Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 279-356-6 | CAS number: 79953-85-8
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
![](https://echa.europa.eu/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
Toxicity to reproduction
Administrative data
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From Nov 2021 to Oct 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Cross-reference
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From Nov 2021 to Oct 2022
- 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)
- Version / remarks:
- 29 July 2016
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- TEST MATERIAL
Batch no. 0004079400
CAS no. 79953-85-8
Purity 99.4 wt%
Expiry/Retest date 27 August 2022
Appearance Yellow powder
Storage conditions Room temperature - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Wistar Hannover rat
- Details on species / strain selection:
- The Wistar Hannover rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.
The oral route was selected as it is a possible route of exposure of the test item in man and has been specifically requested by the Regulatory Authorities. - Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females: nulliparous and non-pregnant
- Age at study initiation: 9 - 11 weeks old
- Weight at acquisition: 212-230 g for males and 182-193 g for females
- Housing arrival - mating: animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm, nesting material was provided inside suitable bedding bags and changed at least twice a week
- Housing during mating: animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor, each cage tray held absorbent material which was inspected and changed daily
- Housing after mating: the males were re-caged as they were before mating, females were transferred to individual solid bottomed cages for the gestation period, birth and lactation, nesting material was provided inside suitable bedding bags, additional suitable nesting material was provided as necessary, nesting material was changed at least twice a week
- Diet: ad libitum, laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy)
- Water: ad libitum, via water bottles
- Acclimation period: approx 2 weeks
DETAILS OF FOOD AND WATER QUALITY:
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-60
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 21 Dec 2021 (allocation) To: 17 Feb 2022 (last day of necropsy) - Route of administration:
- oral: gavage
- Vehicle:
- CMC (carboxymethyl cellulose)
- Remarks:
- 0.5% CMC in water, softened by reverse osmosis
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Dose volume: 10 mL/kg body weight, dose volumes for males and females were adjusted once per week for each animal according to the last recorded body weight, dose volumes for females during the gestation and lactation periods were calculated according to the last recorded body weight
- Dosing preparation: preparations were made daily, preparations were kept under magnetic stirring for at least 16 hours at room temperature and up to completion of dosing - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The analytical method was validated in a separate study in the range from 10 to 100 mg/mL. In the same study, a 28 hour stability at room temperature and a 8-day stability at 2- 8°C were verified in the range from 10 to 100 mg/mL. Samples of the formulations prepared during the current study (the first and the last week of treatment where possible) were analysed to check the concentration and homogeneity. Results of the analyses were within the acceptability limits for suspensions (85-115% for concentration and CV < 10% for homogeneity).
- Duration of treatment / exposure:
- Males were treated for 2 consecutive weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 39 days.
Females were treated for 2 consecutive weeks prior to pairing, during pairing and throughout the gestation and lactation periods until day 13 post partum, for a period comprising from 51 to 55 days. - Frequency of treatment:
- daily
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Group 1
Control - Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- Group 2
Low-level dose - Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Remarks:
- Group 3
Medium-level dose - Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Remarks:
- Group 4
High-level dose - No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: dose levels of 100, 300 and 1000 mg/kg/day were selected based on information from subacute oral toxicity studies with structurally related pigments. 1000 mg/kg bw is the maximimum dose prescribed in the OECD testing guideline.
- Rationale for animal assignment: rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights., individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed 5 of one sex per cage
- Fasting period before blood sampling for clinical biochemistry: yes - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Mortality: checked twice daily
- Clinical signs: once before commencement of treatment and at least once daily during the study
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of treatment and at least once a week thereafter
- Observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions (see table No. 1 for observed parameters and evaluation scale)
- Animals were examined in an open arena for a minimum of three minutes
BODY WEIGHT: Yes
- Time schedule for examinations: males were weighed weekly from allocation to termination, females were weighed weekly from allocation to positive identification of mating and on days 0, 7, 14 and 20 post coitum, dams were also weighed on days 1, 4, 7, 13 post partum and just before to necropsy
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- the weight of food consumed by each cage of males and females were recorded weekly (whenever possible) during the pre-mating period starting from day 1 of dosing up to mating
- Individual food consumption for mated females were measured on gestation days 7, 14 and 20 post coitum starting from day 0 post coitum and on day 7 and 13 post partum starting from day 1 post partum
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment period
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes (except for coagulation test in males)
- How many animals: 5 males and 5 females (with viable litters if possible), randomly selected
- Parameters checked in table No. 2 were examined
- Coagulation parameters investigated: prothrombin time and activated partial thromboplastin time
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment period
- Animals fasted: Yes
- How many animals: 5 males and 5 females (with viable litters if possible), randomly selected
- Parameters checked in table No. 3 were examined
SERUM HORMONES: Yes
- Time of blood sample collection: at termination, timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days
- Animals fasted: Yes
- How many animals: all parental animals, each litter (1 sample for males and 1 sample for females, when possible), blood samples from different pups was pooled to obtain the required volume
- Hormones analyzed: serum levels of total thyroxine (total T4) and thyroid stimulating hormone (TSH)
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: once during study towards end of treatment, for males on day 39 and for females (with viable litters) on day 12 post partum
- Number of animals: 5 males and 5 females randomly selected from each dose group
- Studies conducted: grip strength and sensory reactivity to stimuli, motor activity assessment
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
- Necropsy: the clinical history of adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices), changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination
- All females were examined also for the following: number of visible implantation sites (pregnant animals), number of corpora lutea (pregnant animals)
- Pups: all pups found dead in the cage were examined for external and internal abnormalities, all culled pups sacrificed at day 4 post partum were subjected to an external examination, all live pups sacrificed on day 14 post partum were examined for external abnormalities, gonads were inspected from all pups in order to confirm the sex previously determined by external examination
ORGAN WEIGHTS:
- Parental animals: from all animals completing the scheduled test period, the organs indicated in table No. 4 were dissected free of fat and weighed, the ratios of organ weight to body weight was calculated for each animal
- Pups at day 14 post partum: thyroid was weighed from one male and one female pup selected for blood collection and preserved in 10% neutral buffered formalin, the thyroid weight was determined after fixation
HISTOPATHOLOGY: Yes
- Samples of all tissues (parental animals) required for histopathological examination are listed in table No. 4
- Samples of the tissue were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol)
- After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin, in addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS), the morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed
The examination was restricted as detailed below:
(i) Sexual organs (cervix, clitoral gland, ovaries, uterus and vagina) and thyroid glands from all parental females
(ii) Sexual organs (coagulation gland, epididymides, preputial gland, prostate gland, seminal vesicles and testes) and thyroid glands from all parental males
(iii) Tissues specified in section 4.5.7 from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term
(iv) Morphological evaluation of the seminiferous epithelium (staging of the spermatogenic cycle) from all males in control and high dose groups
(v) All abnormalities in all groups - Other examinations:
- PARTURITION AND GESTATION LENGTH:
- Parturition check from day 20 - 25 post coitum, three rimes a day during working day or twice daily during weekends and Public Holidays
- Females which did not give birth after 25 days of post coitum period were sacrificed shortly after
- Gestation length calculated as the time between the day of successful mating (day 0 post coitum) and the day of birth
- Day of birth was defined as day 0 post partum
PUPS IDENTIFICATION, WEIGHT AND OBSERVATIONS:
- As soon as possible after parturition was considered complete (day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified
- Live pups were individually weighed on days 1, 4, 7 and 13 post partum
- Pups dying during the lactation period were weighed before the despatch to necropsy
- Observation was performed once daily for all litters from day 0 post partum until termination
- After culling, all pups were sacrificed with the dams on Day 14 post partum
ANOGENITAL DISTANCE (AGD):
- For each pup measured on day 1 post partum
- AGD was normalized to the cube root of body weight collected on day 1 post partum
NIPPLE COUNT CHECK:
- presence of nipples/areolae in male pups was checked on day 13 post partum
REPRODUCTIVE INDICES:
- Males: copulation index, fertility index
- Females: copulatory index, fertility index, pre-natal loss, post-natal loss at day 0, 4 and 13 post partum
- Pre coital interval
- Sex ratio of litter - Statistics:
- Standard deviations were calculated as appropriate.
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t-test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off. - Clinical signs:
- no effects observed
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One control female was sacrificed for humane reason on Day 22 of gestation phase due to difficulties in parturition.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Body weight and body weight gain for male and female animals were comparable between treated and control groups through the study. The statistically significant decrease in body weight gain recorded in high dose males and mid-dose females were considered incidental.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Food consumption was unaffected by treatment in both genders during the study.
- Food efficiency:
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- he statistically significant differences between contrTol and treated animals (neutrophils in males and platelets in females) were recorded only in animals dosed at 300 mg/kg/day, therefore they were considered to be incidental.
- Clinical biochemistry findings:
- no effects observed
- Endocrine findings:
- no effects observed
- Description (incidence and severity):
- The determination of T4 and TSH was performed on:
- Samples from all parental males from all groups
- Samples from pups on Day 14 post partum.
No treatment-related changes were recorded. Thyroid stimulating hormone was statistically significantly lower than controls in parental males (31%). This is due to 2 control animals, which showed values above the range of historical data, therefore the differences observed has no biological/toxicological significance.
The oestrous cyclicity of the treated females monitored during the pre-mating period, for a total of 14 days, was not affected by treatment. The mean number of oestrous cycles observed in treated animals and control group was comparable.
No differences were recorded in pre-coital interval and copulation plugs between treated and control groups. Copulatory index and fertility index did not show any treatment-related differences. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- Observation of treated animals at removal from the cage and in an open arena did not reveal significant changes when compared to controls.
No alterations in motor activity, grip strength, landing footsplay and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related macroscopic observations at the end of the treatment period. Any macroscopic observations were within the range of occasionally observed and expected spontaneous changes in rats of the same age and therefore considered unrelated to treatment.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related microscopic observations at the end of the treatment period. There were no test item-related microscopic observations in the testis (stage were evaluation on PAS-stained slides). Any microscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- effects observed, treatment-related
- Dose descriptor:
- NOAEL
- Remarks:
- systemic toxicity
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Critical effects observed:
- no
- Conclusions:
- The oral administration of the test substance by gavage to male and female Wistar rats revealed no signs of systemic toxicity up to the highest tested dose level. Therefore, a NOAEL for general systemic toxicity was set to 1000 mg/kg bw/day.
- Executive summary:
Under the conditions of the OECD TG 422 and in compliance with GLP, a combined repeated dose toxicity study with the reproductive/developmental screening test was conducted in male and female Wistar rats. The test substance was administered daily as a solution to groups of 10 male and 10 female Wistar rats (P0 animals) by gavage at dose levels of 0 mg/kg bw/d (control; test group 1), 100 mg/kg bw/d (test group 2), 300 mg/kg bw/d (test group 3) and 1000 mg/kg bw/d (test group 4). 0.5% carboxymethylcellulose served as vehicle, control animals were dosed daily with the vehicle only. All doses were administered at a constant volume of 10 mL/kg body weight. For males, the duration of treatment covered a 2-week premating period and pairing with females until the day before necropsy (a total of 39 days). Females were treated for 2 consecutive weeks prior to pairing, during pairing and throughout the gestation and lactation periods until day 13 post partum, for a period comprised from 51 to 55 days.
No test-item related mortality occurred throughout the study and no treatment-related clinical signs were noted during the study. No signs of neurotoxicity (weekly observations at removal from the cage and in an open arena, alterations in motor activity, grip strength and sensory reactivity to stimuli) were observed during the study in parental males and females. No differences in body weight and food consumption were observed in treated animals, compared to the control group. No treatment-related changes were observed in haematological (including coagulation and blood clotting time) or clinical chemistry parameters. Thyroid hormone evaluation in parental animals and pups sacrificed on day 14 post partum did not show treatment-related changes. No treatment-related changes were observed in terminal body weight or absolute and relative organ weights of treated animals of both sexes, when compared to the controls. No treatment-related changes were observed at post mortem macroscopic observations and microscopic evaluation. No treatment-related changes were observed in reproductive and developmental parameters.
Copulatory and fertility indices did not show any treatment related differences among treated and control groups. Parturition, lactation, implantation, litter data and sex ratio did not show changes. No differences in the anogenital distance (normalised value) and no nipple were seen between control and treated groups both for male and female pups. Necropsy findings and thyroid weight in pups did not reveal any treatment-related effect.
Therefore, the no observed adverse effect level (NOAEL) for general systemic toxicity was determined to be 1000 mg/kg bw/day for male and female Wistar rats.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 022
- Report date:
- 2022
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- 29 July 2016
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
Test material
- Reference substance name:
- 3,3'-[(2-chloro-5-methyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-[2-(4-chlorophenoxy)-5-(trifluoromethyl)phenyl]benzamide]
- EC Number:
- 279-356-6
- EC Name:
- 3,3'-[(2-chloro-5-methyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-[2-(4-chlorophenoxy)-5-(trifluoromethyl)phenyl]benzamide]
- Cas Number:
- 79953-85-8
- Molecular formula:
- C55H37Cl5F6N8O8
- IUPAC Name:
- 3,3'-{(2-chloro-5-methyl-1,4-phenylene)bis[imino(1,3-dioxobutane-2,1-diyl)diazene-2,1-diyl]}bis{4-chloro-N-[2-(4-chlorophenoxy)-5-(trifluoromethyl)phenyl]benzamide}
- Test material form:
- solid: nanoform, no surface treatment
- Details on test material:
- BET = 71.4 m2/g
Batch 0004079400
Purity 99.4%
Storage stability: The stability of the test substance under storage conditions was guaranteed until 27 Aug 2022 as indicated by the sponsor, and the sponsor holds this responsibility.
Constituent 1
- Specific details on test material used for the study:
- TEST MATERIAL
Batch no. 0004079400
CAS no. 79953-85-8
Purity 99.4 wt%
Expiry/Retest date 27 August 2022
Appearance Yellow powder
Storage conditions Room temperature
Test animals
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- Wistar Hannover rat
- Details on species / strain selection:
- The Wistar Hannover rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.
The oral route was selected as it is a possible route of exposure of the test item in man and has been specifically requested by the Regulatory Authorities. - Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females: nulliparous and non-pregnant
- Age at study initiation: 9 - 11 weeks old
- Weight at acquisition: 212-230 g for males and 182-193 g for females
- Housing arrival - mating: animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm, nesting material was provided inside suitable bedding bags and changed at least twice a week
- Housing during mating: animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor, each cage tray held absorbent material which was inspected and changed daily
- Housing after mating: the males were re-caged as they were before mating, females were transferred to individual solid bottomed cages for the gestation period, birth and lactation, nesting material was provided inside suitable bedding bags, additional suitable nesting material was provided as necessary, nesting material was changed at least twice a week
- Diet: ad libitum, laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy)
- Water: ad libitum, via water bottles
- Acclimation period: approx. 2 weeks
DETAILS OF FOOD AND WATER QUALITY:
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-60
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 21 Dec 2021 (allocation) To: 17 Feb 2022 (last day of necropsy)
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- CMC (carboxymethyl cellulose)
- Remarks:
- 0.5% CMC in water, softened by reverse osmosis
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Dose volume: 10 mL/kg body weight, dose volumes for males and females were adjusted once per week for each animal according to the last recorded body weight, dose volumes for females during the gestation and lactation periods were calculated according to the last recorded body weight
- Dosing preparation: preparations were made daily, preparations were kept under magnetic stirring for at least 16 hours at room temperature and up to completion of dosing - Details on mating procedure:
- - M/F ratio per cage: monogamous (one male to one female)
- Length of cohabitation: female was paired with the same male until positive identification of mating
- Proof of pregnancy: spermidentification, vaginal plug in situ or copulation plugs found in the cage tray, day of successful mating was referred to as day 0 post coitum
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm), nesting material was provided inside suitable bedding bags, additional suitable nesting material was provided as necessary, nesting material was changed at least twice a week - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The analytical method was validated in a separate study in the range from 10 to 100 mg/mL. In the same study, a 28 hour stability at room temperature and a 8-day stability at 2- 8°C were verified in the range from 10 to 100 mg/mL. Samples of the formulations prepared during the current study (the first and the last week of treatment where possible) were analysed to check the concentration and homogeneity. Results of the analyses were within the acceptability limits for suspensions (85-115% for concentration and CV < 10% for homogeneity).
- Duration of treatment / exposure:
- Males were treated for 2 consecutive weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 39 days.
Females were treated for 2 consecutive weeks prior to pairing, during pairing and throughout the gestation and lactation periods until day 13 post partum, for a period comprising from 51 to 55 days. - Frequency of treatment:
- daily
- Details on study schedule:
- - Age at mating of the mated animals (P0 generation) in the study: approx. 13-14 weeks (approx. 11-12 weeks old at start of administration, followed by 2 weeks pre-mating phase)
Doses / concentrationsopen allclose all
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Group 1
Control
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- Group 2
Low-level dose
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Remarks:
- Group 3
Medium-level dose
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Remarks:
- Group 4
High-level dose
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: dose levels of 100, 300 and 1000 mg/kg/day were selected based on information from a preliminary non-GLP compliant study
- Rationale for animal assignment: rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights., individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed 5 of one sex per cage
- Fasting period before blood sampling for clinical biochemistry: yes
Examinations
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Mortality: checked twice daily
- Clinical signs: once before commencement of treatment and at least once daily during the study
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of treatment and at least once a week thereafter
- Observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions (see table No. 1 for observed parameters and evaluation scale)
- Animals were examined in an open arena for a minimum of three minutes
BODY WEIGHT: Yes
- Time schedule for examinations: males were weighed weekly from allocation to termination, females were weighed weekly from allocation to positive identification of mating and on days 0, 7, 14 and 20 post coitum, dams were also weighed on days 1, 4, 7, 13 post partum and just before to necropsy
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- the weight of food consumed by each cage of males and females were recorded weekly (whenever possible) during the pre-mating period starting from day 1 of dosing up to mating
- Individual food consumption for mated females were measured on gestation days 7, 14 and 20 post coitum starting from day 0 post coitum and on day 7 and 13 post partum starting from day 1 post partum
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment period
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes (except for coagulation test in males)
- How many animals: 5 males and 5 females (with viable litters if possible), randomly selected
- Parameters checked in table No. 2 were examined
- Coagulation parameters investigated: prothrombin time and activated partial thromboplastin time
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment period
- Animals fasted: Yes
- How many animals: 5 males and 5 females (with viable litters if possible), randomly selected
- Parameters checked in table No. 3 were examined
SERUM HORMONES: Yes
- Time of blood sample collection: at termination, timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days
- Animals fasted: Yes
- How many animals: all parental animals, each litter (1 sample for males and 1 sample for females, when possible), blood samples from different pups was pooled to obtain the required volume
- Hormones analyzed: serum levels of total thyroxine (total T4) and thyroid stimulating hormone (TSH)
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: once during study towards end of treatment, for males on day 39 and for females (with viable litters) on day 12 post partum
- Number of animals: 5 males and 5 females randomly selected from each dose group
- Studies conducted: grip strength and sensory reactivity to stimuli, motor activity assessment
IMMUNOLOGY: No - Oestrous cyclicity (parental animals):
- - Vaginal smears were taken in the morning from day 1 of dosing up to positive identification of mating
- The vaginal smear data was examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
- Before despatch to necropsy: vaginal smears were also taken from all females and the oestrous cycle phase recorded - Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4 pups per sex per litter (partial adjustment was possible); excess pups were killed, and at least one culled male and one culled female per litter were selected for hormone determination
- No pups were eliminated when the litter size dropped below the culling target (8 pups per litter in total)
- If there was only one pup available above the culling target, only one pup was eliminated and used for blood collection for possible hormone determination
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
(i) Number and sex of pups
(ii) Stillbirths, live births
(iii) Body weight measured on days 1, 4, 7 and 13 post partum
(iv) Anogenital distance measured on day 1 post partum, AGD was normalized to the cube root of body weight collected on day 1 post partum
(v) Nipple count check examined in male pups on day 13 post partum
(vi) Necropsy
GROSS EXAMINATION OF DEAD PUPS: Yes
- All pups found dead in the cage were examined for external and internal abnormalities
- All culled pups sacrificed at day 4 post partum were subjected to an external examination
- All live pups sacrificed on day 14 post partum were examined for external abnormalities
- Gonads were inspected from all pups in order to confirm the sex previously determined by external examination
- All pups with abnormalities were retained in a 10% neutral buffered formalin
ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: sacrifice after the mating of all females on day 40 of the study
- Maternal animals: females with live pups were killed on day 14 post partum, females showing no evidence of copulation were killed 25 days after the last day of the mating session
GROSS PATHOLOGY: Yes
- Necropsy: the clinical history of adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices), changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination
- All females were examined also for the following: number of visible implantation sites (pregnant animals), number of corpora lutea (pregnant animals)
ORGAN WEIGHTS:
- Parental animals: from all animals completing the scheduled test period, the organs indicated in table No. 4 were dissected free of fat and weighed, the ratios of organ weight to body weight was calculated for each animal
HISTOPATHOLOGY: Yes
- Samples of all tissues (parental animals) required for histopathological examination are listed in table No. 4
- Samples of the tissue were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol)
- After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin, in addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS), the morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed
The examination was restricted as detailed below:
(i) Sexual organs (cervix, clitoral gland, ovaries, uterus and vagina) and thyroid glands from all parental females
(ii) Sexual organs (coagulation gland, epididymides, preputial gland, prostate gland, seminal vesicles and testes) and thyroid glands from all parental males
(iii) Tissues specified in section 4.5.7 from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term
(iv) Morphological evaluation of the seminiferous epithelium (staging of the spermatogenic cycle) from all males in control and high dose groups
(v) All abnormalities in all groups - Postmortem examinations (offspring):
- SACRIFICE
- Pups (F1 offspring) that had completed the scheduled test period (days 4 or 14 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal
- Pups selected for blood collection for hormone determination were killed on the day of blood sampling
GROSS PATHOLOGY
- All pups found dead in the cage were examined for external and internal abnormalities
- All culled pups sacrificed at day 4 post partum were subjected to an external examination
- All live pups sacrificed on day 14 post partum were examined for external abnormalities
- Gonads were inspected from all pups in order to confirm the sex previously determined by external examination
ORGAN WEIGHTS
- Pups at day 14 post partum: thyroid was weighed from one male and one female pup selected for blood collection and preserved in 10% neutral buffered formalin, the thyroid weight was determined after fixation - Statistics:
- Standard deviations were calculated as appropriate.
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t-test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off. - Reproductive indices:
- MALES:
Copulation index (%) = (no. of males with confirmed mating/no. of males cohabitated) X 100
Fertility index (%) = (no. of males which induced pregnancy/ no. of males cohabitated) X 100
MALES AND FEMALES:
Pre coital Interval = the number of nights paired prior to detection of mating
FEMALES:
Copulation index (%) = (no. of females with confirmed mating/no. of females cohabitated) X 100
Fertility index (%) = (no. of pregnant females/ no. of females cohabitated) X 100
Pre-natal loss was calculated as a percentage from the formula:
(no. of visible implantations - live litter size at birth/no. of visible implantations) X 100
Pre-natal loss at day 0 post partum was calculated as a percentage from the formula:
(total litter size - live litter size/total litter size) X 100
Post-natal loss at day 4 post partum (before culling) was calculated as a percentage from the formula:
(live litter size at birth - live litter size at day 4 (before culling)/live litter size at birth) X 100
Post-natal loss at day 13 post partum (after culling) was calculated as a percentage from the formula:
(live litter size on day 4 (before culling) - live litter size at day 13/live litter size on day 4 (before culling)) X 100 - Offspring viability indices:
- Sex ratios was calculated at birth, on day 4 and on day 14 post partum and was presented as the percentage of males per litter.
Results and discussion
Results: P0 (first parental generation)
General toxicity (P0)
- Clinical signs:
- no effects observed
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One control female was sacrificed for humane reason on Day 22 of gestation phase due to difficulties in parturition.
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Food consumption was unaffected by treatment in both genders during the study.
- Food efficiency:
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Endocrine findings:
- no effects observed
- Description (incidence and severity):
- The determination of T4 and TSH was performed on
- Samples from all parental males from all groups
- Samples from pups on Day 14 post partum.
No treatment-related changes were recorded. Thyroid stimulating hormone was statistically si
gnificantly lower than controls in parental males (31%). This is due to 2 control animals, which sh
owed values above the range of historical data, therefore the differences observed has no biological/
toxicological significance.
The oestrous cyclicity of the treated females monitored during the pre-mating period, for a total of
14 days, was not affected by treatment. The mean number of oestrous cycles observed in treated
animals and control group was comparable.
No differences were recorded in pre-coital interval and copulation plugs between treated and control
groups. Copulatory index and fertility index did not show any treatment-related differences. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- Observation of treated animals at removal from the cage and in an open arena did not reveal significant changes when compared to controls.
No alterations in motor activity, grip strength, landing footsplay and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related microscopic observations at the end of the treatment period. There were no test item-related microscopic observations in the testis (stage were evaluation on PAS-stained slides). Any microscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.
- Histopathological findings: neoplastic:
- no effects observed
Reproductive function / performance (P0)
- Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- The oestrous cyclicity of the treated females monitored during the pre-mating period, for a total of
14 days, was not affected by treatment. The mean number of oestrous cycles observed in treated
animals and control group was comparable.
No differences were recorded in pre-coital interval and copulation plugs between treated and control
groups. Copulatory index and fertility index did not show any treatment-related differences. - Reproductive performance:
- no effects observed
- Description (incidence and severity):
- Implantation, pre implantation loss, pre-natal loss data and gestation length of females: Mean gestation period was comparable between control and treated animals, being of 22/23 days. Corpora lutea, number of implantations sites and live litter size did not show dose-related or treatment-related differences, compared to controls. Pre-natal loss (percentage) was similar between control and treated groups.
Effect levels (P0)
open allclose all
- Dose descriptor:
- NOAEL
- Remarks:
- reproduction toxicity
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Dose descriptor:
- NOAEL
- Remarks:
- systemic toxicity
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
Target system / organ toxicity (P0)
- Critical effects observed:
- no
Results: F1 generation
General toxicity (F1)
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No treatment-related clinical signs were observed in pups during the 14-day observation period.
- Body weight and weight changes:
- no effects observed
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Anogenital distance (AGD):
- no effects observed
- Nipple retention in male pups:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- No differences were observed in the weight of thyroid in treated pups, when compared to controls.
- Histopathological findings:
- not examined
Developmental neurotoxicity (F1)
- Behaviour (functional findings):
- not examined
Developmental immunotoxicity (F1)
- Developmental immunotoxicity:
- not examined
Effect levels (F1)
- Dose descriptor:
- NOAEL
- Remarks:
- developmental toxicity
- Generation:
- F1
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
Target system / organ toxicity (F1)
- Critical effects observed:
- no
Overall reproductive toxicity
- Reproductive effects observed:
- no
Any other information on results incl. tables
Tab. 5: Summary of reproduction data (group data)
Observations | 0 | 100 | 300 | 1000 |
No. Dams Inseminated | 10 | 8 | 10 | 10 |
Oestrus cycle (mean number) | 4 | 4 | 3 | 3 |
No. Dams That Conceived | 10 | 6 | 10 | 8 |
Percent Dams Conceived | 100.0 | 60.0 | 100.0 | 80.0 |
Conceiving (1-5 days) | 10 | 6 | 9 | 8 |
Conceiving (6-14 days) | 0 | 0 | 1 | 0 |
No. Dams Died During Study | 1 | 0 | 0 | 0 |
No. Dams with Resorptions only | 0 | 0 | 0 | 0 |
No. of Litters | 9 | 6 | 10 | 8 |
Total No. Implantation | 110 | 66 | 107 | 95 |
Mean No. Implants/Pregnancy | 11.0 | 11.0 | 10.7 | 11.9 |
Pre natal Loss (%) | 11.0 | 17.9 | 10.7 | 22.4 |
Post natal Loss (%) | 0.0 | 1.9 | 0.0 | 0.0 |
Tab. 6: Summary of litter data (group data)
Pairs started (no.) | 10 | 10 | 10 | 10 | |
Dams with live pup at birth (no.) | 9 | 6 | 10 | 8 | |
Dams with live pups at Day 4 pp (no.) | 9 | 6 | 10 | 8 | |
Live litter size at birth (mean) | 9.4 | 8.8 | 9.6 | 9.3 | |
Live litter size at Day 4 (mean) | 9.2 | 8.7 | 9.6 | 9.3 | |
Sex ratio (%) at birth (mean) | 55.1 | 60.4 | 53.7 | 52.4 | |
Sex ratio (%) at Day 4 (mean) | 54.0 | 60.8 | 53.7 | 52.4 | |
Litter weight at Day 1 (mean) | 63.7 | 60.5 | 66.6 | 62.4 | |
Litter weight at Day 4 (mean) | 96.9 | 94.5 | 103.5 | 94.9 | |
Pup weight at Day 1 (mean) | 6.9 | 6.9 | 7.2 | 6.8 | |
Pup weight at Day 4 (mean) Pup weight at the Day of AGD (Day 1pp) |
10.6 | 10.9 | 11.3 | 10.4 | |
Pub weight at the Day of AGD (Day 1pp) mean males | 7.0 | 7.1 | 7.4 | 7.0 | |
Pub weight at the Day of AGD (Day 1pp) mean females | 6.8 | 6.7 | 7.0 | 6.7 | |
Pup AGD normalized (Day 1pp) mean males | 2.4 | 2.5 | 2.5 | 2.5 | |
Pup AGD normalized (Day 1 pp) mean females | 1.5 | 1.4 | 1.5 | 1.4 | |
Male pup nipple present at Day 13 (no.) | 0 | 0 | 0 | 0 | |
Pup weight at Day 13 (mean) | 29.0 | 29.2 | 30.7 | 28.6 | |
PUPS WITH MAJOR ABNORMALITIES AT NECROPSY | |||||
Dams with 0 | 9 | 6 | 10 | 8 | |
Dams with 1 | 0 | 0 | 0 | 0 | |
Dams with>2 | 0 | 0 | 0 | 0 |
Table 7: Sum of pre- and postnatal loss
Observations | 0 | 100 | 300 | 1000 | ||
Pre-natal loss (implantations minus live births)(no.) | ||||||
Females with 0 | 3 | 1 | 3 | 0 | ||
Females with 1 | 2 | 3 | 4 | 3 | ||
Females with 2 | 3 | 1 | 2 | 1 | ||
Females with>3 | 2 | 1 | 1 | 4 | ||
Post-natal loss (live births minus live at Day 1) (no.) | ||||||
Females with 0 | 7 | 5 | 10 | 8 | ||
Females with 1 | 2 | 1 | 0 | 0 | ||
Females with 2 | 0 | 0 | 0 | 0 | ||
Females with>3 | 0 | 0 | 0 | 0 | ||
Post-natal loss (live births at Day 4 after culling minus live Day 13) (no.) | ||||||
Females with 0 | 9 | 6 | 10 | 8 | ||
Females with 1 | 0 | 0 | 0 | 0 | ||
Females with 2 | 0 | 0 | 0 | 0 | ||
Females with>3 | 0 | 0 | 0 | 0 |
Applicant's summary and conclusion
- Conclusions:
- The oral administration of the test substance by gavage to male and female Wistar rats revealed no signs of reproduction and developmental toxicity up to the highest tested dose level. Therefore, a NOAEL for general systemic toxicity, reproductive performance as well as for developmental toxicity was set to 1000 mg/kg bw/day.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
![ECHA](/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/echa_logo.png)