4-fluoroaniline

Endpoint summary

Description of key information

Read-across approach to structural analogue substance 3,5 -Difluoroaniline (CAS 372 -39 -4) was used. In the conducted combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test, the NOAEL(rat)  for systemic toxicity was determined to be 10 mg/kg bw.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

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)

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.

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

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.

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)

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.

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'

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and guideline compliant study. The test substance was a structural analogue substance, please refer to IUCLID section 13 for read across justification.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No data with the test substance is available. Thus, data from the similar substance 3,5- difluoroaniline (CAS 372-39-4) was summarised to cover these endpoints. For further justification see IUCLID section 13.

Key study

BASF SE, Study No. 95R0050/99018, 2001

Read-across approach to structural analogue substance 3,5 -Difluoroaniline (CAS 372 -39 -4) was used.

In the combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test, the test substance was administered to groups of 10 male and 10 female healthy young adult rats (parental generation) and 5 female healthy young adult virgin rats (satellite animals) by gavage at doses of 2, 10 and 50 mg/kg body weight/day throughout the whole study period. The test substance was administered as an oily solution in a standard dose volume of 5 mL/kg body weight. The control groups were dosed with the vehicle only (olive oiI). At least 14 days after the beginning of treatment, parental animals were mated to produce a litter. Mating pairs were from the same dose group. To guarantee an administration period of about 3 months in order to enable a detailed examination of systemic toxicity the parental males and the satellite females were sacrificed 91/92 days after the first test substance administration. In contrast, the parental females were sacrificed 46 days after the first test substance administration and the pups were killed on day 4 p.p. (post partum).

 

The satellite animals, the parents and the pups state of health was checked each day, and parental animals were examined for their mating and reproductive performances; this included determinations of the number of implantation and the calculation of the post implantation loss.

 

Detailed clinical observations in an open field were conducted prior to the start of the administration period and weekly thereafter. A functional observational battery (FOB) and motor activity measurement (MA) were carried out after 86-87days of treatment.

 

Food consumption of the parents was determined regularly during premating, after the mating period and - in dams with litters - during gestation and lactation periods. For the satellite females it was determined over the whole study period.

 

In general, body weights of all animals were determined once weekly. However, during gestation and lactation parental females were weighed on days 0, 7, 14 and 20 of gestation, on the day of parturition, and on day 4 after birth.

 

The pups were sexed and were weighed on the day after birth and on day 4 post partum. Their viability was recorded. All pups were examined macroscopically at necropsy for external and visceral findings.

 

Clinicochemical and hematological examinations were carried out in the surviving parental males and satellite females at the end of the administration period.

 

All parental animals and satellite females were assessed by gross pathology (including weight determinations of several organs of the parental males. All animals of the control and the high dose and individual animals of the 2 and 10 mg/kg groups were subjected to a histopathological examination, special attention being paid to the organs of the reproductive system.

The following adverse substance-related findings were observed:

Test group 3 (50 mg/kg body weight/day)

Parental male and female animals

- statistically significantly decreased food consumption in parental females during weeks 0 - 1 of premating and days 14 - 20 of gestation

- initial body weight loss in parental females during weeks 0 - 1 of premating as well as retarded body weight gain and statistically significant reduced body weights on day 20 of gestation

- decreased red blood cells, hemoglobin and hematocrit in male rats (parental females were not investigated)

- significantly increased mean absolute and relative spleen weights in male rats (parental females were not investigated)

- increased incidence and graded severity of extramedullary haemopoiesis in the spleen of both sexes

- increased amounts of haemosiderin deposition in the spleen of both sexes

- congestion of the spleen in both sexes

 

Satellite females

- decreased red blood cells, hemoglobin and hematocrit

- increased mean absolute and relative spleen weights

- increased incidence and graded severity of extramedullary haemopoiesis in the spleen

- increased amounts of haemosiderin deposition in the spleen

- congestion of the spleen

Under the conditions of this modified combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, the oral administration of the test substance via gavage at a dose level of 50 mg/kg body weight to parental male and female satellite for a period of about 3 months and to parental females for a period of 46 days caused a hemolytic anemia including increased levels of hemosiderin deposition and extramedullary haemopoiesis in the spleen in all animals. Furthermore, transiently reduced food consumption, initial body weight loss and retarded body weights gains were noted in the parental females. No treatment-related findings were observed in the animals given 2 mg/kg body weight and 10 mg/kg body weight of the test compound.

The test substance had no adverse effects on reproductive performance or fertility of the parental animals.

There were no signs of developmental toxicity in the progeny of the parents up to and including the high dose group.

Thus, the NOAEL (no observed adverse effect level) for systemic toxicity was 10 mg/kg body weight/day for the parental animals and the satellite females.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP and guideline compliant study. The test substance was a structural analogue substance, please refer to IUCLID section 13 for read across justification.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)
The available study is considered reliable and suitable for classification purposes under Directive 67/548/EEC. As a result the substance is considered not to be classified for repeated dose toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.

 

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

As a result the substance is considered not to be classified for repeated dose toxicity under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation (EC) No 605/2014.