N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

Endocrine disrupter mammalian screening – in vivo (level 3)

Administrative data

Endpoint:

endocrine disrupter mammalian screening – in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 March 1997 to 07 April 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

Rats were randomly allocated to 3 groups (control, 500 or 1500 ppm test substance, concentrations employed in the carcinogenicity study performed previously. The effects of the administration for 3 weeks on the oestrus cycle and various plasma hormone levels were investigated in male and female rats. The was given in the diet because the oral route is the most likely anticipated for human exposure.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

EC Number:

410-690-9

EC Name:

N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)-phenyl-aminocarbonyl]-2,6-difluorobenzamide

Cas Number:

103055-07-8

Molecular formula:

C17 H8 Cl2 F8 N2 O3

IUPAC Name:

1-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]-3-(2,6-difluorobenzoyl)urea

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10 weeks
- Weight at study initiation: 248 - 282 g for females and 363 - 445 g for males.
- Housing: The animals were housed, three to a translucent polypropylene cage, on hardwood chip bedding. The cages were exchanged twice weekly at 3 or 4 day intervals with replacement of soiled Beta chips.
- Diet: powder MF diet, ad libitum
- Water: Drinking water, ad libitum via bottles.
- Acclimation period: 2 weeks, during which body weights, health conditions for both sexes and estrus cycle for females were monitored. Only after confirmation of normal status were they used for the study at the age of 12 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: FROM 12 March 1997 TO: 07 April 1997

Administration / exposure

Route of administration:

other: Oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION AND ANALYSIS
The test material was added to powder MF diet to the required levels and mixed in a mixer for 30 minutes. Corn oil was added to all diets including control diet at a concentration of 2 %.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity was assessed at the first preparation, 100 g samples each were collected from the top, middle and bottom layer of the mixer. Content analysis data were mean values calculated using the three values for homogeneity.

Duration of treatment / exposure:

3 weeks

Frequency of treatment:

Continously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm (nominal)

Remarks:

Group 2. Dietary concentration; equivalent to 30.5 and 39.4 mg/kg bw/day for males and females, respectively

Doses / concentrations 2

Dose / conc.:

1 500 ppm (nominal)

Remarks:

Group 3. Dietary concentration; equivalent to 92.5 and 120.1 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

15

Control animals:

yes

Examinations

Observations and examinations performed and frequency:

SURVIVAL AND CLINICAL OBSERVATIONS
All animals were checked twice a day for mortality and clinical signs, and findings were individually recorded.

BODY WEIGHT
Body weights were individually measured using an electric balance at the commencement of the study and weekly thereafter. Final body weights were determined at the termination of the treatment.

FOOD CONSUMPTION AND TEST MATERIAL INTAKE
Two-day consumption per cage was measured weekly and mean daily food consumption values per rat were calculated using the number of animals housed in one cage and the number of days. Test material intake was calculated from the nominal dietary levels, group mean food consumption and body weight data.

INVESTIGATION OF THE FEMALE OESTRUS CYCLE
Every morning (9:00~12:00 am) during the administration, vaginal smears were taken from 10 animals of each group and stained with Giemsa solution for microscopic observation after air-drying on slide glasses. Proestrus, oestrus, metestrus and dioestrus stages were judged, and vaginal cytology was examined. Each smear was scored for the relative density of cornified and nucleated cells (2=heavy density, 1=moderate density, 0=light density to none present) observed on each day.

Sacrifice and pathology:

PLASMA HORMONE LEVELS
At necropsy, 10 mL blood samples were collected from 10 animals of each group into heparinized tubes from the abdominal aorta. Plasma samples separated by centrifugation were stored in a Freezer (-20 °C).
The hormones examined were as follows: Oestradiol (E2), Progesterone, Corticosterone, Aldosterone, Prolactin, Luteinizing hormone (LH), Follicle stimulating hormone(FSH), Adrenocorticotropic hormone (ACTH), Testosterone.

HORMONE LEVELS IN THE TISSUE SAMPLES
The left ovaries, adrenals and testes from 10 animals of each group were removed, dipped into liquid nitrogen and stored in a deep-freezer (-80 °C) Analyses will be performed when required by the sponsor.

PATHOLOGY
Pathological examination was as follows for 10 animals of each group.

GROSS PATHOLOGY
Female animals were scheduled.to be sacrificed the day after displaying the proestrus stage of their oestrous cycle whenever possible after a minimum of 21-days dosing. Male animals were killed after 21-days administration. Blood samples (10 mL) were collected from the abdominal aorta under ether anaesthesia and the animals were then killed by exsanguination (refer to Plasma hormone levels). The organs and tissues in the thoracic and abdominal cavities were examined macroscopically and the uterus, ovaries (with oviduct), vagina, testes, adrenals and pituitary were removed and preserved in buffered formalin solution.

ORGAN WEIGHTS
At autopsy, the weights of the uterus, ovaries (with oviduct), testes, adrenals and pituitary (after fixation) were measured using an electric balance and organ to body weight ratios were calculated using the final body weights.
HISTOPATHOLOGY
The organs in section Gross pathology were embedded in paraffin wax, sectioned, stained with haematoxylin and eosin (HE), and microscopically observed.

CHOLINESTERASE ACTIVITIES
After 3-weeks administration, cholinesterase activity in plasma, erythrocyte and brain sample prepared from 5 rats of each group was determined using an automatic analyser.

Statistics:

STATISTICAL ANALYSIS
The significances of differences between control and treated groups for each parameter, excluding food consumption and test material intake, were analysed, and evaluated at P<0.05 or P<0.0l. Analysis of variance for each group was performed using the F- test. In cases of equal variance the Student’s t—test was applied and where this was not the case the Welch—test was used. The Significance of differences in incidence data were analysed using the Fisher’s exact test.

Results and discussion

Endocrine disrupting potential:

negative

Maximum tolerated dose level exceeded:

no

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No change of general conditions related to test substance administration were observed.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No deaths related to test substance administration were observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No differences in mean body weights of either sex were observed between the groups of rats given test substance and the control groups with the exception of body weight gain retardation in the 1500 ppm female group at week 3.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no difference between the food consumption of test and control animals of both sexes.

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

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No statistically significant differences in any of the samples were noted between the groups of rats given test substance and the controls.

Endocrine findings:

no effects observed

Description (incidence and severity):

Oestrus cycles: No statistically significant differences in mean oestrus cycle length were found between the treated groups and control, although there were two cases in the 500 ppm group of 7 and 10 days length respectively and one case in the 1500 ppm group of 4.6 days length. There was no statistically significant difference in the relative density of cornified and nucleated epithelial cells, between the treated and control animals.
Plasma hormone levels: Statistically significant elevation of mean prolactin, FSH and ACTH levels was noted for the 1500 ppm male group. For prolactin, 8 out of 10 of the 1500ppm animals had plasma levels that were within the control range, therefore the higher mean value for this group was not considered to be biologically significant. The slightly higher FSH levels in the 1500 ppm group were also not considered large enough to be biologically significant. The increase in ACTH levels is relatively small, and may indicate a stress response in these animals that is unrelated to treatment. No other statistically significant differences in hormone levels were found between any of the groups given test substance and controls.
No effects on cholinesterase were found.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No gross pathological abnormalities were observed in any of the groups, other than inner dilatation of the uterus in one rat in the 1500 ppm group. Significantly lower relative uterus weights were noted in the 500 ppm group compared to controls. This was unlikely to be treatment-related as the effect was slight and there was no dose dependence. There were no statistically significant differences between test and control animals in the weights of the ovaries, testes, adrenals or pituitary.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Angiectasis in the adrenals was observed in one to three female rats in each group and one male rat fed 1500 ppm test substance. Fatty metamorphosis in the adrenals was also noted in one to two male rats in each group. Inner dilatation of the uterus was observed in one rat each of the control group and the 1500 ppm group. No abnormal histopathological findings were noted for the pituitary, ovaries, vagina or testes in any of the groups.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 500 other: ppm (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

1500 ppm Dietary equivalent to 92.5 and 120.1 mg/kg bw/day in males and females, respectively

Any other information on results incl. tables

Table 1 Intergroup comparison of body weights (g)

	Dietary Concentration of Test substance (ppm)
	Males			Females
week	0	500	1500	0	500	1500
0 3	409.9 471.5	408.9 476.0	419.9 485.1	265.9 299.0	266.9 301.0	267.7 286.9*

* Statistically significant difference from control group mean, p<0.05

Table 2 Overall mean dose received (mg/kg/day)

Sex	Dietary Concentration of Test substance (ppm)
	0	500	1500
Males	0	30.5	92.5
Females	0	39.4	120.1

Table 3 Intergroup comparison of hormone level data – selected parameters

	Dietary Concentration of Test substance (ppm)
	Males			Females
Parameter	0	500	1500	0	500	1500
Prolactin	20.200	22.635	33.420*	85.610	<80.460	95.870
FSH	19.4	21.64	22.47**	18.26	16.72	21.50
ACTH	41.35	50.95	67.88**	115.22	130.57	118.29
Corticosterone	295.30	325.15	336.46	950.10	977.20	953.30

* Statistically significant difference from control group mean, p<0.05

** Statistically significant difference from control group mean, p<0.01

Table 4: Organ to bodyweight ratio data –females - selected parameter

Organ	Dietary Concentration of Test substance (ppm)
	0	500	1500
Uterus	0.2143	0.1899*	0.2386

* Statistically significant difference from control group mean, p<0.05

Applicant's summary and conclusion

Conclusions:

Administering the test substance at 500 ppm (30.5 and 39.4 mg/kg bw/day for males and females, respectively) or 1500 ppm (92.5 and 120.1 mg/kg bw/day for males and females, respectively) to rats showed no effect on the endocrine system in either sex, thereby focusing on the pituitary, adrenal and genital organs.

Executive summary:

In the current study, without an applicable guideline and not carried out in accordance to GLP, the effects of treatment with two dose levels of test substance for 3 weeks on the oestrus cycle and various plasma hormone levels were investigated in male and female rats. A total of 45 female and 45 male rats [Crj: Sprague-Dawley (SD)] were randomly allocated to 3 groups: control, 500 (30.5 and 39.4 mg/kg bw/day for males and females, respectively) or 1500 ppm (92.5 and 120.1 mg/kg bw/day for males and females, respectively) test substance. These dose levels have been employed in the carcinogenicity study performed previously. All animals were checked twice a day for mortality and clinical signs. Body weights were recorded at the start of the study and weekly thereafter. Two-day food consumption per cage was measured weekly and mean daily food consumption values per rat were calculated.

There were no effects of test substance on mean oestrus cycle length or relative density of cornified and nucleated epithelial cells. Furthermore, no treatment-related changes were observed on histopathological assessment of the female genital system. These results suggest that test substance administration does not result in any adverse effects on the female genital system. The lack of any effect of test substance treatment on plasma hormone levels in females indicates that it does not modify the endocrine system.

In males, the only biologically significant change was an increase in mean ACTH at 1500 ppm. However, it is considered that this increase is more likely to be as a result of a slight stress response in some animals, rather than an effect of test substance. In the absence of any other changes in either hormone levels or pathology that may correlate with this effect, it is not considered to indicate any modification of the endocrine system.

The results of this investigation of the effects of test substance administration on the rat endocrine system, focused on the pituitary, adrenal and genital organs, suggest that there is no effect on the endocrine system in either sex. This conclusion is supported by the in vivo reproduction studies, which show no effect of test substance on any reproductive endpoint.