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

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Apr - 05 Sep 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Version / remarks:

Adopted in 1983

Deviations:

no

GLP compliance:

yes

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

Details on species / strain selection:

Details on strain: Crl: (WI) WU BR
Wistar rats have been used for reprotoxicological studies at Bayer Health Care AG for a number of years.
Historical data of Wistar rats on the test parameters, including reproductive parameters, are available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age of parental (P) animals at study initiation: males: 8 weeks, females: 14 weeks
- Mean body weight at study initiation: (P) males: 217 g (range:194 - 248 g), females: 196 g (range: 177 - 214 g)
- Housing: Animals were conventionally housed in Makrolon cages type IIIh on low-dust soft-wood shaving (Source: Ssniff GmbH, Soest, Germany). Animals were singly housed; except for mating period. Females were provided with nesting material (coarse wood shavings) shortly before parturition.
- Diet: fixed-formula standard diet Provimi Kliba 3883.9.25 meal (Provimi Kliba AG, Kaiseraugst, Switzerland), ad libitum
- Water: tap water of drinking water quality, ad libitum
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 Apr 2005 To: 05 Sep 2005

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: standard diet

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): once a week
- Mixing appropriate amounts with (Type of food): fixed-formula standard diet Provimi Kliba 3883.9.25 meal (Provimi Kliba AG, Kaiseraugst, Switzerland)
- Storage temperature of food: at room temperature

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Animals were co-housed over night at a maximum of 12 times during a 3-week mating period.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after unsuccessful attempts: Yes. Parental females found sperm-positive after the first mating but not shown to be pregnant (lack of weight gain within 14 days following insemination) were co-housed again over one week with the same male without checking insemination or measuring body weight.
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance was quantified using a liquid chromatographic method. For analytical investigations, samples from the test substance formulation covering the test substance concentration range used in the study, were taken. These samples were extracted and diluted with methanol and subsequently quantified by reversed phase (C-18) high-performance liquid chromatography (HPLC) with UV-detection (DAD; wavelength: 238 nm) in case of test substance concentration >10 ppm. Feed formulations with a test substance concentration <10 ppm were extracted and diluted with a mixture of acetonitrile/aqueous 0.05 % formic acid (4:1) and subsequently quantified by reversed phase (C-18) HPLC with mass selective detection (MSD; SIM-ions: 154).
Homogeneity, stability and content of test substance in the formulation were confirmed at the beginning of the study, twice during the in-life phase, and at termination of the study.

Duration of treatment / exposure:

(P) males: 10 weeks pre-treatment, 3 weeks mating period
(P) females: 2 weeks pre-treatment, 3 weeks mating period, 3 weeks gestation, 4 weeks lactation

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

- Age at mating of the mated animals in the study: (P) males: 18 weeks, females: 16 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1.5 ppm

Remarks:

actual test substance intake: males: 0.104 mg/kg bw/day, females: 0.137 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

15 ppm

Remarks:

actual test substance intake: males: 1.075 mg/kg bw/day, females: 1.421 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

150 ppm

Remarks:

actual test substance intake: males: 10.547 mg/kg bw/day, females: 15.499 mg/kg bw/day

No. of animals per sex per dose:

25 animals/sex/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels for the present study were selected based on a 5-week dose-range finding study (Lanxess, 2005). Dietary exposure of rats to 100 ppm N-Isopropyl-4-fluoroaniline for 5 weeks resulted in macroscopical (blackish discoloration) and microscopical changes of the spleen. Microscopical changes included increased blood content in dilated sinuses of the red pulp in females and increased extramedullary hematopoiesis in both sexes. In addition, spleens of treated animals appeared swollen or enlarged, and spleen weight values were increased compared to control. Repeated oral or inhalation exposure of animals to higher dose levels resulted in nephrotoxicity, hepatotoxicity and marked hematotoxicity (for details please see respective repeated-dose toxicity studies within this dossier, Lanxess, 2005 and 2006).

- Fasting period before blood sampling for clinical biochemistry: no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: twice daily
- Cage side observations included: prolonged parturition, morbidity and mortality
The persistence of those cage side noticeable clinical signs, which had been already noted during weekly detailed clinical observation were recorded daily but not reported separately.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: once weekly
- Detailed clinical observations included: observation of the general state of health, behavior, condition of the fur, and the orifices as well as excretory products. During gestation periods females were clinically examined on day 0, 7, 14, 20, and during lactation on day 0, 4, 7, 14, 21, and 28.

BODY WEIGHT: Yes.
- Time schedule for examinations: once weekly

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.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes.

HEMATOLOGICAL INVESTIGATIONS
Blood sampling for hematological investigations were taken at following time points:
(P) males: In week 11 of pre-mating period in the first 10 males per group.
(P) dams: On day 21 - 24 post parturition in the first 5 - 10 females per group.
These samples were taken each in the morning from the retroorbital venae plexus of non fasted rats, which had been narcotized with diethyl ether.
The following parameters were measured: leukocytes, erythrocytes, hemoglobin, hematocrit, reticulocytes, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes.
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

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. A lung flotation in water was performed during the necropsy of pups found dead on the day of the first litter inspection to determine whether pups had breathed at birth or not.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals as soon as they were not required for further treatment.
- Maternal animals: All surviving animals after the last litter was weaned.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Liver, kidneys and spleen were first weighed and then prepared for microscopic examination, respectively. In addition, epididymides, ovaries with oviducts, pituitary gland, prostate, seminal vesicles (incl. coagulating glands), testes, uterus with cervix, and vagina were examined histopathologically in high dose group and control group animals. Organs of animals with suspected infertility and all macroscopic findings were also histopathologically assessed.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed at 28 - 30 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:
Animals were macroscopically inspected after opening the body cavities, with particular attention on the organs of reproduction. This included also visible skeletal abnormalities as far as possible.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:

Analysis of Variance (ANOVA) and in case of significant results Dunnett's test as post hoc test for:
- Body weights in male and female animals
- Body weight gains in male and female animals
- Feed consumption of male and female animals
- Number of implantation sites per female
- Number of viable pups per female
- Organ weights at necropsy
- Time to insemination

2 by N CHI2 test; in case of significant differences Fisher's exact test with Bonferroni correction for:
- Number of post-implantation losses per group based on the number of implantation sites
- Number of viable pups per group based on the number of implantations
- Insemination rate
- Fertility rate
- Gestation rate

Kruskall-Wallis test and in case of significant differences Dunnett's test for:
- Number of prenatal losses per litter

Hematology data were evaluated using a variance analysis followed by a Dunnett test, or a Kruskal-Wallis test followed by an adjusted U-test or an adjusted Welsh test (reticulocytes).

Differences between the control and 4-Fluor-N-isopropylanilin-treated groups were considered significant when p <0.05. Significant differences from the control are indicated with* for p <0.05 and ** for p <0.01.

Reproductive indices:

Reproduction indices were calculated for each dose group using the following formulae:

Insemination index (%) = (No. of sperm positive females* / No. of females co-housed with a male) x 100

Fertility index (%) = (No. of pregnant females / No. of sperm positive females*) x 100

Gestation index (%) = (No. of females with live pups / No. of pregnant females) x 100

Rearing index (%) = (No. of females reared a litter up to day 21 post partum / No. of females born a litter) x 100

* including pregnant females that were not sperm positive

Offspring viability indices:

Reproduction indices were calculated for each dose group using the following formulae:

Live birth index (%)* = (No. of live pups at birth / Total No. of pups born) x 100

Viability index (%)* = (No. of live pups on day 4 pre-culling / No. of live pups born) x 100

Lactation index (%)* = (No. of live pups after three weeks / No. of live pups after four days (after culling)**) x 100


* index calculation from litter means
** moribund pups that died during the course of culling were not included

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related changes of body weight values were observed in any treatment group during premating, gestation and/or lactation.
Female animals of the high dose group showed slightly higher body weights compared to control. These animals ingested slightly more diet during premating and lactation on some dates. This finding was not considered as toxicologically relevant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related changes of food consumption and test substance intake were observed in treated animals.
Males of all dose groups and females of the low and mid dose group showed no change in food intake compared to control. Accordingly, the mean test substance intake agreed to the theoretical dose factor in these groups. Females of the high dose group ingested slightly more diet and test substance as expected.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The results of the hematological examinations revealed effects on red blood parameters of parental animals. Several red blood parameters were statistically significantly affected at 15 ppm and above. In male and female animals of the high dose group (150 ppm) statistically significantly reduced erythrocyte counts, reduced hemoglobin and hematocrit concentrations, as well as increased reticulocyte counts were seen. Additionally, in female animals mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) values were statistically significantly increased whereas mean corpuscular hemoglobin concentration (MCHC) was decreased. In the high dose group females leucocyte counts were increased as well. Only in females of the mid dose group (15 ppm) statistically significantly reduced erythrocyte counts, reduced hemoglobin and hematocrit concentrations, and increased MCV and MCH mean values were observed. There were no changes in red blood parameters in male animals of the mid dose group (15 ppm) and in animals of the low dose group (1.5 ppm) in both sexes.

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

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examinations of the spleen revealed that at 1.5 and 15 ppm the level of the occurrence of iron pigment deposits in the spleen was slightly elevated when compared to the control group, whereas a clear increase in deposition of Prussian Blue-positive iron containing pigment was observed in both sexes dosed with 150 ppm. This correlates to the dark discoloration in the spleen observed macroscopically in these animals (at 150 ppm). Increased iron deposition was accompanied with an increased incidence of hematopoiesis in the red pulp (females) and congestion/ectatic sinuses of the red pulp (both sexes) in the 150 ppm dose group. These findings are regarded to be treatment-related and adverse.
The slightly elevated incidence of increased iron pigment deposits in the spleen at 1.5 and 15 ppm was considered to be not an adverse effect as other morphological correlates of an increased turnover of erythrocytes were absent in these groups.
There was no evidence of any dose-correlated finding in reproductive organs of parental male and female animals.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

The insemination, fertility, gestation and rearing indices as well as the mean duration of pregnancy and implantation site count were not affected at dose levels up to and including 150 ppm when compared to control.
There were some parental females (3-5-5-12 with ascending dose), which had been found to be sperm-positive after the first day of co-housing, but failed to become pregnant. According to experience, this could happen, if an inexperienced male, co-housed with a female for the first time, inseminated the female outside the estrus.
This assumption is obviously correct, since after these females had been remated with the same male for one week few (1-1-1-3 with ascending dose) of them had no pups (and no implantation sites).
There were no toxicologically relevant changes on mating performance up to 150 ppm. The relative low value for the mean day of mating period when insemination occurred or the relative low value for mating days until day 0 p.c. visible at 150 ppm is based on the fact that in this group several females were found to be sperm positive after the first co-housing possibly when outside their estrus. As mentioned above, nearly all these females got pregnant after re­mating with their male. A treatment-related effect is, therefore, not concluded.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

150 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: No effects up to and including the highest tested dose of 150 ppm, which corresponded to actually ingested 10.547 mg/kg bw/day for males, and 15.499 mg/kg bw/day for females.

2

Key result

Dose descriptor:

NOAEL

Effect level:

1.5 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: No effects observed at the lowest tested dose of 1.5 ppm, which corresponded to actually ingested 0.104 mg/kg bw/day for males, and 0.137 mg/kg bw/day for females.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

15 ppm

System:

haematopoietic

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 150 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL corresponding to the highest dose tested

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present study, the NOAEL of the test substance for reproductive toxicity was established at the highest tested dose of 150 ppm for both parental male and female animals and the progeny, corresponding to 10.547 and 15.499 mg/kg bw/day actual substance intake in male and female parental animals, respectively.
The NOAEL of the test substance for systemic toxicity was established at the lowest tested dose level of 1.5 ppm for parental rats, corresponding to 0.104 and 0.137 mg/kg bw/day actual substance intake in male and female animals, respectively.