Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Specific investigations: other studies

Administrative data

Endpoint:

specific investigations: other studies

Remarks:

Liver enzyme induction in mice

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

31 Aug 1995 to 12 Oct 1995

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:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study was intended to identify and phenotype a prospective inducing effect and its reversibility of the test article on the cytochrome P450 system and other xenobiotic metabolising enzymes of the Tif:MAGf (SPF) mouse. From 8 mice/sex/group, liver homogenates were prepared, and following fractions were separated: 100g supernatant, microsomal fraction, and cytosolic fraction. Investigations included: protein content of all fractions, microsomal cytochrome P450 content, microsomal 7-ethoxyresorufin- and 7-pentoxyresorufin-O-dealkylase activity (EROD, PROD), microsomal lauric acid 11- and 12-hydroxylation (La-11-OH and La-12-OH), peroxisomal fatty acid beta-oxidation (FAO), cytosolic glutathione S-transferase activity (GST), immunoblots with antibodies for CYP1A, CYP3A and CYP4A isoenzymes.

GLP compliance:

yes (incl. QA statement)

Type of method:

in vivo

Endpoint addressed:

carcinogenicity

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

EC Number:

276-696-7

EC Name:

Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Cas Number:

72490-01-8

Molecular formula:

C17 H19 N O4

IUPAC Name:

ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Test animals

Species:

mouse

Strain:

other: Tif:MAGf(SPF)

Sex:

male/female

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Duration of treatment / exposure:

14 days

Frequency of treatment:

Continuously

Post exposure period:

28 days (control and high dose group only)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 ppm

Remarks:

Low dose. Dietary equivalent to 10.0 and 10.1 mg/kg bw/day for males and females, respectively

Doses / concentrations 2

Dose / conc.:

500 ppm

Remarks:

Mid dose. Dietary equivalent to 92.9 and 91.7 mg/kg bw/day for males and females, respectively

Doses / concentrations 3

Dose / conc.:

2 000 ppm

Remarks:

High dose. Dietary equivalent to 365.0 and 361.6 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Results and discussion

Details on results:

The major treatment-related biochemical alterations were an increase in cytochrome P450 content up to 166% and 147% of control at the highest dose in male and female animals, respectively. This was paralleled by a strong increase in protein cross reacting with the monoclonal antibody clo4 specific for rat CYP4A isoenzymes, as well as a strong induction of lauric acid 12-hydroxylation up to 753% and 1254% of control at the highest dose for male and female animals, respectively. The peroxisomal fatty acid ß-oxidations was moderately induced at 500 and 2000 ppm and attained 203% and 243% of control at the highest dose for male and female animals, respectively.
Minor biochemical alterations comprised a slight to moderate induction of lauric acid 11-hydroxylase activity up to 245% and 226% of control as well as slight increases in ethoxyresorufin O-de-ethylase activity up to 224% and 186% of control and pentoxyresorufin O-depentylase activity up to 463% and 177% of control at the highest dose for male and female animals, respectively. In addition, slightly increased microsomal protein contents (114-118% of control) were observed in males at 500 and 2000 ppm.
The liver enzyme activity prole in treated male and female mice, i.e. prominent increase in lauric acid 12-hydroxylation and moderate increase in fatty acid ß-oxidation together with a strong increase of protein detected with the monoclonal antibody for rat liver CYP4A isoenzymes, indicate that the test substance is a strong inducer of hepatic xenobiotic metabolising enzymes in the mouse and can be classified as a peroxisome proliferator type inducer.

Any other information on results incl. tables

Mean daily food consumption and body weight development were comparable in all experimental groups. No clinical signs of toxicity were observed. Treatment had no effect on the carcass weight. No macroscopic changes were observed at necropsy at any concentration. Absolute as well as relative liver weights were dose-dependently and reversibly increased in both sexes. There was no effect on absolute or relative lung weights. Biochemical liver parameters were investigated in 8 out of 20 male and female animals of each dose group.

The major treatment-related biochemical alterations were an increase in cytochrome P450 content up to 166% and 147% of control at the highest dose in male and female animals, respectively. This was paralleled by a strong increase in protein cross reacting with the monoclonal antibody clo4 specific for rat CYP4A isoenzymes, as well as a strong induction of lauric acid 12-hydroxylation up to 753% and 1254% of control at the highest dose for male and female animals, respectively. The peroxisomal fatty acid ß-oxidations was moderately induced at 500 and 2000 ppm and attained 203% and 243% of control at the highest dose for male and female animals, respectively.

Minor biochemical alterations comprised a slight to moderate induction of lauric acid 11-hydroxylase activity up to 245% and 226% of control as well as slight increases in ethoxyresorufin O-de-ethylase activity up to 224% and 186% of control and pentoxyresorufin O-depentylase activity up to 463% and 177% of control at the highest dose for male and female animals, respectively. In addition, slightly increased microsomal protein contents (114-118% of control) were observed in males at 500 and 2000 ppm.

Treatment had no effect on protein contents of 100g supernatant and cytosolic liver fractions, glutathione S-transferase and protein detected with two monoclonal antibodies specific for rat CYP1A and CYP3A isoenzymes.

All treatment related effects were reversible after the 28-day recovery period.

Table 1. Protein contents

DOSE [ppm]	GROUP	SEX	SUPERNATANT		MICROSOMAL		CYTOSOLIC FRACTION
			[mg/g	liver]	[mg/g	liver]	[mg/g	liver]
			Mean	SD	Mean	SD	Mean	SD
TREATMENT
0	1	m	197	14	19.5	2.6	101.6	4.4
50	3	m	201	9	18.8	2.6	101.8	4.4
500	4	m	209	12	23.0*	2.4	100.6	4.8
2000	5	m	203	14	22.2	2.8	95.8	6.5
TREATMENT/RECOVERY GROUPS
0/0	2	m	181	20	20.3	4.1	104.8	5.8
2000/0	6	m	189	14	17	2.1	98.6*	5.6
TREATMENT
0	7	f	184	6	20.8	1.3	97.5
50	9	f	182		19.3	2.1	90.2	6.8
500	10	f	196	8	22.1	2	95.7	4.9
2000	11	f	180	16	21.9	1.8	87.8*	5.3
TREATMENT/RECOVERY GROUPS
0/0	8	f	180	20	17.8	1.7	96	6.6
2000/0	12	f	177	15	17.9	0.9	94.4	4.2

Two-sided Dunnett’s : * = p<0.05; ** = p<0.01; *** = p<0.001

Table 2. Cytochrome P450 content and alkoxyresorufin dealkylase activities

DOSE	GROUP	SEX	P450		EROD		PROD
[ppm]	No.		[nmol/g	liver]	[nmollmin/g	liver]	[nmol/min/g	liver]
			Mean	SD	Mean	SD	Mean	SD
TREATMENT
0	1	m	15.1	1.5	3.1	0.4	0.44	0.07
50	3	m	17.7	2.3	4.5**	1.3	0.57	0.14
500	4	m	23.1***	4	6.1***	1.1	1.22***	0.32
2000	5	m	25.1***	2.9	6.9***	1.4	2.02***	0.42
TREATMENT/RECOVERY GROUPS
0/0	2	m	13.7	2.7	3.5	1	0.53	0.15
2000/0	6	m	12.8	1.4	3.8	0.8	0.63	0.13
TREATMENT
0	7	f	16.4	2.9	5	1.2	1.98	0.46
50	9	f	15.2	1.5	4.5	0.5	1.73	0.28
500	10	f	20.7*	3.7	8.2***	1.8	2.80**	0.76
2000	11	f	24.0***	2.5	9.3***	1.9	3.50***	0.53
TREATMENT/RECOVERY GROUPS
0/0	8	f	11	1.7	3.9	0.7	2.05	0.54
2000/0	12	f	10.4	2.1	3.6	0.7	1.98	0.45

Two-sided Dunnett’s : * = p<0.05; ** = p<0.01; *** = p<0.001

Table 3. Lauric Acid Hydroxylase activitys

DOSE	GROUP	SEX	LA-11-OH		LA-12-OH
[ppm]	No.		[nmol/min/g	liver]	[nmol/min/g	liver]
			Mean	SD	Mean	SD
TREATMENT
0	1	m	18.6	2.6	17.7	3.6
50	3	m	23.9*	3.4	23.3	5.9
500	4	m	36.6**	7.6	73.2***	26.2
2000	5	m	45.6***	4.9	133.3***	26.8
TREATMENT/RECOVERY GROUPS
0/0	2	m	21	4.3	20.9	5.6
2000/0	6	m	21.9	3.7	35.2	27.9
TREATMENT
0	7	f	30.9	8.6	23.9	11.6
50	9	f	42.1*	4.9	40.0*	11.6
500	10	f	58.8***	8.5	153.6***	21.7
2000	11	f	69.8***	17.4	299.8***	76.8
TREATMENT/RECOVERY GROUPS
0/0	8	f	31.1	6.6	25.1	5.8
2000/0	12	f	31.4	4.4	23.2	7.2

Two-sided Dunnett’s : * = p<0.05; ** = p<0.01; *** = p<0.001

Table 4. Fatty acid ß-oxidation and glutathione S-transterase activity

DOSE	GROUP	SEX	FAO		GST
[ppm]	No.		[nmol/min/g	liver]	[µmol/min/g	liver]
			Mean	SD	Mean	SD
TREATMENT
0	1	m	1002	73	480	71
50	3	m	1097	154	500	137
500	4	m	1473***	101	464	61
2000	5	m	2036***	355	410	119
TREATMENT/RECOVERY GROUPS
0/0	2	m	907	276	497	144
2000/0	6	m	948	249	474	78
TREATMENT
0	7	f	1124	158	178	40
50	9	f	1102	148	161	31
500	10	f	1524***	102	158	15
2000	11	f	2736***	272	153	20
TREATMENT/RECOVERY GROUPS
0/0	8	f	1150	150	210	74
2000/0	12	f	1103	165	163	11

Two-sided Dunnett’s : * = p<0.05; ** = p<0.01; *** = p<0.001

Table 5. Densitometric quantification of cytochrome P450 isoenzymes on immunoblots of liver microsomes

DOSE	GROUP	SEX	BAND INTENSITY [RELATIVE AREA UNITS]
[ppm]	No.		ANTIBODY
			d15	p6	clo4
TREATMENT
0	1	m	1595	0/1302	450
50	3	m	1616	0/1973	415
500	4	m	1684	0/1748	2891
2000	5	m	1261	0/2393	2333
TREATMENT/RECOVERY GROUPS
0/0	2	m	1759	0/1874	845
2000/0	6	m	1817	0/1679	1121
TREATMENT
0	7	f	906	718/204	564
50	9	f	451	622/384	477
500	10	f	527	587/337	2993
2000	11	f	367	839/187	4207
TREATMENT/RECOVERY GROUPS
0/0	8	f	627	580/379	845
2000/0	12	f	466	395/525	304

Values are the results from densitometric scans of single immunoblots and represent the area of the protein bands given in relative area units. 400, 200 and 25 µg liver tissue equivalents of microsomal protein were applied per lane the detection with antibodies and clo4, respectively. The microsomal fractions were mixtures of equal volumes of the microsomal suspension from all eight animals per experimental group selected for determination of biochemical parameters

Applicant's summary and conclusion

Conclusions:

The liver enzyme activity prole in treated male and female mice, i.e. prominent increase in lauric acid 12-hydroxylation and moderate increase in fatty acid ß-oxidation together with a strong increase of protein detected with the monoclonal antibody for rat liver CYP4A isoenzymes, indicate that the test substance is a strong inducer of hepatic xenobiotic metabolising enzymes in the mouse and can be classified as a peroxisome proliferator type inducer.

Executive summary:

This study was intended to identify and phenotype a prospective inducing effect and its reversibility of the test article on the cytochrome P450 system and other xenobiotic metabolising enzymes of the Tif:MAGf (SPF) mouse. Groups of 20 young adult male and female mice were treated for 14 consecutive days with the test substance at dietary concentrations of 0, 50, 500 and 2000 ppm corresponding to mean daily doses of 0, 10.1, 92.9 and 365.0 mg/kg bw/day for males and 0, 10.0, 91.7 and 361.6 mg/kg bw/day for female animals. In order to test for the reversibility of possible treatment-related effects groups of 20 animals received 0 or 2000 ppm for 14 days followed by a 28-day recovery period.

Mean daily food consumption and body weight development were comparable in all experimental groups. No clinical signs of toxicity were observed. Treatment had no effect on the carcass weight. No macroscopic changes were observed at necropsy at any concentration. Absolute as well as relative liver weights were dose-dependently and reversibly increased in both sexes. There was no effect on absolute or relative lung weights. Biochemical liver parameters were investigated in 8 out of 20 male and female animals of each dose group.

The major treatment-related biochemical alterations were an increase in cytochrome P450 content up to 166% and 147% of control at the highest dose in male and female animals, respectively. This was paralleled by a strong increase in protein cross reacting with the monoclonal antibody clo4 specific for rat CYP4A isoenzymes, as well as a strong induction of lauric acid 12-hydroxylation up to 753% and 1254% of control at the highest dose for male and female animals, respectively. The peroxisomal fatty acid ß-oxidations was moderately induced at 500 and 2000 ppm and attained 203% and 243% of control at the highest dose for male and female animals, respectively.

Minor biochemical alterations comprised a slight to moderate induction of lauric acid 11-hydroxylase activity up to 245% and 226% of control as well as slight increases in ethoxyresorufin O-de-ethylase activity up to 224% and 186% of control and pentoxyresorufin O-depentylase activity up to 463% and 177% of control at the highest dose for male and female animals, respectively. In addition, slightly increased microsomal protein contents (114-118% of control) were observed in males at 500 and 2000 ppm.

Treatment had no effect on protein contents of 100g supernatant and cytosolic liver fractions, glutathione S-transferase and protein detected with two monoclonal antibodies specific for rat CYP1A and CYP3A isoenzymes.

All treatment related effects were reversible after the 28-day recovery period.

In conclusion, the liver enzyme activity prole in treated male and female mice, i.e. prominent increase in lauric acid 12-hydroxylation and moderate increase in fatty acid ß-oxidation together with a strong increase of protein detected with the monoclonal antibody for rat liver CYP4A isoenzymes, indicate that the test substance is a strong inducer of hepatic xenobiotic metabolising enzymes in the mouse and can be classified as a peroxisome proliferator type inducer.

The lowest dose of 50 ppm is a minimal effect level. Hydroxylation of lauric acid at the 12-position was slightly induced in the female animals and at the 11-position in both sexes. A minimal increase of the ethoxyresorufin O-de-ethylase activity at this dose in male animals wasneither followed by a substantial further increase at the two higher doses nor paralleled by an increase of proteins. The minimal increases of the absolute and relative liver weights in female animals at the lowest dose were in the of 10% above control and are regarded as borderline. In a cell proliferation study in male mice, dietary administration of the test substance at 500 ppm caused an increase of the relative and absolute liver weight after 14 days treatment. However, after treatment for 42 days at the same dose level, this increase was reduced and no more statistically significant, indicating a certain adaptation. Correspondingly, the minimal effects at the lowest dose of 50 ppm in the female as observed in this study might be transient.