4-fluoroaniline

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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:

equivalent or similar to guideline

Guideline:

other: EPA Health Effects Test Guideline OPPTS 870.3650, JuIy 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

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 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

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

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:

91/92 days (male and satellite female animals)
46 days (female parents)

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:
nominal in diet

No. of animals per sex per dose:

10 (parental generation)
5 (only female for satellite animals)

Control animals:

yes, concurrent no treatment

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.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
- Cage side observations checked: posture, tremor, convulsions, abnormal movements, impairment of gait

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily
- Nesting, littering, and Iactation behavior

BODY WEIGHT: Yes
- Time schedule for examinations: Male and female parental animals as well as the female satellite animals was determined once a week at the same time of the day. Additionally at the day of sacrifice the body weight was determined in the Pathology.
- During the mating period the parental females were weighed on the day of positive evidence of sperm (day 0 p.c.) and on days 7, 14 and 20 post coitum
- Females with litter were weighed on the day of parturition (day 0 p.p.) and on days 4 post partum.

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
Food consumption was not determined during the mating period (e.g.; between week 2 and 3 for parental males).
Food consumption of the females with evidence of sperm was determined for days 0 - 7, 7 - 14 and 14 - 20 p.c.
Food consumption of females, which gave birth to a litter was determined for days 0-4 p.p.

CLINICAL PATHOLOGY
Blood was taken from the retroorbital venous plexus in the morning from fasted parental male and satellite female animals without anesthesia.

The following examinations were carried out in 5 animals per test group and sex at the end of the application period.
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter.

Hematology
Ieukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count

Clinical chemistry
alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-y-glutamyltransferase, sodlum, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

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..

Open fleld observation (OFO)
Open field observations were performed for all male and satellite animais once before the administration (day -3), as weIl as on weeks 1 - 12, each time starting usually at the same weekday at about 10:00 a.m..

The following parameters were examined:
behavior during "handling", fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

Motor activity assessment (MA)
MA was measured on the same day and with the same animals as FOB was performed.

Litter observations:

STANDARDISATION OF LITTERS
All surviving pups were sacrificed (by means of CO2) on day 4 p.p.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.

Postmortem examinations (parental animals):

The parental male animals and satellite female animals were sacrificed about 3 months after beginning of administration.

SACRIFICE
- The parental male animals and satellite female animals were sacrificed about 3 months after beginning of administration.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic.
- After sacrifice of the female animais, the uteri and ovaries were removed (including the uteri of apparently non-pregnant animais) for further investigations.

HISTOPATHOLOGY
The tissues indicated were prepared for microscopic examination and weighed, respectively.
The following organs or tissues of all parental animals and of all female satellite animals that were killed intercurrently or at study termination.

All gross lesions, target organs, brain, thyroid glands/parathyroid glands, thymus, trachea, lungs, heart, liver, spleen, kidneys, adrenal glands, ovaries (with oviducts), uterus and vagina, prostate gland, seminal vesicles and coagulation glands with their fluids, stomach, duodenumjejunum, ileum, cecum, colon, rectum, urinary bladder, lymph nodes (mesenteric and mandibular nodes), sciatic nerve, bone marrow (femur), spinal cord (cervical, thoracic and lumbar)

ORGAN WEIGHTS
Male parental animals:
Anaesthetised animais, testes, epididymides, liver, kidneys, adrenal glands, thymus, spIeen, brain, heart

Female animals of the satellite groups:
Anaesthetised animals, liver, kidneys, adrenal glands, thymus, spIeen, brain, heart, ovaries

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations macroscopic examination as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and viscernal examinations.

Statistics:

Statistical analyses used:

DUNNETT-test (two-sided)
FISHER'S EXACT
WILCOXON-test (one-sided)
KRUSKAL-WALLIS test (two-sided)
MANN- WHITNEY U-test (two sided)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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.

Clinical observations for parental females during gestation:
Transient salivation after treatment occurred in total in 4 out of 10 parental females of the high dose group. The individual animais displayed this treatment-related finding only on single days of gestation.
One sperm positive female of the control group, 3 of the mid dose group and one of the high dose group did not deliver any pups.
Due to the scattered occurrence without relation to dosing this was considered to be not treatment-related.

Clinical observations for parental females during Iactation
With the exception of transient salivation after treatment in one out of 10 mid dose and and 5 out of 10 high dose females no further treatment-related effect was noted during lactation in any of the other parental females.

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.

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 spieen 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.


Female satellite group animals
Weight parameters were only obtained from female rats.
Due to the premature sacrifice of a control female, the number of animals (n = 4) was too small and made the statistical test used too insensitive. Consequently no levels of significance were indicated for any ot the weight parameters in this segment of the study.

Absolute weights
The mean weight of the spleen was increased in the high dose group (+ 21%). The other mean absolute weight parameters did not differ considerably from the control group.

Relative weights (related to terminal body weight)
The mean weight of the spleen was increased in the high dose group (+ 23%). The other mean relative weight parameters did not differ considerably from 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.

Similarly in one male animal of the control group, lesions in the lungs (deposition and adhesion), thoracic cavity (effusion), and pericardium (thickening of wall), were suggestive for a survived gavage error. The other gross lesions in this animal were considered to be indicative of chronic iliness and included erosion/ulcer in the glandular stomach, reduced organ sizes of spleen, thymus and seminal vesicle, enlarged mediastinal Iymph nodes, and focal oedema in the skin of the cervical region.
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.

In the reproductive organs of the female rats, only incidental flndings were recorded, microscopically.


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 animais, when compared to the controls the incidence and graded severity of extramedullay haemopoiesis and the degree of haemosiderin deposition were greater in animais 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.

In the reproductive organs of the female rats, the following incidental flndings were recorded, microscopically:
In the ovaries, a unilateral follicular cyst was recorded in one animal of the high dose group.

All other microscopic findings recorded in the other organs of the parental animals were either single observations, occurred in control animals or at a 10w incidence only, or were
recorded at comparable incidence and graded severity in control and high dose animals.


Male cohabitation data
There was no substance-related effect on the male mating or fertility index.

Female reproduction and delivery data
The administration of the test substance did not adversely affect reproduction and delivery data of the parental females.

Dose descriptor:

NOAEL

Remarks:

reproductive performance and fertility

Effect level:

> 50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Transiently reduced food consumption, initial body weight loss and retarded body weights gains were noted in the parental females.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
No substance-induced effects on pup viability/mortality were recorded. The viability index, as an indicator for the viability of the pups during the first 4 days after birth and the mean number of live pups/litter on days 0 and 4 p.p. did not show any difference of
biological relevance between the substance-treated groups and the control group.
Thus, there were no substance-related differences concerning pup viability and mortality.

Pup number and status at delivery
The mean number of delivered pups/dam and the rate of liveborn and stillborn pups were not affected by the administration of the test substance. The differences observed were not statistically significant and of no biologically relevance.

CLINICAL SIGNS (OFFSPRING)
The pups did not show any clinical signs.

BODY WEIGHT (OFFSPRING)
Mean body weights/body weight gains were not influenced by the test substance administration. The differences observed were without any biological relevance and showed no dose-response relationship.

SEXUAL RATIO (OFFSPRING)
The sex distribution and sex ratios of live pups on the day of birth and on day 4 p.p. did not show any substantial differences between controls and treated groups; all differences observed were regarded to be spontaneous in nature.

The mean number of delivered pups/dam and the rate of liveborn and stillborn pups were not affected by the administration of the test substance. The differences observed were not statistically significant and of no biologically relevance.

GROSS PATHOLOGY (OFFSPRING)
The macroscopic examination of all pups did not reveal any differences between the test groups neither in the type nor in the number of pup necropsy observations.

OnIy spontaneous findings were seen at necropsy (e.g. post mortem autolysis, dilated renal pelvis and perforation of colon) in a few of the large number of examined pups of all groups including the controls generally without a dose-response relationship.

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

> 50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Reproductive 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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

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.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No 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, developmental/ repoductive toxicity screening test the test substance was administered continuously at doses of 0, 2, 10, and 50 mg/kg body weight/day by gavage in this modified combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in Wistar rats. The test substance was administered for about 3 months to male parental and female satellite animals to enable the examination of subchronic toxicity, while the female parental animals received the test substance for about 46 days.

 

The clinical examinations showed statistically significantly decreased food consumption in parental females during weeks 0-1 of premating and days 14-20 of gestation and an initial body weight less during weeks 0-1 of premating as well as retarded body weight gain and statistically significant reduced body weights an day 20 of gestation in these animals. Despite these no further substance-related adverse effects were noted within the clinical examinations. Transient salivation after the daily gavage on single or several days of the treatment period were noted in most of the high dose parental males and satellite females as well as in some parental females within premating, gestation and lactation.

This finding occurred also to a dose-dependently lesser extent in the mid dose parental males and females (during lactation only) and satellite females as well as in the low dose parental males and satellite females. Since the transient salivation was not associated with any adverse effect (e.g. irritation of the gastrointestinal tract) and there was no homogenous occurrence in the individual animals or within the different groups, it is not assessed as sign of toxicity but treatment related possibly due to unpleasant physical/chemical properties. Two animals of the high dose group died or were killed prematurely, respectively. Both animals revealed microscopic findings indicative of a gavage error and consecutive alterations to a chronic injury. As these two premature decedents belong to the high dose group, it can be suggested that the test compound might have influenced the development of gavage error. Rather than a direct toxic effect, however, the most likely explanation is that the physical/chemical properties of the test substance had influenced the animals during the application procedure to the extent that they were difficult to handle thus provoking a gavage error.

Detailed clinical examinations, functional observational battery as weIl as measurement of motor activity revealed no signs of neurotoxicity.

 

Regarding hematology, administration of the test compound at a dose level of 50 mg/kg body weight produced a mild anemia in both sexes having slightly lower red blood cell counts, haemoglobin concentrations and hematocrit values. However, no changes in erythrocyte indices and in red blood cell morphology were observed. 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 hemalytic anemia.

 

Pathology showed increased levels of haemosiderin deposition and extramedullary haemopoiesis in the spleen, findings that were consistent with a physiological response to haemalytic anaemia. On the one hand, the greater amounts of iron pigment resulting from increased red cell destruction are stored as haemosiderin while the increasedamounts of haemopoietic tissue in the splenic red pulp act to restore the number of erythrocytes.Splenic congestion was another histopathologic finding noted in animals of the high dose group. lts aetiology is not evident, however, no such finding was noted in lower dose groups. All three microscopic findings have contributed to the increased mean absolute and relative spleen weights.

 

Finally, all other gross lesions and microscopic findings recorded in the other organs were either single observations, occurred in control animals only, or were recorded at comparable incidence and graded severity in control and high dose parental animals or females of the satellite groups.

There were no indications from the clinical and pathological examinations, that the administration had adverse effects on reproductive performance or fertility of the parental animals of all substance-treated groups. Mating behavior, conception, gestation, parturition and lactation as well as the determined sexual organ weights, gross and histopathological findings of these organs were similar between the substance-treated rats and the corresponding controls. With the exception of few male and female parental rats in the control group, the mid dose group and the high dose group all F0 parental rats proved to be fertile. In total, in the five mating pairs in which no offspring were generated no treatment-related morphologic alteration was found that may explain the animals' status “animal not pregnant/impaired fertility". In one animal, a severe uterine dilation may have contributed to the animals' infertility, however, this finding as all the other gross lesions and microscopic findings noted in these animals were regarded to be of spontaneous origin. Thus, the observable difference was regarded to be incidental in nature and not of toxicological concern.

 

There were no signs of developmental toxicity in the progeny of the parents up to and including the high dose group (50 mg/kg body weight/day). The mean number of delivered pups/dam, the percentage of live born pups, the viability index, the mean number of live pups/litter on days 0 and 4 p.p., the sex ration, pup body weight/body weight gain as well as clinical and necropsy observations did not show any difference of biological relevance between the substance-treated groups and the control group. Thus, the NOAEL (no observed adverse effect level) for systemic toxicity was 10 mg/kg body weight/day.

The NOAEL for reproductive performance and fertility is greater than 50 mg/kg body weight/day.

The NOAEL for developmental toxicity is greater than 50 mg/kg body weight/day.

Short description of key information:
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 NOAEL for reproductive and developmental toxicity was determined to be > 50 mg/kg bw.

Justification for selection of Effect on fertility via oral route:
GLP and guideline compliant study. The test substance was a structural analogue substance, please refer to IUCLID section 13 for read across justification.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

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 reproductive and developmental 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 reproductive and developmental toxicity under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation (EC) No 605/2014.