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EC number: 206-735-5 | CAS number: 371-40-4
- 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
Repeated dose toxicity: oral
Administrative data
- Endpoint:
- short-term repeated dose toxicity: oral
- Remarks:
- combined repeated dose and reproduction / developmental screening
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1999-02-09 to 1999-11-15
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 001
- Report date:
- 2001
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- March 22, 1996
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EPA Health Effects Test Guideline OPPTS 870.3650, JuIy 2000
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- 3,5-difluoroaniline
- EC Number:
- 206-752-8
- EC Name:
- 3,5-difluoroaniline
- Cas Number:
- 372-39-4
- Molecular formula:
- C6H5F2N
- IUPAC Name:
- 3,5-difluoroaniline
- Test material form:
- solid: crystalline
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Boehringer Ingeheim, Pharma KG, Biberach/Riss, Germany
- Age at study initiation: Nulliparous
- Weight at study initiation: Parental male animals: 375 g / Satellite female animals: 243 g / Parental female animals: 246 g
- Housing: Individually in type DK III stainless steel wire mesh cages supplied by BECKER & CO., Castrop-Rauxel, Germany; with the following exceptions: for the overnight mating the females were put into the cages of the males; from day 18 p.c. until sacrifice, the pregnant animals and their litters were housed in Makrolon type M III cages.
- Diet: Kuba Iaboratory diet rat/mouse/hamster, supplied by Provimi Kliaba SA, Kaiseraugst, Switzerland, ad libitum
- Water: Water bottles ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-70 %
- Air changes: Fully air-conditioned rooms
- Photoperiod: 12 hours light from 6.00 a.m. to 6.00 p.m. and 12 hours darkness from 6.00 p.m. to 6.00 a.m.
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- olive oil
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test substance and the olive oil were mildly heated up to approx. 50°C until the test substance was completely dissolved. Then the specific amount of the test substance related to the dose group was weighed and topped up with heated olive oil in a calibrated beaker and was mixed thoroughly with the Ultraturrax.
The preparations for the high and mid concentrations were prepared twice a week, every 96 hours latest. The preparations for the low concentration were also prepared twice a week for about the first three weeks of application.
VEHICLE
- Amount of vehicle: 5 mL/kg body weight - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Samples of all concentrations of the oily test substance solutions were sent to the analytical laboratory at the beginning and towards the end of the study for verification of the concentrations. A sample of the low concentration was sent additionally after about three weeks after the beginning of application.
The test substance solutions were analyzed by HPLC. - Duration of treatment / exposure:
- 3 months
- Frequency of treatment:
- once a day at approx. the same time of day (in the morning) using 3 mL/5 mL-syringes
Doses / concentrations
- Remarks:
- Doses / Concentrations:
2, 10, 50 mg/kg bw/day
Basis:
actual ingested
- No. of animals per sex per dose:
- 10 (parental generation)
5 (only female for satellite animals) - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
2 mg/kg body weight/day: as the expected no observed adverse effect level
10 mg/kg body weight/day: as the intermediate dose level
50 mg/kg body weight/day: as the dose level with possible toxic effects
The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.
- Rationale for animal assignment: This strain was selected since extensive historical control data were available on Wistar rats and the rat is the preferred animal species for reproduction studies.
The assignment of the animals to the different test groups was carried out using a randomization program (NIJENHUIS, A. and WILF, H.S.; 1978), according to their weight before the beginning of the administration period.
Examinations
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily a check was made for dead or moribund animals. If animals were in a moribund state, they were sacrificed and necropsied.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All parental animals and satellite animals were checked daily for clinically evident signs of toxicity.
BODY WEIGHT: Yes
- Time schedule for examinations: In general, the body weight of the male and female parental animais as well as the female satellite animals was determined once a week at the same time of the day (in the morning). Additionally at the day of sacrifice the body weight was determined in the Pathology.
FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Generally food consumption was determined once a week (in general for a period of 7 days) for parental animals and satellite female animals.
HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: 5
- Parameters checked: Ieukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was taken from the retroorbital venous plexus in the morning from fasted parental
male and satellite female animals without anesthesia at the end of the application period.
- Animals fasted: Yes
- How many animals: 5 animals per test group and sex
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-y-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium
NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: Motor activity
OTHER:
Food analyses
The food used in the study was assayed for chemical and for microbiological contaminants.
Drinking water analyses
The drinking water is regularly assayed for chemical contaminants by the municipal authorities as for the presence of microorganisms by a contract Iaboratory.
Bedding analyses
The bedding is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).
Open fleld observation (OFO)
Open field observations were performed for all male and satellite animals once before the administration (day -3), as weIl as on weeks 1 - 12.
Functional observational battery (FOB)
FOBs were performed in 5 male parental animals and 5 female satellite animals per group on day 86 (males) and 87 (females), starting at about 10:00 a.m.. The FOB consisted of 4 parts, starting with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
The animals were sacrificed by decapitation under CO2 anaesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The animals that died prematurely were necropsied and assessed as soon as possible after death to prevent autolysis.
Organ weights:
The following weight parameters of the male parental animals sacrificed at scheduled dates were determined:
1. anaesthetised animals
2. testes
3. epididymides
4. liver
5. kidneys
6. adrenal glands
7. thymus
8. spIeen
9. brain
10. heart
The following weight parameters of the female animals of the satellite groups sacrificed at scheduled dates were determined:
1. anaesthetised animais
2. liver
3. kidneys
4. adrenal glands
5. thymus
6. spIeen
7. brain
8. heart
9. ovaries
HISTOPATHOLOGY: Yes
The following organs or tissues of all parental animais and of all female satellite animals that were killed intercurrently or at study termination were fixed in 4% formaldehyde solution:
1. all gross lesions
2. targetorgans
3. brain
4. thyroid glands/parathyroid glands
5. thymus
6. trachea
7. lungs
8. heart
9. liver
10. spleen
11. kidneys
12. adrenal glands
13. ovaries (with oviducts)
14. uterus and vagina
15. prostate gland, seminal vesicles and coagulation glands with their fluids
16. stomach
17. duodenum, jejunum, ileum
18. cecum, colon, rectum
19. urinary bladder
20. lymph nodes (mesenteric and mandibular nodes)
21. sciatic nerve
22. bone marrow (femur)
23. spinal cord (cervical, thoracic and lumbar)
Testes and epididymides of males that were killed intercurrently or at study termination were fixed in Bouin's solution and embedded in paraplast thereafter.
The testes and epididymides, of the males that died prematurely, were fixed in 4% formaldehyde solution.
After the organs were fixed, histotechnical processing was performed on all male and female parental animals and female satellite animals as outlined in the following table.
Examination by light microscopy and assessment of findings was performed. - Statistics:
- Statistical analyses were performed according to:
Food consumption: DUNNETT-test
Male and female mating index: FISHER'S EXACT
WILCOXON-test
KRUSKAL-WALLIS test (two-sided)
MANN- WHITNEY U-test (two sided) for the hypothesis of equal medians
Statistics of clinical pathology
Means and standard deviations of each test group were calculated for several parameters
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided)
Statistics of pathology
KRUSKAL-WALLIS test (two-sided)
Results and discussion
Results of examinations
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Food efficiency:
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, treatment-related
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Details on results:
- CLINICAL SIGNS AND MORTALITY
There were no substance-induced mortalities in any of the male and female animals in any of the groups.
A total of 9 out of 10 male parental animals of the high dose group, 6 out of 10 of the mid dose group and 2 out of 10 of the low dose group showed transient salivation after treatment. In the satellite females a total of 4 out of 5 of the high dose group, 3 out of 5 of the mid dose group and 4 out of 5 of the low dose group as well as 3 out 10 of the female parental animals of the high dose group displayed this finding. However, the observed salivation occurred only sporadically and persisted in the respective animals only for a few minutes after the actual gavage had taken place. There was no homogeneous occurrence, individual animals displayed this finding on one or several days of the administration period.
The animals that were sacrificed moribund or the one that was found dead showed several clinical findings (e.g., chromodacryorrhea, labored respiration, piloerection, swelling of limb, fur smeared with urine, urine discolored) reflecting their impaired health status as a consequence of misgavage or incidental injury.
The few other clinical findings that occurred in individual animals within the different groups are considered to be spontaneous in nature.
Detailed clinical observations (Open Field Observations)
Despite incidental or misgavage related effects only few other findings in a single control group and one mid dose animal were noted at individual intervais. However, due to the isolated occurrence and the lack of a dose-response relationship this was assessed as biologically not relevant.
Detailed clinical observations (Open Field Observations)
Despite incidental or misgavage related effects only few other findings in a single control group and one mid dose animal were noted at individual intervals. However, due to the isolated occurrence and the lack of a dose-response relationship this was assessed as biologically not relevant.
Functional observational battery
As all findings were equally distributed between treated and control animals or occurred in single animals only, they were clearly incidental in nature.
Motor activity measurement
Regarding the overall motor activity no significant deviations were seen.
BODY WEIGHT AND WEIGHT GAIN
The mean body weights of the high dose parental females were below the corresponding control values during the entire treatment period.
Mean body weights/body weight gains of the parental females of the 2 and 10 mg/kg groups revealed no substance-induced impairment.
Body weights/body weight gains of all substance-treated parental males and all satellite fermales (2, 10 and 50 mg/kg body weight/day) were not influenced by the test substance administration. All differences in body weights and body weight gains observed for these rats were without any biological relevance.
Thus, the reduced mean body weights and retarded mean body weight gain of the high dose parental females was assessed to be substance-related.
FOOD CONSUMPTION AND COMPOUND INTAKE
The food consumption of the low and mid dose parental females as well as of the parental males and the satellite females of all dose groups was not influenced by the test substance administration. All differences between these substance-treated groups and the concurrent control groups were without any biological relevance.
Thus, the slightly and transiently statistically significantly reduced food consumption of the high dose parental females was considered to be substance-related.
HAEMATOLOGY
After 3 months of test substance administration significantly decreased red blood ceII counts and hemoglobin concentrations were observed in the high dose parental males and females of the satellite groups. Hematocrit values were also reduced in the high dose animals of either sex. These findings, however, were seen only as a trend toward decreased values. No treatment-related changes were found in the other hematology parameters of the high dose rats and in the animals given 2 mg/kg body weight and 10 mg/kg body weight of the test compound.
Since it is known that aniline - the basic substance - is associated with red blood cell destruction and, thus, produces hemolysis, the anemic process is classified as a hemolytic anemia.
CLINICAL CHEMISTRY
Compound-related differences in clinical chemistry parameters were not evident at any dose level in either males or females.
NEUROBEHAVIOUR
Functional abservational battery as weIl as measurement of motor activity revealed no signs of neurotoxicity
ORGAN WEIGHTS
Weight parameters were only obtained for male parental rats.
Absolute weights
The mean weight of the spleen was slightly (p =< 0.05) but significantly increased in the high dose group (+ 25%).
The other mean absolute weight parameters for male parental animals of the main groups (F1) did not show significant differences when compared with the control group.
Relative weights (related to terminal body weight)
The mean weight of the spleen was slightly (p < 0.05) but significantly increased in the high dose group (+ 22%). The other mean relative weight parameters for male parental animals of the main groups (F1) did not show significant differences when compared with the control group.
GROSS PATHOLOGY
Throughout the sections of the pathology report, when inter-group differences are referred to as "significant" it implies that the differences have attained statistical significance (p =< 0.05) when compared with the control group.
Gross lesions
Major gross lesions were noted in two high dose male animals, which died or had to be killed prematurely as a consequence of gavage error. They comprised: yellow discoloration of the mucosa in the glandular stomach, adhesion of the diaphragm to the or deposition of a yellow white material in the lungs, deposition of a yellow white material and effusion of cloudy or bloody fluids in the thoracic cavity, thickening of the pericardial wall and effusion of a bloody fluid into the cavity of the pericardium, and a small focus in the pituitary gland.
In the animals that were killed at the scheduled sacrifice, a few incidental gross lesions were noted in the glandular stomach (black erosion/ulcer in the mucosa of a control and low dose female rat), liver (yellow focus in a control female), kidneys (retraction in a low and high dose male), ovaries (cyst in a low dose animal), uterus (dilation in a mid dose rat), and adrenal cortex (focus in a low dose female animal). They were all regarded as spontaneous in origin and unrelated to treatment.
Female satellite group animals
Major gross lesions were noted in a control female animal that was killed prematurely 46 days after start of treatment. They comprised a large abscess in the mammary gland, enlarged spleen, liver lymph node and axillary lymph node (unilateral) and a few red foci in the liver. They were all considered to have arisen fortuitously.
In animals that were killed at the scheduled sacrifice, only a few gross lesion were noted in the oesophagus (abscess in a mid dose animal), liver (reduced organ size in a mid dose female), ovaries (unilateral cyst in a control rat) and uterus (dilation in a low dose animal).
All of these gross lesions were considered to have arisen spontaneously.
HISTOPATHOLOGY: NON-NEOPLASTIC
The majority of the gross lesions could be correlated with a meaningful microscopic finding. Only a few grossly noted lesions lacked a microscopic correlate.
However, regardless of whether or not they had a microscopic correlate all the gross lesions were considered to have developed spontaneously and to be unrelated to treatment.
Treatment-related microscopic findings were confined to the spleen of both sexes of the high dose group. When compared to the controls the incidence and graded severity of extramedullary hemopoiesis and the degree of haemosiderin deposition were greater in animals given the highest dose than in controls. In addition, minimal to slight splenic congestion was present in eight of ten males and nine of ten females given the highest dosage.
Female satellite group animals
The majority of the gross lesions could be correlated with a meaningful microscopic finding. OnIy a few grossly noted lesions Iacked a microscopic correlate.
However, regardless of whether or not they had a microscopic correlate all the gross lesions were considered to have developed spontaneously and to be unrelated to treatment.
Treatment-related microscopic findings were confined to the spleen of both sexes of the high dose group. As in the parental animals, when compared to the controls the incidence and graded severity of extramedullay haemopoiesis and the degree of haemosiderin deposition were greater in animals given the highest dose than in controls.
In addition, slight splenic congestion was present in all females given the highest dosage.
One animal of the control group was killed prematurely 46 days after start of treatment. Major microscopic findings were an abscess on the mammary gland and reactive hyperplasia in the associated axillary lymph node.
All other microscopic findings recorded in the other organs of female satellite animals were either single observations, occurred in control animals or at a low incidence only, or were recorded at comparable incidence and graded severity in control and high dose animals.
Effect levels
- Dose descriptor:
- NOAEL
- Effect level:
- 10 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: see 'Remark'
Target system / organ toxicity
- Critical effects observed:
- not specified
Any other information on results incl. tables
Stability analyses
The stability of test substance solution in olive oil over a period of 96 hours at ambient temperature was demonstrated within a previous study (project no. 19R0050/99003). Since the first concentration control analysis of the low concentration (40 mg/100 mL) showed unexpectedly a recovery rate of only about 55.5% an additional stability analysis of the low concentration was performed shortly after the results became available. The results demonstrated that the oily test substance solution of the low concentration (40 mg/100 mL) was stable over a period of at least 4 hours either after storage at ambient temperature or in a refrigerator. Thereafter, the recovery rates declined slowly to values of about 81% after 70 hours storage at ambient temperature and of about 82% after 70 hours storage in a refrigerator. Consequently, the frequency of test substance preparation was changed from twice a week to daily after about three weeks after the beginning of test substance application.
Concentration control analyses
The concentration control analyses of the oily test substance solutions of the high and mid dose samples taken at the beginning and towards the end of the application period yielded values of about 96.5% - 103.3% of the nominal concentrations. Thus, the correctness of the prepared concentrations was confirmed.
Food analyses
On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants the food was found to be suitable.
Drinking water analyses
On the basis of the analytical findings the drinking water was found to be suitable.
Bedding analyses
On the basis of the findings of analysis the bedding was found to be suitable.
Applicant's summary and conclusion
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.
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