biological variation with respect to behaviour, reactions to different type of stimuli or manipulations.
No toxicologically significant changes, or effects considered adverse or related to test item administration were noted in the landing foot splay or grip strength tests. When compared to Control, there were no statistically or toxicologically significant differences in the mean grip strength (g) or landing foot splay (cm) values of the forelimbs or hind limbs in both the Main and Recovery animals.
Ophthalmology: No test item related changes compared to pre-treatment were noted during ophthalmoscopy examination of 5 male and 5 female Control and High dose Main animals during the last week of treatment prior to necropsy (males, Day 26 pm, females, PPD 3 pm), thus no additional evaluation was required in other dose groups or Recovery animals.
Body weight and body weight gain: No adverse effects or test item related changes were noted on the mean body weight and body weight gain values following daily administration of the test substance at dose levels of up to and including 250 mg/kg bw/day.
At 1000 mg/kg bw/day, slightly lower than control body weight gain values were noted, up to approximately -4% in the males and up to approximately -7% in the females, generally without attaining statistical significance. The body weight gain however was similarly lower than control in both High dose Main males and females, but attaining statistical significance when evaluated for the whole period of treatment between Days 0 and 28 in the 1000 mg/kg bw/day Main male (96.75 g vs. 115.50 g, p< 0.05), or during the gestation period between GD0 and GD20 in the 1000 mg/kg bw/day Main females (133.50 vs. 161.1 g, p<0.05, mainly based on possible test item related effects during the second half of pregnancy).
In the Recovery animals, the body weights remained lower than controls, with mean values up to approximately -5%, p<0.05 on Days 28 and 35 in the males, and up to approximately -4%, p<0.01, on Day 55 in the females. No statistically significant differences occurred in the body weight gain of the Recovery animals.
Food consumption: There were no test item-related differences to Control in the mean daily food consumption in any test-item treated Main or Recovery group (62.5, 250, or 1000 mg/kg bw/day) when compared to the Control.
The mean values were either higher than control in the test-item treated groups, or, if lower, the differences noted were minor, showed no dose response, and/or were associated with variations within the group, changes in the study schedule including mating, delivery, or fasting before blood collection for clinical pathology evaluation, unrelated to treatment and with no statistical or toxicological significance.
Clinical pathology
Haematology: No test item related effects, or changes considered toxicologically significant were noted in the haematology parameters evaluated in the Main animals at up to and including 250 mg/kg bw/day dose levels.
At 1000 mg/kg bw/day, WBC increases considered possibly related to the test substance administration were noted in the Main animals evaluated on Day 14, up to 45% and 50% higher than control, p<0.01, in the males and females, respectively. The mean values remained 92% higher than control, p<0.01, in the Recovery males evaluated on Day 56, to become comparable with the physiological values in the Recovery females.
Variations were noted in a few parameters, on occasion attaining statistical significance, including for example, statistically higher MPV in the Main males at all dose levels, but without a clear dose response or a similar response in the Main or Recovery females, lower PT up to -6%, especially in the High dose Main males, but remaining within the physiological ranges, or higher PLT in the High dose Main females only. Evaluation of the mean and individual results in comparison with the Control data did not reveal any test-item related cause of the changes noted, and/or no consistent dose or gender-related response was observed. Therefore, these differences observed between the Control and treated groups were considered to be incidental or individual findings, which were not related to treatment, were generally comparable with the expected physiological range or were with no toxicological significance.
Clinical chemistry: In the Main animals evaluated at the completion on Day 14, an apparent total bilirubin T BIL increase was noted in the 1000 mg/kg bw/day Main High dose males and females (174% and 166%, p<0.05 and 0.01, respectively). The bile acids were also higher than controls in both males and females at 250 and 1000 mg/kg bw/day, attaining statistical significance in the Mid and Main High dose males (43%, p<0.05 and 105% higher than control, p<0.01, at 250 and 1000 mg/kg bw/day) and in the Main High dose females (180%, p<0.01). After a 14-day recovery period, the T-BIL remained up to 36.2% higher than control in the High dose Recovery males and females. This was considered to be related to a possible spectral interference with the analytical method caused by the discolouration of the serum by the test item and not to reflect an adverse effect on the liver function.
In addition, lower albumin/globulin ratio, up to -20%, and lower ALT , up to -23%, were recorded in the Main High dose animals, effects considered potentially related to test item administration at 1000 mg/kg bw/day dose level; the ALT mean value remained up to -53% lower than control in the Recovery animals, however, without attaining statistical significance .
Other clinical chemistry parameters showed on occasion statistically significant variations, however, there was no dose or gender response or the values were within the physiological ranges (including, for example slightly higher than control, but statistically significant Cl- in the Main males at all dose levels, but not in the females, or slightly lower Ca++ in the Main High dose females, but not in the males or in the Recovery animals). For this reason, these variations were not considered toxicologically significant or related to treatment.
Urinalysis: Pink urine was noted in all animals evaluated from the Main Mid dose 250 mg/kg bw/day group (5/5 males and 5/5 females, subgroup B) and red urine, in all the High dose 1000 mg/kg bw/day animals examined (5/5 males and 5/5 females, subgroup B Main and in 5/5 male and 5/5 female Recovery animals), at urinalysis performed prior to necropsy after the animals were placed overnight in metabolic cages for urine collection. These changes were ascribed to elimination of the test substance or its metabolites through urine (urine collection in metabolic cages) and an expected staining effect. The few other minor variations observed did not attain statistical significance and/or were regarded as normal background changes, without toxicological relevance.
Oestrus cycle, reproductive ability assessment and indices: There were no statistically significant differences between the Control and test item-treated groups with regard to reproductive ability or in the mating or gestation indices, or effects considered adverse or toxicologically significant in correlation with the test substance administration.
In all groups, the mating indices were 100%, the fertility indices, 83%, 100%, 92% and 92% due to 2, 0, 1 and 1/12 non-pregnant females, respectively, in Groups 1, 2, 3 and 4, and the gestation indices, 100%, with the exception of the High dose group in which the gestation index was incidentally 91%, due to 1/11 pregnant, but not delivered females in this group, values which are comparable with concurrent control data in Wistar rats.
Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 6 days of pairing (cohabitation).
Evaluation of the gestation, parturition and post-partal period: No test item effect on the duration of pregnancy or abnormalities in the gestation outcome ascribed to the treatment were observed.
The mean duration of pregnancy was similar in the control and test item treated groups and varied from 22.00 days (22 days) in the Controls, 22.17 days (22 to 23 days) in the Low 62.5 mg/kg bw/day group, 22.45 days (p<0.05, 22 to 23 days) in the Mid dose 250 mg/kg bw/day group, to 22.09 days (22 to 23 days) in the 1000 mg/kg bw/day High dose group. These values were comparable with the contemporaneous historical control data (gestation length: 22.40 ± 0.53 days, range: 22 to 23 days, n = 22) and the minor differences to control were not regarded as toxicologically relevant, although the mean duration of pregnancy attained statistical significance in the Mid 250 mg/kg bw/day group only. All the parturitions were normal.
The number of corpora lutea and implantation sites were comparable in the treated groups up to and including 1000 mg/kg bw/day with the mean value recorded in the Control group.
There were no statistically significant differences or effects that could be ascribed to treatment on the pre/post-implantation, post-natal or total mortality values (%) at up to and including 250 mg/kg bw/day.
At 1000 mg/kg bw/day, the intrauterine mortality, post-natal mortality and subsequently the total mortality were higher than control, however, only the post-natal mortality attaining statistical significance (intrauterine mortality: 28.19% vs. 11.44%, 146.4% higher than control; post-natal mortality: 16.34%, p<0.05, vs. 0.71%; total mortality: 33.65% vs. 12.03%, 179.6% higher than control in the High dose vs. the control group), effects considered possibly related to the test substance administration at the High dose level, although a role of the individual variability cannot be excluded (females 4501, 4503 and 4506).
Macroscopic Findings: Test item-related pink discoloration was observed in numerous organs/tissues including the stomach, small and large intestine, kidneys, pancreas, testes, seminal vesicles, prostate, epididymides, vagina, uterus and cervix, mandibular salivary gland, mandibular and mesenteric lymph nodes, thymus, tongue, trachea, urinary bladder, eye, lacrimal gland, skin, oral, nasal and conjunctival mucosa, fatty (subcutaneous abdominal and thoracic) and connective tissues (subcutaneous abdominal, thoracic, intramuscular) at necropsy. Affected animals included 24/24 rats from the High Dose group.
Red fluid content (urine) in the urinary bladder was noted in 13/24 High dose Main animals (12/12 males and 1/12 female) and red discolored digestive content was observed in 2/24 animals (2/12 males) in the stomach, small and large intestine also regarded as test item-related.
In the Mid dose animals, pink discoloration of the stomach (1/24), kidneys (1/24) or trachea (5/24) was also seen with low incidence.
These changes were considered due to the expected coloring effect of the test item (dye). All other macroscopic changes were regarded as incidental.
Microscopic Findings: Test item-related findings were microscopically observed in the kidneys and stomach, and were in correlation with the observations noted at necropsy. The changes were characterized as minimal to mild degeneration/regeneration of the cortical tubules in the kidneys and epithelial cells of the glandular mucosa in the stomach.
In the kidneys, tubular degeneration was associated with eosinophilic intracytoplasmic droplets of variable size and/or vacuolated cytoplasm of the epithelial cells. Regenerating tubular epithelium was characterized by basophilia, surrounded by minimal mononuclear infiltrate and regenerating cells showed lower cuboidal forms. Affected animals included 22/24 from the High Dose groups (10 males, 12 females). In addition, minimal tubular degeneration was also noted in 3/12 Mid Dose males. No tubular degeneration was seen in the Mid Dose females examined or in any rats of the Low Dose animals.
In the stomach, eosinophilic droplets of variable size were present in the cytoplasm of glandular epithelial cells. In addition, these alterations were accompanied with minimal to mild influx of inflammatory cells in the glandular submucosa and mucosa in majority of animals, and sporadically with formation of submucosal lymphoid follicles. The regeneration of the epithelial cells lining glandular mucosa resulted in transition into lower cuboidal epithelial cells with the increase of cytoplasmic basophilia and mitotic figures. Altered animals included 24/24 from the High Dose groups.
At additional histopathological evaluation of the stomach from Low and Mid Dose groups, minimal to mild epithelial degeneration/regeneration of the glandular mucosa was noted in 5/12 Mid Dose males and 3/12 Mid Dose females. This change was accompanied with minimal inflammatory infiltrate in 5/12 Mid Dose males and lymphoid follicle formation in the male 3001. In 2/12 Low Dose males, minimal epithelial degeneration/regeneration was also observed however without influx of inflammatory cells.
There was no evidence of Reactive Red F03-0318-related histopathological findings in the High Dose animals in the reproductive organs. Histopathological evaluation of the male gonads as well as testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoons were similar in Control and High Dose males. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both Control and High Dose females.
All other macroscopic changes were regarded as incidental or terminal procedure-related.
RECOVERY (DAY 56)
Macroscopic Findings: Pink discoloration related to the administered test item was macroscopically noted in variety of organs/tissues including the stomach, small and large intestine, adrenal gland, kidneys, pancreas, testes, seminal vesicles, prostate, epididymides, vagina, uterus and cervix, mandibular salivary gland, mandibular and mesenteric lymph nodes, thymus, tongue, trachea, urinary bladder, eye, lacrimal gland, skin, oral, nasal and conjunctival mucosa, fatty (subcutaneous abdominal and thoracic) and connective tissues (subcutaneous abdominal, thoracic, intramuscular).
In addition, red digestive content of the stomach, small and large intestines and red fluid content in the urinary bladder were also considered to be test item-related.
All other macroscopic changes were regarded as incidental or terminal procedure-related.
Microscopic Findings: A residual effect of the test item administration was still present in the kidneys and stomach, and correlated with the macroscopic findings noted at necropsy. Minimal to mild degeneration/regeneration of the cortical tubules in the kidneys was noted in 10/10 High Dose rats. Minimal degree of severity of degeneration/regeneration of the glandular mucosa in the stomach was in 8/10 High Dose animals. The mitotic index was decreased and the influx of the inflammatory cells or formation of the lymphoid follicles were not recorded. All other changes with low incidence and/or severity, observed in control and/or treated animals were considered to be incidental or procedure-related.
In summary, a daily oral (gavage) administration of Reactive Red F03-0318 to Wistar RJHan:WI rats under the conditions of this study results in pink/red discoloration of numerous organs/tissues observed at necropsy and degeneration/regeneration of the cortical tubules in the kidneys and epithelial cells of the glandular mucosa in the stomach, microscopically present. The severity and/or incidence of these changes indicated a relationship to dose.
In the kidneys, test item-related findings mentioned above were present at dose levels of 250 and 1000 mg/kg bw/day however there was no evidence of degeneration/regeneration in the Mid Dose females (250 mg/kg bw/day).
In the stomach, the findings associated with the administration of the test item were also recorded at dose levels of 250 and 1000 mg/kg bw. There was clear dose responded decrease in the severity when compared High Dose (1000 mg/kg bw/day) and Mid Dose (250 mg/kg bw/day) animals. Also, minimal to mild inflammatory influx in the submucosa was decreased to minimal degree in Mid Dose (250 mg/kg bw/day) animals.
The formation of the submucosal lymphoid follicles was not present in rats dosed at 250 mg/kg bw/day. During additional histopathological examination, minimal epithelial degeneration/regeneration without influx of inflammatory cells was observed in two Low Dose males.
After a 14 day of recovery period, a residual effect of treatment was microscopically seen in the kidneys and stomach in the High Dose groups. In the kidneys, decreased severity of the test item-related microscopic alterations from mild to minimal was more evident in the males compared to the females. Cessation of treatment for 14 days resulted in almost completed regression of the test item-related lesions in the stomach. The decrease of the severity from mild to minimal was observed in all High Dose animals. Also, mitotic index has been decreased and inflammatory influx as well as formation of the submucosal lymphoid follicles, were no more observed.
Organ weights: In the Main animals, evaluated immediately after completion of the treatment, at necropsy on Day 28 (males) or PPD5 (females), there were no toxicologically relevant changes in the organ weight values, or effects considered adverse or related to the test substance administration at up to and including 250 mg/kg bw/day.
At 1000 mg/kg bw/day, slight increases in the kidneys, adrenals or liver weights were noted in both males and females, without attaining statistical significance in the Main animals at evaluation of the absolute mean group values. As the adrenals and liver weights showed no dose response and the mean values were comparable with the physiological values and no microscopic findings were noted at histopathological evaluation, their weight variations were regarded as incidental, not toxicologically significant and unrelated to treatment.
The absolute kidneys weight however was 14% higher than control in the High dose Main males, without attaining statistical significance, but with a relative weight related to the body weight 20% higher than control, p<0.01, and 15% higher, p<0.05, when adjusted for the brain weight. No similar effects were observed in the High dose Main females, however, kidneys weights were higher than control in both male and female Recovery animals. In view of the microscopic findings recorded, these kidneys weight variations were considered to reflect a possible test substance-effect at 1000 mg/kg bw/day administered under the conditions of this study.
Other absolute organ weights or relative to the body and/or brain weights were similar in the control and test item treated groups, or showed minor variations, ascribed to biological variability.
At 1000 mg/kg bw/day, the mean number of viable pups was -26.1% lower than control on PND0 and -30.4% lower on PND4, attaining statistical significance when evaluated for the total number of pups, results correlated with the higher post-natal and total mortality observed in the High dose dams. Survival index was -18.3% lower than control in the High dose group on PND0 and -26.2% on PND4, although without attaining statistical significance.
The pups found dead and cannibalised were counted and sex determined if possible, but not further examined macroscopically. No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. Findings such as cold to touch, occasional cannibalization or lack of suckling were noted with low incidence, within the physiological range expected in the population of Wistar rats and considered without toxicological significance, or to reflect a test item or adverse effect.
In the Control group (131 pups examined), 3 pups were found dead on PND0, with negative pulmonary floating test (possibly stillborn), and another pup was found dead on PND2 without displaying any clinical signs prior to their death.
In the 62.5 mg/kg bw/day Low dose group with 155 pups examined, 3 pups were found dead on PND0, with negative pulmonary floating test (possibly stillborn), 4 were not suckled on PND0, then survived with no clinical signs until the scheduled necropsy on PND4, and another pup was found dead on PND3 without displaying any clinical signs prior to their death.
In the 250 mg/kg bw/day Mid dose group, with 141 pups examined, 4 pups were found dead on PND0, with negative floating test (possibly stillborn), one was found dead and had positive pulmonary floating test, 5 were cannibalized and 2 cold or not suckled.
In the 1000 mg/kg bw/day High dose group with 113 pups examined, 7 pups were found dead on PND0, with negative floating test (possibly stillborn), 2 were found dead on PND0, with positive pulmonary floating test, 1 was found dead on PND1, and 6 were cold, not suckled and/or cannibalized.
There were no adverse effects, or effects considered related to test item administration on the sex ratios, and no statistical or toxicological significant differences to control were noted.
Body weight and body weight gain: There were no effects considered adverse on the offspring weight or weight gain following administration of the test substance at 62.5 and 250 mg/kg bw/day to parental generation under the conditions of this study. When evaluated per litter basis or for all pups, the mean litter weights on PND 0 and 4, pups body weight and/or body weight gain evaluated on PND 0 and 4 showed no differences compared to controls in the F1 generation that could be considered toxicologically relevant or related to test item administration.
At 1000 mg/kg bw/day, when evaluated per litter basis, there were no statistically significant differences to control, however, the mean litter weights were lower than control on both PND0 and PND4 (on PND0, 11 litters, 65.7 g, vs. 10 litters, 90.1 g, thus -27.1% lower than controls mean litter weight in the High dose group; on PND4, 9 litters, 112.9 g, vs. 10 litters, 140.1 g, thus -19.4% lower than controls mean litter weight in the High dose group).
When evaluated for all pups, the mean body weights were statistically significant, slightly lower up to -7% than control: on PND0, 6.39 g vs. 6.88 g, and on PND4, 10.36 g vs. 11.03 g. As the differences were minor and the dose response was not clear, a test item related adverse effect on the offspring development following administration of 1000 mg/kg bw/day to the parental generation in the conditions of this study could not be ascertained, although it cannot be excluded.
Pathology evaluation
FOUND DEAD
F1 Generation: Twenty three pups were found dead between Days 0-3 post partum. Necropsy was performed on 4, 4, 5 and 10 pups from the Control, Low, Mid and High Dose groups, respectively.
Negative floating test was observed in 3/4, 3/4, 4/5 and 7/10 animals from the Control, Low, Mid and High Dose groups. Positive floating test was observed in 1/5 and 2/10 Mid and High dose of the pups found dead on PND 0 (no floating test was required on the remaining pups which died between PND1-3). White liquid/creamy material in the stomach was present in 1/4, 1/4 and 1/10 pups from Control, Low and High Dose. The stomach was empty in 1/5 and 2/10 offsprings from the Mid and High Dose groups. A cause of death of these pups could not be determined.
TERMINAL (MAIN)
F1 Generation: Macroscopic Findings:
No macroscopic changes were seen in F1 offspring generation euthanized and examined externally at scheduled termination on PND 4.
Measured concentrations of the dosing solutions
Analytical occasion |
Nominal concentration mg/mL |
Measured concentrations with the 95% confidence intervals, mg/mL |
Measure concentration in the percentage of the nominal |
29 April 2011 (first week of treatment) |
6.25 |
6.18±0.52 |
99 |
25 |
25.7±0.66 |
103 |
|
100 |
102±2.83 |
102 |
|
20 May 2011 (mid term) |
6.25 |
6.09±0.19 |
97 |
25 |
25.6±1.19 |
102 |
|
100 |
107±1.03 |
107 |
|
09 June 2011 (last week of treatment) |
6.25 |
6.07±0.12 |
97 |
25 |
23.9±1.23 |
95 |
|
100 |
99.8±5.38 |
100 |
Selected Clinical Chemistry Parameters, Main animals
|
P (Parent) Male – Main |
P (Parent) Female – Main |
|||||||
DOSE GROUP |
ALT U/L |
T-BILμmol/L |
A/G |
Bile acidsμmol/L |
ALT U/L |
T-BILμmol/L |
A/G |
Bile acidsμmol/L |
|
Control (1) |
MEAN |
51.40 |
8.48 |
1.28 |
16.68 |
57 |
7 |
2 |
13 |
SD |
5.03 |
4.05 |
0.08 |
5.34 |
8 |
4 |
0 |
7 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
62.5 mg/kg bw/day (2) |
MEAN |
48.00 |
6.36 |
1.22 |
15.96 |
55 |
8 |
1 |
21 |
SD |
10.02 |
2.88 |
0.11 |
2.18 |
7 |
2 |
0 |
4 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
±% |
-7 |
-25 |
-5 |
-4 |
-2 |
14 |
-8 |
61 |
|
250 mg/kg bw/day (3) |
MEAN |
54.00 |
7.44 |
1.24 |
23.78 |
49 |
8 |
2 |
22 |
SD |
8.15 |
0.95 |
0.09 |
2.44 |
7 |
2 |
0 |
9 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
±% |
5 |
-12 |
-3 |
43 |
-14 |
10 |
1 |
71 |
|
1000 mg/kg bw/day (4) |
MEAN |
41.00 |
23.20 |
1.06 |
34.20 |
44 |
19 |
1 |
36 |
SD |
3.94 |
6.47 |
0.05 |
9.59 |
8 |
3 |
0 |
10 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
±% |
-20 |
174 |
-17 |
105 |
-23 |
166 |
-20 |
180 |
|
|
* |
* |
** |
** |
* |
** |
** |
** |
|
DN |
U |
DN |
U |
DN |
DN |
DN |
DN |
REMARKS:
±% = Percent Deviation Versus Control
NS = Not Significant
* = p<0.05
** = p<0.01
U = Mann-Whitney U – test Versus Control
DN = Duncan’s multiple range test
Selected Clinical Chemistry Parameters, Recovery Main animals
|
Male – Recovery |
Female – Recovery |
|||||||
DOSE GROUP |
AST U/L |
T-BILμmol/L |
A/G |
Bile acidsμmol/L |
AST U/L |
T-BILμmol/L |
A/G |
Bile acidsμmol/L |
|
Control (1) |
MEAN |
142.60 |
4.92 |
0.94 |
9.44 |
122 |
6 |
1 |
8 |
SD |
15.13 |
0.77 |
0.05 |
1.97 |
28 |
1 |
0 |
2 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
1000 mg/kg bw/day (4) |
MEAN |
117.00 |
6.70 |
1.10 |
9.04 |
98 |
7 |
1 |
8 |
SD |
21.68 |
0.35 |
0.10 |
2.15 |
14 |
0 |
0 |
2 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
±% |
-18 |
36.2 |
17 |
-4 |
-20 |
20 |
13 |
6 |
|
|
NS |
** |
* |
NS |
NS |
* |
NS |
NS |
REMARKS:
±% = Percent Deviation Versus Control
NS = Not Significant
* = p<0.05
** = p<0.01
T - test versus control
TABULAR SUMMARY REPORT
|
Dose (mg/kg bw/day) |
|||
|
Control |
52.5 |
250 |
1000 |
Pairs started (N) |
12 |
12 |
12 |
12 |
Surviving females showing evidence of copulation (N) |
12/12 |
12/12 |
12/12 |
12/12 |
Females achieving pregnancy (N) |
10/12 |
12/12 |
11/12 |
11/12 |
Conceiving days 1 – 5 (N) |
12 |
12 |
11 |
12 |
Conceiving days 6 (N) |
0 |
0 |
1 |
0 |
Pregnancy≤21 days (N) |
0 |
0 |
0 |
0 |
Pregnancy = 22 days (N) |
10 |
10 |
6 |
10 |
Pregnancy≥23 days (N) |
0 |
2 |
5 |
1 |
Dams with live young born (N) |
10/10 |
12/12 |
11/11 |
10/11 |
Dams with live young at PN4 (N) |
10/10 |
12/12 |
11/11 |
9/11 |
Corpora lutea/dam (mean) |
19.40 |
20.92 |
22.36 |
19.45 |
Implantations/dam (mean) |
14.50 |
14.17 |
14.00 |
13.36 |
Live pups/dam at birth (mean) |
12.80 |
12.67 |
12.45 |
9.64 |
Live pups/dam at day 4 (mean) |
12.80 |
12.58 |
12.00 |
8.91 |
Sex ratio at birth (mean) |
42.87 |
53.95 |
50.14 |
47.05 |
Sex ratio at PN4 (mean) |
42.87 |
53.72 |
49.27 |
34.24 |
Litter weight at birth (mean) |
90.1 |
85.9 |
91.9 |
65.7 |
Litter weight at day 4 (mean) |
140.1 |
134.2 |
144.4 |
112.9 |
Pup weight at birth (litter mean) |
6.90 |
6.74 |
7.18 |
6.54 |
Pup weight at day 4 (litter mean) |
11.12 |
10.85 |
12.21 |
10.12 |
STRUCTURALLY ABNORMAL PUPS |
||||
Dams with 0/ Dams with live born |
10/10 |
12/12 |
11/11 |
10/11 |
Dams with 1 or≥2 |
0 |
0 |
0 |
0 |
LOSS OF OFFSPRING |
||||
Pre-implantation (corpora lutea minus implantations) |
||||
Females with 0 |
2/11# |
1/12 |
1/11 |
2/11@ |
Females with 1 |
2/11# |
1/12 |
0/11 |
2/11@ |
Females with 2 |
1/11# |
0/12 |
0/11 |
1/11@ |
Females with≥3 |
6/11# |
10/12 |
10/11 |
6/11@ |
Pre-natal/post-implantation (implantation’s minus live births) (intrauterine) |
||||
Females with 0 |
1/10# |
3/12 |
4/11 |
4/11@ |
Females with 1 |
4/10# |
5/12 |
3/11 |
1/11@ |
Females with 2 |
3/10# |
2/12 |
2/11 |
3/11@ |
Females with≥3 |
2/10# |
2/12 |
2/11 |
3/11@ |
Post-natal (live births minus alive at post-natal day 4) |
||||
Females with 0 |
9/10 |
11/12 |
9/11 |
7/11@ |
Females with 1 |
1/10 |
1/12 |
1/11 |
2/11@ |
Females with 2 |
0/10 |
0/12 |
0/11 |
0/11@ |
Females with≥3 |
0/10 |
0/12 |
1/11 |
2/11@ |
# females 1502 had incidentally total pre-implantation mortality (14 corpora lutea and no implantation sites), excluded from statistics.
@ female 4501 has 21 corpora lueta, 14 implantation sites and 1 stillborn.
There were no intercurrent deaths in the control-, low- and mid-dose group animals. In the high dose group (1000 mg/kg body weight), 1 male and 1 female animal was found dead early with unknown pathogenesis. In addition, further 6/28 males and 4/27 females were found dead or had to be killed on human grounds from study week 6-7 onwards. Animal No. 128 was killed by mistake on day 51.
Behavior and health status was not affected in low- and mid-dose group animals with the exception of 4 males exhibiting broken off incisors from week 6 onwards. Several high-dose animals had broken off- and white-discolored incisors, generally starting to occur from study week 6 onwards. Some of those animals developped general clinical signs (stilted gait, hypoactivity, coat bristling, irregular respiration, respiratory sounds diarrhea, snout encrusted blood colored or swollen etc.) and some of those ended up in a general poor condition.
Blue discolored feces were observed in all P-generation male and female animals of the 250 and 1000 mg/kg body weight groups.
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight gain was significantly decreased for high dose animals that had dental problems.
Those high dose animals that were found dead from week 6 onwards or were killed on human grounds did not take up food a few days before death. Mean absolute food consumption in all remaining animals of the high dose group (1000 mg/kg) was slightly to moderately decrerased. This was in line with the lower body weight gains recorded for this group. Hence, relative food consumption was generally comparable in all groups throughout the study, except for high dose females, who exhibited a significant decrease of relative food consumption during the lactation period
REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no test item related differences in the estrous cycle.
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Due to the lower food consumption resulting from broken-off incisors the pregnancy index was lower in high-dose females
The mean number of implantations counted, mean live pups/litter, birth index were comparable in all groups. In addition, supernumerary
implantation sites, percentage of implantations, were not influenced by administration of the test compound.
Mean gestation length was comparable in all groups.
ORGAN WEIGHTS
In high dose males, liver, kidney, testes, epididymides, prostate and seminal vesicles weight were slightly lower, with statistical significance, which was due to the reduction of terminal body weight and hence, not related to target organ toxicity.
The same applied for high dose females, where liver, kidney and uterus weight was slightly lower, with statistical significance.
GROSS PATHOLOGY (PARENTAL ANIMALS)
Males and females from the mid-dose group exhibited kidneys with dark brown discolorations. In addition, the kidneys of one male in this groups was bluish discolored.
The main relevant findings were discolorations in several organs animals of the high dose group. Further major alterations were white discolored or broken incisors in nearly all animals of this group.
HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological findings in parental animals of the high-dose group at terminal killing revealed intratubular pigment in kidneys in 10 male and 5 female animals. Single animals exhibited degenerations or necrosis of tubular cells. Increased number of necrotic/apoptotic cells were found in the liver. Mixed cellular infiltrations in the submucosal area of the stomach were found particular in males.
no effects
CLINICAL SIGNS (OFFSPRING)
no effects
BODY WEIGHT (OFFSPRING)
Mean body weight of live pups during lactation was significantly decreased in the high dose offspring (1000 mg/kg bw.) from day 14, post partum onwards. Mean body weight was not affected in any other group
This study is read across to the structural analogue 02, which is considered to be structurally equivalent to the substance to be registered. The read-across justification can be found in section 13.
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
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
No teratogenic or developmental effects were noted in reproductive studies with structural analogues.
A modified one-generation study was carried out with structural analogue 02. This test substance contained a higher content (0.3%) of fluoride as impurity in the test substance resulting in fragility of the incisors and a very low food uptake in high-dose dams. Consequently, the survival rate in the high-dose group was rather low. However, daily oral administration of the test substance to rats during the premating, mating, gestation and lactation period at dose levels of 62.5 or 250 mg/kg body weight/day, where the absolute fluoride uptake was distinctly lower, did not affect food consumption, body weight development, male or female mating/reproductive performance, fertility, gestation length as well as development of their progeny. Daily oral administrations of 1000 mg/kg body weight were well tolerated in rats within the first 5 weeks of treatment, but thereafter, from week 6 onwards, caused mortality due to dental lesions with subsequent disability of food uptake and starvation (clinical picture of dental fluorosis). Consequently, due to the fact that no fluoride is contained in Reactive Red F66813, a NOAEL of 1000 mg/kg body weight is expected for the test substance for parental reproductive performance and their offspring.
In a developmental screening study with structural analogue 04, the only effects noted were unspecific toxic effects resulting in higher intrauterine, postnatal and total foetal mortality at 1000 mg/kg bw/day. In this study, for reproductive toxicity, the no observed effect level (NOEL) for parental/adult effects is considered to be 1000 mg/kg bw/day for the males and 250 mg/kg bw/day for the females, due to the higher intrauterine, postnatal and total foetal mortality at 1000 mg/kg bw/day, correlated with a NOEL of 250 mg/kg bw/day for F1 offspring, based on a slight secondary effect on pups survival and birth weight at 1000 mg/kg bw/day. No structural adverse effects were observed in the surviving pups at up to and including 1000 mg/kg bw/day, under the conditions of this study. The relation to test substance administration is questionable and this effect is not considered to be an adverse effect related to substance toxicity, hence, the NOAEL was considered to be 1000 mg/kg bw/day for male and female rats and their offspring.
Effects on developmental toxicity
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 1 000 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
No classification necessary
Additional information
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