Benzenamine, 4-fluoro-N-(1-methylethyl)-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Feb - 22 Mar 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

The present study is a dose-range-finding study for a one-generation study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Exploratory, dose-range-finding study for a one-generation reproduction toxicity study. Repeated dose toxicity was assessed after 5 weeks administration of the test substance via diet.
- Short description of test conditions: Test conditions were similar to OECD TG 407 without detailed examinations.
- Parameters analysed / observed: clinical signs of toxicity, body weight, food intake, gross necropsy, organs weights of spleen, liver and kidney, histopathology of spleen, liver, and kidney

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

448-100-7

EC Name:

-

Cas Number:

70441-63-3

Molecular formula:

C9H12FN

IUPAC Name:

4-fluoro-N-(propan-2-yl)aniline

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: about 6 weeks
- Mean body weight at study initiation: males: 146 g (range: 133 - 161 g); females: 119 g (range: 108 - 145 g)
- Housing: Animals were conventionally housed in Makrolon cages type IV on low-dust wooden granulate (Source: Ssniff Spezialdiäten Inc., Soest, Germany). Animals were group housed; 5 animals per cage. Environmental enrichment in form of wooden blocks was in place.
- Diet: fixed-formula standard diet Provimi Kliba 3883.9.25 meal (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: 2 days

DETAILS OF FOOD AND WATER QUALITY
The nutritive composition and contaminant content of the standard diet were routinely checked and analyzed. The tap water complied with the current German Drinking Water Ordinance (December 1990, supplemented May 2001).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): ≥10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 Feb 2005 To: 22 Mar 2005

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: offered in diet

Details on oral exposure:

PREPARATION OF DOSING FORMULATION
The test substance was mixed into the diet intended for the following week according to the dose scheme. A mixing granulator (Lödige, Paderborn, Germany) was used. The mixtures were not longer used than 8 days. Stability of the test substance in the diet from 10 to 12000 ppm was demonstrated and amounts to 15 days.

DIET PREPARATION
- Rate of preparation of diet (frequency): once weekly
- Mixing appropriate amounts with (Type of food): Provimi Kliba 3883.9.25 meal
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

5 weeks

Frequency of treatment:

continuously in the diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 ppm

Remarks:

actual test substance intake: male: 8 mg/kg bw/d; female: 10 mg/kg bw/d

Doses / concentrations 2

Dose / conc.:

500 ppm

Remarks:

actual test substance intake: male: 42 mg/kg bw/d; female: 48 mg/kg bw/d

Doses / concentrations 3

Dose / conc.:

2 500 ppm

Remarks:

actual test substance intake: male: 191 mg/kg bw/d; female: 180 mg/kg bw/d

Doses / concentrations 4

Dose / conc.:

10 000 ppm

Remarks:

actual test substance intake: male: 564 mg/kg bw/d; female: 470 mg/kg bw/d

No. of animals per sex per dose:

5 animals/sex/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The present study was a exploratory dose-range finding study. The dose levels were selected according to results of previous studies in rats.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: Twice daily, once daily on weekends and public holidays.
- Cage side observations checked in table (table 1) were included.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: Weekly.

BODY WEIGHT: Yes.
- Time schedule for examinations: Weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each group determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No.

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY: No.

CLINICAL CHEMISTRY: No.

URINALYSIS: No.

NEUROBEHAVIOURAL EXAMINATION: No.

IMMUNOLOGY: No.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.
All animals sacrificed pre-scheduled or in moribund condition were killed by exsanguination under deep diethyl ether anesthesia and necropsied. Their orifices, external and internal organs and tissues were subjected to thorough gross pathological examination. Any changes were described in terms of localization, size, color, and consistency whenever present.
The organ weights of the liver, spleen, and kidneys were recorded.

HISTOPATHOLOGY: Yes.
Histopathological examinations were performed for the liver, spleen, and kidneys.

Statistics:

Treatment groups were compared with the control group.
Statistical evaluations on body weight and organ weight data were done using the Dunnett-test in connection with a variance analysis.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 10000 ppm all rats exhibited pallor, emaciation, and poor general condition.
No remarkable clinical findings were observed at dose levels of 2500 ppm and below.

Mortality:

mortality observed, treatment-related

Description (incidence):

Because of poor general condition, all animals receiving 10000 ppm were sacrificed pre-scheduled on study day 22 (test substance administration started on study day 1).
No mortality occured at dose levels of 2500 ppm and below.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 10000 ppm, a clear body weight reduction was seen in male and female rats, which resulted in emaciation and need for pre-scheduled sacrifice.
No relevant changes in body weights were noted at dose levels of 2500 ppm and below. In fact, slightly but statistically significantly reduced or increased mean values of body weight found in males of the 100, 500, and 2500 ppm dose groups were incidential and, therefore, not toxicologically relevant (table 2).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 10000 ppm reduced food intake was observed for both sexes. As a consequence of the reduced food intake at 10000 ppm the test substance intake was less than expected in this group.
No relevant changes in food intake were observed at dose levels of 2500 ppm and below.
At doses up to 2500 ppm the test substance intake increased roughly as expected from the dose factor (table 3).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute and relative spleen weights were dose-dependently increased at 100, 500, and 2500 ppm, which was statistically significant from 500 ppm onwards (table 4).
There were no test substance-related changes in absolute and relative liver and kidney weights up to 2500 ppm. The mean relative liver weight of 100 ppm dose group males was statistically significantly reduced. However, this finding is of no toxicological relevance, because of the single occurrence and because a dose-dependency is lacking.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Blackish discoloration of the spleen (starting at 100 ppm), kidneys (starting at 500 ppm), and liver (only 10000 ppm) were noted.
At dose levels ≥ 500 ppm the spleen frequently appeared swollen and/or enlarged (500 ppm: male: 4/5, female: 5/5; 2500 ppm: male: 5/5, female: 5/5;10000 ppm: male: 4/5, female: 5/5).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Spleen:
- Increased blood content in dilated sinuses of the red pulp in 2/5 females of the 100 ppm dose group and in all animals of the higher dosages.
- Increased extramedullary hematopoiesis at all dose levels.
- Increased deposition of a brownish pigment (most likely of ferruginous origin) at dose levels ≥ 500 ppm.

Liver:
- Pigmented Kupffer cells preferably located in the periportal areas starting in males at dose levels ≥500 ppm and in females ≥2500 ppm.
- Periportal cytoplasmic change and, in one case, periportal degeneration and bile duct proliferation at 2500 ppm.
- Centrilobular cytoplasmic change and hypertrophy in males at 500 ppm.

Kidneys:
- Deposition of a brownish pigment in all animals at 2500 ppm.
- Tubular degeneration/regeneration in 1/5 males at 2500 ppm.

Histopathological findings: neoplastic:

not specified

Effect levels

Key result

Dose descriptor:

LOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 ppm

System:

other: spleen

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Any other information on results incl. tables

Table 2: Body weight (g)

Sex	m	m	m	m	m	f	f	f	f	f
Dose (ppm)	0	100	500	2500	10000	0	100	500	2500	10000
Study day
1	143	144	147	147	149	120	117	120	121	118
8	163	171	172	174	129**	140	140	141	136	107**
15	178	190	190	201*	127**	156	160	157	153	105**
22	239	214*	210**	224	125**	175	176	168	164	100**
29	233	227	223	240		181	185	175	170
34	276	271	269	267		194	200	187	182

* p ≤0.05, ** p ≤0.01

Table 3: Calculated test substance intake (mg test substance/kg body weight/day)

Sex	m	m	m	m	m	f	f	f	f	f
Dose (ppm)	0	100	500	2500	10000	0	100	500	2500	10000
Study day
8	-	8	48	213	603	-	12	47	186	201
15	-	8	42	203	515	-	10	48	189	658
22	-	8	41	193	573	-	9	47	185	551
29	-	7	35	170		-	9	45	157
34	-	9	42	175		-	9	54	184

Table 4: Absolute and relative spleen weights

Sex	m	m	m	m	m	f	f	f	f	f
Dose (ppm)	0	100	500	2500	10000	0	100	500	2500	10000
Number of animals	5	5	5	5	-	5	5	5	5	-
Final body weight (g)	276	271	269	267		194	200	187	182
SPLEEN
absolute weight (mg)	575	614	821**	1606**		427	488	668**	1215**
relative weight (mg/100 g bw)	208	227	307**	602**		219	245	358**	668**

* p ≤0.05, ** p ≤0.01

Applicant's summary and conclusion

Conclusions:

Since treatment-related effects, especially regarding the spleen, were noticed from the lowest tested dose level of 100 ppm, 100 ppm is to be considered as Lowest Observed Adverse Effect Level (LOAEL) and no No Observed Adverse Effect Level (NOAEL) could be established in the present range finding study.