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

Carcinogenicity

Description of key information

- The NOAEL for toxicity and carcinogenicity was established at 50 ppm (dietary equivalent to 5.75 and 5.33 mg/kg bw/day for males and females, respectively), based on the incidence of lung tumours and hepatic tumours, mice, feeding study, OECD 451, Bachmann 1995

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Nov 1992 to 26 May 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

1981

Qualifier:

according to guideline

Guideline:

EPA OPP 83-2 (Carcinogenicity)

Version / remarks:

1982

Qualifier:

according to guideline

Guideline:

other: Notification of Director—General, Agricultural Production Bureau, Ministry of Agriculture, Forestry and Fisheries, Japan, 59 Noh San No. 4200, dated January 28, 1985: "Guidance on Toxicology Study data for Application of Agricultural Chemical Registration

Version / remarks:

1985

GLP compliance:

yes (incl. QA statement)

Species:

mouse

Strain:

Tif:MAGf

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approx. 4-5 weeks
- Weight at study initiation: 23.80- 30.20 g in males and 20.00- 25.80 g in females
- Housing: The male animals were housed individually in macrolon cages type 2, and the females
in groups of 5 animals in macrolon cages type 3, both with wire mesh tops and sterilized, granulated soft wood
- Diet: Pelleted, certified standard diet , ad libitum
- Water: Tap water was given ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): 16-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 16 Nov 1992 to 26 May 1994

Route of administration:

oral: feed

Vehicle:

acetone

Remarks:

(evaporated)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Fresh diets were prepared at 4- week intervals throughout the study and stored in stainless steel containers at room temperature until used. For each group, an aliquot of the test article dissolved in acetone was added to a fixed amount of bolus alba and pulverized food. A further quantity of acetone was added to equalize the acetone concentration for all groups. Following thorough mixing and evaporation of the acetone, each group's premix was incorporated into further quantities of diet to achieve the required concentration of test substance. Approximately 25% water was added to ensure pellet quality. The pellets were subsequently airdried. The animals in the control group (group 1) were fed with similarly pelleted food without the test substance.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diets were analysed for test substance homogeneity and stability. Periodically during the study, diets were analysed for test substance concentration.

Duration of treatment / exposure:

18 months

Frequency of treatment:

Continuously

Dose / conc.:

10 ppm

Remarks:

Group 2: Mean dietary equivalent to 1.10 and 1.04 mg/kg bw/day for males and females, respectively

Dose / conc.:

50 ppm

Remarks:

Group 3: Mean dietary equivalent to 5.75 and 5.33 mg/kg bw/day for males and females, respectively

Dose / conc.:

500 ppm

Remarks:

Group 4: Mean dietary equivalent to 55.4 and 51.5 mg/kg bw/day for males and females, respectively

Dose / conc.:

2 000 ppm

Remarks:

Group 5: Mean dietary equivalent to 222 and 201 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

60

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected by the sponsor based on the following previously conducted study: 80 week carcinogenicity / toxicity study in mice.
- Two experimental groups were used: Group I for the evaluation of the carcinogenic potential and survival analysis and group II for the evaluation of haematological paramters

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: daily (a.m. and p.m. on working days, a.m. on weekends and holidays)
- Cage side observations checked: Viability

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily, records at least weekly if clinical signs are present; records at the beginning and end of treatment, if no signs are observed

BODY WEIGHT:
- Time schedule for examinations: Weekly (midweek) for the first 3 months and
monthly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule: Weekly for the first 3 months and monthly thereafter.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake: An overall mean value was calculated based on the nominal content of the test article in the food. Additionally, this value was corrected for the analytically determined content

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: Yes

HAEMATOLOGY:
- Time schedule for collection of blood: Week 52 and week 79
- How many animals: 10 animals per sex
- Parameters checked: Erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, leukocyte count, differential leukocyte count, neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unstained cells and thrombocyte count

Sacrifice and pathology:

GROSS PATHOLOGY:
- Weights: Body (exsanguinated), brain, liver, kidneys, adrenals, gonads (testes/ovaries), spleen (weights)
- The following organs and tissues were preserved in neutral buffered 4% formalin: Skin, mammary area, spleen, mesenteric lymph node, axillary lymph node, sternum with bone marrow, femur with joint, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland (both), liver, gall bladder, pancreas, esophagus, stomach, small intestine ,large intestine, kidney (both), urinary bladder prostate, seminal vesicle, testis (both), epididymis (both), uterus, vagina, Ovary (both), pituitary gland, adrenal gland (both), thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord, eye with optic nerve (both), orbital gland (both), extraorbital lacrimal gland (both), Zymbal gland (both), muzzle, tongue and any gross lesions, masses


HISTOPATHOLOGY:
After the fixation, organ samples listed below were taken, embedded in paraplast, sectioned at 3-5 microns, stained with haematoxylin and eosin, and subjected to a microscopical examination: Skin, mammary area, spleen, mesenteric lymph node, axillary lymph node, sternum with bone marrow, femur with joint, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland (both), liver, gall bladder, pancreas, esophagus, stomach, small intestine, large intestine, kidney (both), urinary bladder, prostate, seminal vesicle, testis (both), epididymis (both), uterus, vagina, ovary (both), pituitary gland, adrenal gland (both), thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord, eye with optic nerve (both), orbital gland (both), muzzle, tongue and any gross lesions, masses.
- Satellite animals used for haematology (10 per group) were excluded from micropscopical examination.
- An external peer review was performed. See 'Any other information on materials and methods incl. tables' for details.

Statistics:

For each time point and parameter an univariate statistical analysis was performed. Nonparametric methods <1> were applied, to allow for non-normal as well as normal data distribution. Each treated group was compared to the control group by Lepage's <2> two- sample test and tested for increasing or decreasing trends from control up to the respective dose group by Jonckheere’s test for ordered alternatives <3>. The Lepage test is a combination of Wilcoxon and Ansari- Bradley statistics, i.e. a combined test for location and dispersion. The Lepage test has a good power against the more general alternative that the distributions differ not only in location but also in dispersion. The Jonckheere test is sensitive to monotone dose-related effects. Two- sided asymptotic p—Values are reported in the "statistics" tables. Flags for significant differences between groups or trends over groups (+ or -) are given in the "means" tables according to the specified significance level. Statistical tests and flags used are indicated in the header of each table. Survival analysis was performed by the regression model (partial likelihood) introduced by Cox <4> in order to compare survival time of treated animals (experimental group I) with control animals. Analysis of pathology data is described in chapter "Pathology (statistical evaluation)" given in Appendix A. Statistical significance does not necessarily imply biological relevance. Hence, the responsible scientist may not comment on statistically significant values lying within the physiological range and on the other hand may on values, which differ substantially from the expected normal values although this difference was not statistically significant

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The clinical signs observed were typical for mice of this colony on long- term feeding studies. The incidences recorded showed no treatment-related influences.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

An exceptionally low mortality was recorded for control females whereas for treated females the usual level of mortality was recorded for all groups. As mortality among treated male groups was similar to that of the male controls, it was concluded that treatment had not influenced the survival of the mice

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gains for treated male groups were similar to those of the control group throughout the study. Mean body weights for females of groups 4 and 5 (500 and 2000 ppm) deviated from control values from about week 18 with lower mean values recorded for the remainder of the study. By the end of the study, mean body weights were and 13% lower than the control value for females of groups 4 and 5 (500 and 2000 ppm), respectively

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the first year of the study the food intakes recorded for treated males were similar to those of the control group. Thereafter, higher values were recorded for males of groups 4 and 5 (500 and 2000 ppm). Although a more frequent occurrence of food spillage was recorded for males of groups 4 and 5 the quantities spilled were insufficient to account for the higher food intakes recorded. Treatment did not influence the food intakes of treated females. The overall food intakes (weeks 1 to 78) for treated female groups were within ± 2% of the control group value.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

During the first 52 weeks of the study, male and female groups had food consumption ratios similar to those of the control group. Thereafter, higher ratios were obtained for males and females of group 5 (2000 ppm) and for females of group 4 (500 ppm).

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the absence of any dose-relationship, no toxicological relevance is attached to the findings in this study of a pathology which occasionally occurs spontaneously in this colony of mice. Treatment did not disturb the haematological profile of the mice. Differences between the means which attained a level of statistical significance were of insufficient magnitude to be of toxicological relevance.

Clinical biochemistry findings:

not examined

Endocrine 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):

Organ weight analysis revealed significantly higher organ to body weight ratios for liver of males and females of groups 4 and 5 (500 and 2000 ppm), for kidneys of group 5 females and for adrenals of group 5 males.
The mean spleen ratio for control females was unduly influenced by mouse (spleen ratio 74.93). Excluding this value and recalculation gave a mean value of 7.93. The higher mean spleen weight and ratio of group 5 males (2000 ppm) was also influenced by the high values recorded for two other mice. Excluding these values and recalculating the gave values within the expected range for mice of this age. Therefore, it was concluded that the spleen was not influenced by treatment. Ovarian means were unduly influenced by the gross weights recorded for some mice in groups 1 (Control), 2 (10 ppm) and 5 (2000 ppm). Excluding these animals and recalculating the means gave similar mean values for all groups.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopical examination revealed an increased occurrence of nodules and masses in the lungs of male groups 4 and 5, and of female group 5. These findings mostly correlated with pulmonary neoplasia.
The incidence of enlargement of the liver was increased in male and female group 5, and the occurrence of hepatic masses in male groups 4 and 5. The hepatic enlargement in males and females correlated with a variety of microscopical changes such as congestion, extramedullary haematopoiesis, fatty change, or occasional infiltration by reactive or neoplastic white blood cells. Since none of these changes was distributed among the groups in a dose-related manner, no toxicological relevance was attributed to the increased incidence of hepatic enlargement in male and female groups 5.
Furthermore, in male animals macroscopical examination revealed an enlargement of the spleen distributed among the groups in an apparently dose-related manner.
The incidence of macroscopically enlarged ovaries was decreased in female group 5, which microscopically showed an increased incidence of ovarian atrophy

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A decrease in the incidence of hepatic fatty change in males of groups 4 and 5 (500 and 2000 ppm) and a decrease in the incidence of adrenal cortical atrophy probably represented a non-specific reaction to stress. In females of groups 4 and 5 (500 and 2000 ppm) a variety of non-neoplastic changes was detected which associated with the suppression of body weight gain and included lower incidences for fatty atrophy of bone marrow and hepatic fatty change. In group 5 females a lower incidence of pancreatic fatty atrophy and a higher incidence of ovarian atrophy were detected.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

LUNGS
Microscopical examination revealed an increase of pulmonary neoplasia in males and females of groups 4 and 5 (500 and 2000 ppm). Pulmonary neoplasias were classified as adenoma or carcinoma and considered to originate from the pulmonary “bronchiolo-alveolar" parenchyma.
In comparison to historical control values, in male animals of groups 4 and 5 the incidence of adenomas was slightly above the control range, the incidence of carcinoma and of a common pool of neoplasia was distinctly higher. In female animals of group 5 the incidence of both benign and malignant tumours was distinctly above the control range, while the values of group 4 were comparable with the high control range. Age-adjusted statistical analysis of the incidence and multiplicity of pulmonary neoplasia confirmed a significant increase in male and female groups 4 and 5.

LIVER
In addition, an increase of hepatocellular tumours was detected in males of groups 4 and 5 (500 and 2000 ppm). Hepatocellular neoplasias were classified as benign hepatoma or hepatocellular carcinoma. The occurrence of a few hepatoblastomas in occasional animals of various experimental groups was not considered to represent a treatment-related effect. For benign hepatoma the incidence was within the range of historical control values, whereas the incidence of hepatocellular carcinoma in male groups 4 and 5 was distinctly above the historical control values. The incidence of a common pool of benign and malignant hepatocellular tumours was slightly above the control values for group 4 and distinctly for group 5.
Age-adjusted statistical analysis confirmed the significance of the increased incidence of hepatocellular neoplasia in male groups 4 and 5 . Separate analysis of the incidence of hepatocellular carcinoma indicated also a significantly increased incidence in male group 3 when those tumours were considered fatal, but not when they were considered incidental. Actually, 10 out of 12 males of group 3 bearing hepatocellular carcinoma had survived until the terminal sacrifice, so that these tumours were not observed in fatal context. Therefore, and because the statistical analysis of the common pool of hepatocellular neoplasia did not indicate any effect on male group 3, the tumour incidence in this group is considered incidental and unrelated to treatment. Statistical analysis for the multiplicity of hepatocellular neoplasia revealed a significant increase in male group 5.
In female animals the incidences of hepatocellular neoplasia were quite low and close to those observed in historical controls. Therefore, a relationship to treatment for hepatocellular neoplasia in female animals was not considered established

INCIDENTAL FINDINGS
Additionally, a variety of other changes was found in this study. They naturally occur in our colony of mice, and, neither their incidences nor their distribution and morphologic appearance were considered to represent a toxicologically relevant effect.
These changes included in male animals: a decreased incidence of renal calcification and a decreased incidence of hyperplasia of pancreatic islets in group 5. In female animals: a higher incidence of myelofibrosis of bones in group 5 , and an apparently dose-related distribution among the groups of hemosiderosis of Kupffer cells, of glandular cystic hyperplasia of uterine endometrium, and of deposition of ceroid pigment in the adrenal gland.

Other effects:

no effects observed

Relevance of carcinogenic effects / potential:

The obviously treatment-related increase in the incidence of pulmonary and hepatocellular neoplasia observed in this study substantiates a question whether the neoplasia in both organs could be induced by a common mechanism? In males the incidence of animals with pulmonary tumours occurring without hepatocellular tumours, as well as the incidence of animals with hepatocellular tumours occurring without pulmonary tumours was only minimally increased in high dose groups. In contrast to this, the incidence of animals bearing both pulmonary and hepatocellular tumours appeared to be distinctly increased in male groups 4 and 5, indicating a possible association. In female animals, however, such a coincidence was not evident.
Statistical analysis of the above data, however, did not indicate any significant link between pulmonary and hepatocellular neoplasia.
It is well known that the lung and liver of aging mice may show quite a high incidence of spontaneous neoplasia and that the incidence of hepatocellular and pulmonary tumours in mice depends on various factors, some of which may affect both organs. For instance .a hormonal influence is manifested by the different incidence rates of spontaneous neoplasia in both sexes. Furthermore, the growth of hepatocellular and pulmonary tumours in mice can be experimentally influenced by changing light/dark cycle and, by that means, probably the levels of melatonin which may inhibit tumour growth or of glucocorticoid which may accelerate it.
All gene mutation and clastogenicity evaluations conducted with the test substance would indicate the product is non-genotoxic.
No non-genotoxic AOP is known which would selectively and simultaneously enhance neoplastic proliferation in the mouse liver and lung. Moreover, if such a common enhancing mechanism were implicated in the development of findings observed in the present study, it would be operational only in male, but not in female animals, since in female animals only pulmonary neoplasia were increased.
Alternatively, such a common enhancing mechanism could depend on a dose and sex related difference in susceptibility. Since in the present study the increase in pulmonary neoplasia in both sexes and hepatocellular neoplasia in males was restricted to the high-dose groups 4 and 5, the observed findings appear to represent a reaction of highly susceptible mouse tissues to high exposure levels of the test article, a situation which may be hardly extrapolated to other species.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: Dietary equivalent to 5.75 and 5.33 mg/kg bw/day for males and females, respectively

Key result

Dose descriptor:

NOAEL

Remarks:

Carcinogenicity

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: Dietary equivalent to 5.75 and 5.33 mg/kg bw/day for males and females, respectively

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 ppm

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Verfication of test diet:

The methods and procedure yielded diets with a homogeneous distribution of test article. The results of these analyses revealed concentrations of 85 to 92% of the nominal values.
The test article mixed with rodent diet was stable for at least 5 at room temperature. The results of these analyses revealed concentrations of 88 to 104% of the nominal values.
The amount of test article in the diet was determined analytically during the study. The results of these analyses revealed concentrations of 81 to 106% of the nominal values.

Table 1. Summary of the results

Dose (mg/kg food)	0		10		50		500		2000		dr
	m	f	m	f	m	f	m	f	m	f
Mortality, n=50	11	2 (5)	12	11	11	12	8	13	11	10
(incl. Satellites, n=60)	(15)	(4)	(13)	(13)	(15)	(14)	(8)	(14)	(14)	(11)
Clinical signs				no treatment-related findings ic no treatment-related findings				d/dc i (2)	ic (1)	d/dc i (2)
Body weight (gain)											f
Food consumption											m,f
Haematology
Organ weights -  liver -  kidney -  adrenal							ica/r   ic a	ic r	ica/r   ica/r	ica/r ic r dc a	m,f
											m
Pathology
macroscopy
- lungs: nodules	7/60	1/60	8/60	4/60	3/60	6/59	12/60	4/60	17/60	8/60	m
- lungs: masses	0/60	1/60	3/60	3/60	3/60	1/59	4/60	2/60	4/60	4/60	m
- liver: enlarged	2/60	4/60	1/60	3/60	4/60	6/59	6/60	5/60	16/60	14/60
- liver: masses	18/60	4/60	13/60	2/60	20/60	6/59	26/60	5/60	38/60	5/60	m
- spleen: enlarged	8/60	34/60	12/60	31/60	19/60	29/59	24/60	33/60	26/60	32/60	m
microscopy
neoplastic lesions
- pulmonary adenoma	8/50	1/50	8/50	5/50	4/50	4/49	14/50	6/50	16/50	11/50	m
- pulmonary carcinoma	1/50	2/50	3/50	2/50	1/50	2/49	10/50	3/50	10/50	9/50	m
- pulmonary pooled(3)	9/50	3/50	11/50	7/50	5/50	6/49	21/50(4)	9/50(4)	22/50(4)	20/50(4)	m,f
- benign hepatoma	11/50	2/50	12/50	2/50	9/50	3/49	13/50	1/50	16/50	3/50
- hepatocell. carcinoma	8/50	1/50	4/50	0/50	12/50	2/49	17/50	0/50	21/50	2/50	m
- hepatic tumours pooled	16/50	3/50	13/50	2/50	17/50	5/49	25/50	1/50	29/50(4)	4/50	m
microscopy
non-neoplastic lesions
- liver: foci of cellular change	5/50	2/50	1/50	4/50	7/50	3/49	2/50	3/50	9/50	7/50
- liver: fatty change	39/50	42/50	33/50	34/50	39/50	28/49	26/50	15/50	30/50	8/50	m,f
- adrenal cortex: atrophy	33/50	0/50	25/50	0/50	19/50	0/50	26/50	0/50	15/50	0/50
- bone marrow: fatty atrophy	0/50	36/50	0/50	30/50	0/50	26/49	0/50	23/50	0/50	16/50	f
- pancreas: fatty atrophy	0/50	6/50	0/50	3/50	1/50	2/49	0/50	2/50	0/50	0/50
- ovaries: atrophy		19/50		18/50	22/49			26/50		31/50

dr dose related

dc/ic statistically significantly decreased/increased compared to the controls

d/i decreased/increased, but not statistically significantly compared to the controls

a/r absolute/relative

1 also relative food consumption

2 relative food consumption only, from week 62 onwards

3 shows the number of animals bearing at least one benign or malignant tumour of indicated type

4 multiplicity also increased

5 mortality in control females was exceptionally low, while mortality in treated females was within the normal range.

Conclusions:

Dietary administration of the test substance up to dose levels of 2000 ppm was tolerated by the mice without overt clinical signs or effects on survival. However, the dietary level of 2000 ppm was considered to represent the maximum tolerated dose, producing mean body weights 13% lower in females by the end of the treatment period. Males treated at this dietary level were not similarly affected. Dietary levels of 500 and 2000 ppm resulted in increased incidences of pulmonary tumours in males and females and hepatic tumours in males. Based on these findings, the NOAEL is set at 50 ppm for systemic toxicity and carcinogenicity (dietary equivalent to 5.75 and 5.33 mg/kg bw/day for males and females, respectively)

Executive summary:

 

The purpose of this study was to characterise the carcinogenic potential the test substance following long-term exposure to mice according to OECD TG 451 and GLP principles. Groups of 60 male and 60 female Tif:MAGf mice were fed diets containing 0, 10, 50, 500 and 2000 ppm (dietary equivalent to 0, 1.10, 5.75, 55.4 and 222 mg/kg bw/day for males and 0, 1.04, 5.33, 51.5 and 201 mg/kg bw/day for females, respectively of the test substance for a period of 18 months. In life observations, mortality, body weight, food consumption (ratios), haematology, organ weights and pathology were determined in this study.

Results showed no treatment related mortality and in-life observations. Body weight gains of male groups were not disturbed by treatment. By termination, mean body weights for females of groups 4 and 5 (500 and 2000 ppm) were 9% and 13% lower than the control value, respectively. Higher mean values Were recorded for males of groups 4 and 5 (500 and 2000 ppm) during the last six months of the study. Food intakes by females were not disturbed by treatment. Higher ratios were obtained during the last six months of the study for males and females of group 5 (2000 ppm) and for females of group 4 (500 ppm). The haematological profile of the mice was not disturbed by treatment. Higher organ to body weight ratios were obtained for liver of males and females of groups 4 and 5 (500 and 2000 ppm), for kidneys of group 5 females, for adrenals of group 5 males. Necropsy revealed an increased incidence of nodules/masses in the lungs of males of groups 4 and 5 (500 and 2000 ppm) and females of group 5 (2000 ppm). In addition, an increased incidence of hepatic masses was detected in males of groups 4 and 5 (500 and 2000 ppm). Microscopical examination revealed an increase of pulmonary tumours in males and females of groups 4 and 5 (500 and 2000 ppm). In addition, an increase of hepatocellular tumours was detected in males of groups 4 and 5 (500 and 2000 ppm). These changes were associated with a slight increase in the incidence of hepatic foci of cellular change in males and females of group 5 (2000 ppm). A decrease in the incidence of hepatic fatty change in males of groups 4 and 5 (500 and 2000 ppm) and a decrease in the incidence of adrenal cortical atrophy probably represented a non-specific reaction to stress. In females of groups 4 and 5 (500 and 2000 ppm) a variety of non-neoplastic changes was detected which associated with the suppression of body weight gain and included lower incidences for fatty atrophy of bone marrow and hepatic fatty change. In group 5 females a lower incidence of pancreatic fatty atrophy and a higher incidence of ovarian atrophy were detected.

In conclusion, dietary administration of the test substance up to dose levels of 2000 ppm was tolerated by the mice without overt clinical signs or effects on survival. However, the dietary level of 2000 ppm was considered to represent the maximum tolerated dose, producing mean body weights 13% lower in females by the end of the treatment period. Males treated at this dietary level were not similarly affected. Dietary levels of 500 and 2000 ppm resulted in increased incidences of pulmonary tumours in males and females and hepatic tumours in males. Based on these findings, the NOAEL is set at 50 ppm for systemic toxicity and carcinogenicity (dietary equivalent to 5.75 and 5.33 mg/kg bw/day for males and females, respectively)

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5.33 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Guideline study (OECD TG 451) performed in compliance with GLP

System:

other: Hepatobiliary and lower respiratory system

Organ:

liver

lungs

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Based on an increased incidence of neoplastic lesions in liver and lungs in mice several mechanistic studies were performed to elucidate the mechanisms involved and to assess the human relevance of these effects. Several studies with the test substance were submitted to investigate the liver and lung effects in vivo and in vitro in several species: rats, mice, marmosets, human (see Chapter 5.10.1.3 of the CSR).

 

Liver

Upon sub-chronic oral (feeding) of male and female mice, the test substance was identified as a strong inducer of hepatic xenobiotic metabolising enzymes and was classified as a peroxisome proliferator type inducer (Beilstein 1996, liver enzyme induction in mice). In vitro oxidative metabolism studies with the test substance revealed urethane (ethyl carbamate) formation upon incubations of liver microsomes in the presence of NADPH (Beilstein 1997). Urethane formation was induced by pre-treatment of mice with the test substance (autoinduction). Remarkable species differences in urethane formation rate between mouse and man were observed. Human liver microsomes were 11- to 173-fold and 22- to 353-fold slower than control and pre-treated male mouse liver microsomes, respectively (Beilstein 1998).

Urethane is a known inducer of liver and lung tumours in mice, rats and hamsters. The major mode of the carcinogenic action of urethane is its ability to undergo covalent interactions with DNA after metabolic activation to vinyl carbamate epoxide. Identification and quantification of a possible of formation of urethane-derived DNA adducts after single oral treatment of male mice gave no indication for a genotoxic potential of the test substance mediated by the formation of urethane-type DNA adducts (Sagelsdorff 1998).Therefore it is highly unlikely, that genotoxicity mediated by DNA binding of metabolically released urethane is the mechanism for tumorigenicity of the test substance. 

In conclusion, the increase in liver tumours is considered to be caused by the induction of peroxisome proliferation, a mechanism for which man is less sensitive. Therefore, the liver tumours in mice are not considered relevant for human risk assessment.

 

Lung

Upon sub-chronic oral (feeding) of male and female mice, the test substance was identified as a having no inductive properties on mouse pulmonary xenobiotic metabolising enzymes (Beilstein 1996, lung enzyme induction in mice). This might indicate that these reactive metabolites might be transported to the lung (susceptible organ) after their formation in the liver via the blood circulation. When compared to mice and rats, human liver microsomes showed an at least ten-fold lower formation rate of the reactive metabolites ethyl carbamate and benzoquinone/hydroquinone (Beilstein 1997). This implies that man is more than an order of magnitudes less sensitive to lung tumour formation by the test substance via metabolic activation to ethyl carbamate and/or benzoquinone/hydroquinone.

However, in contrast to these in vitro studies, no indications were found for a genotoxic potential of test substance mediated by the formation of urethane-type DNA adducts in mice (Sagelsdorff 1998). In addition, lung tumours were not observed in rats, and differences in the metabolic pattern of the test substance were noted between rats and mice.

Based on the results of the mechanistic studies, no final conclusion can be drawn on the possible mechanism of lung tumour formation. However, the genotoxicity studies performed clearly indicate that the test substance has no genotoxic potential. Therefore, it is concluded that the formation of the lung tumours in mice, will be based on a non-genotoxic mechanism. For this reason, in the human risk assessment, a threshold based mechanism can be assumed. The DNEL used in the risk assessment is sufficient to allow safe use of the substance.

Justification for classification or non-classification

Lung tumours and hepatic tumours occurred in mice and was treatment related to the test substance. Based on the available data classification for carcinogenicity a Carc. Cat. 2 (H351) is warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.

Additional information

18-month dietary carcinogenicity study in mice, Bachmann 1995 - Key

The purpose of this study was to characterise the carcinogenic potential the test substance following long-term exposure to mice according to OECD TG 451 and GLP principles. Groups of 60 male and 60 female Tif:MAGf mice were fed diets containing 0, 10, 50, 500 and 2000 ppm (dietary equivalent to 0, 1.10, 5.75, 55.4 and 222 mg/kg bw/day for males and 0, 1.04, 5.33, 51.5 and 201 mg/kg bw/day for females, respectively of the test substance for a period of 18 months. In life observations, mortality, body weight, food consumption (ratios), haematology, organ weights and pathology were determined in this study.

Results showed no treatment related mortality and in-life observations. Body weight gains of male groups were not disturbed by treatment. By termination, mean body weights for females of groups 4 and 5 (500 and 2000 ppm) were 9% and 13% lower than the control value, respectively. Higher mean values Were recorded for males of groups 4 and 5 (500 and 2000 ppm) during the last six months of the study. Food intakes by females were not disturbed by treatment. Higher ratios were obtained during the last six months of the study for males and females of group 5 (2000 ppm) and for females of group 4 (500 ppm). The haematological profile of the mice was not disturbed by treatment. Higher organ to body weight ratios were obtained for liver of males and females of groups 4 and 5 (500 and 2000 ppm), for kidneys of group 5 females, for adrenals of group 5 males. Necropsy revealed an increased incidence of nodules/masses in the lungs of males of groups 4 and 5 (500 and 2000 ppm) and females of group 5 (2000 ppm). In addition, an increased incidence of hepatic masses was detected in males of groups 4 and 5 (500 and 2000 ppm). Microscopical examination revealed an increase of pulmonary tumours in males and females of groups 4 and 5 (500 and 2000 ppm). In addition, an increase of hepatocellular tumours was detected in males of groups 4 and 5 (500 and 2000 ppm). These changes were associated with a slight increase in the incidence of hepatic foci of cellular change in males and females of group 5 (2000 ppm). A decrease in the incidence of hepatic fatty change in males of groups 4 and 5 (500 and 2000 ppm) and a decrease in the incidence of adrenal cortical atrophy probably represented a non-specific reaction to stress. In females of groups 4 and 5 (500 and 2000 ppm) a variety of non-neoplastic changes was detected which associated with the suppression of body weight gain and included lower incidences for fatty atrophy of bone marrow and hepatic fatty change. In group 5 females a lower incidence of pancreatic fatty atrophy and a higher incidence of ovarian atrophy were detected.

In conclusion, dietary administration of the test substance up to dose levels of 2000 ppm was tolerated by the mice without overt clinical signs or effects on survival. However, the dietary level of 2000 ppm was considered to represent the maximum tolerated dose, producing mean body weights 13% lower in females by the end of the treatment period. Males treated at this dietary level were not similarly affected. Dietary levels of 500 and 2000 ppm resulted in increased incidences of pulmonary tumours in males and females and hepatic tumours in males. Based on these findings, the NOAEL is set at 50 ppm for systemic toxicity and carcinogenicity (dietary equivalent to 5.75 and 5.33 mg/kg bw/day for males and females, respectively)

2 -year dietary carcinogenicity study in rat, Goodyer 1992 - Supporting

The purpose of the study is to determine the toxicity of the test substance and its effects on the incidence and morphology of tumours according to OECD TG 453 and GLP principles. Groups of 60 male and female Sprague Dawley rats of each sex were fed diets the test substance (orally) at nominal levels of 0, 200, 600 or 1800 ppm for up to 104 weeks. The dose levels, expressed in mg/kg bw/day were as 0, 24.7 ± 5.4, 75.1 ± 16.6 and 217.2 ± 52.6 mg/kg bw/day. After 52 weeks dosing 10 animals from each sex and group were killed and necropsied. All surviving animals were killed after 104 weeks dosing and necropsied. A range of tissues and organs were examined histologically for the control and high dose level animals and the livers were examined for the low and intermediate dose levels. Blood and urine samples were collected and examined during the course of the study. The concentration of the test substance in the diets was analysed in week 1 and then every 13 weeks thereafter. All analyses showed that the actual concentration of the test substance in the diet was close to the nominal concentrations.

Results showed that at the low dose level the survival, clinical condition. incidence of palpable masses, body weight development, food consumption and plasma enzyme activities were similar to the controls. In addition, at both the interim and terminal kills all organ weights were similar to the corresponding control weights. No treatment-related findings were found in the liver at either of the scheduled kills. There was no evidence of any effect on the incidence of any tumour type and the tumours found were considered to represent the normal background pathology of the laboratory rat.

At the intermediate dose level the survival, clinical condition, incidence of palpable masses, body weight development and food consumption were similar to the control. At the week 102 examination GOT, GPT and the alkaline phosphatase activities were higher than the control. The GOT and GPT increases were statistically significant in the males (p<0.01). At the interim kill the mean relative liver weight for males and adjusted liver weight to necropsy body weight for females were increased when compared to the controls by 16% and 10% respectively. At the terminal kill the adjusted liver weight to necropsy body weight was increased when compared to the control for males and females by 11% and 13% respectively. At the interim kill a low incidence of treatment- related liver lesions was seen in 2 out of 10 males characterised by minimal centrilobular hypertrophy of the liver associated with moderate focal necrosis and foci of pigmented histocytes. The livers of the remaining 8 males and all 10 females were comparable with the controls. At the terminal kill 6 out of 34 males showed centrilobular hypertrophy of the liver and an increased incidence (15 out of 34) of focal necrosis of the liver compared with the control. The incidence of all other lesions in the males and all lesions in the females were comparable with the controls. There was no effect of treatment on tumour incidence.

At the high dose level there was no treatment-related effect on survival, clinical condition or incidence of palpable tissue masses. In week 1 a slight reduction in the male mean body weight gain was recorded. Subsequently on frequent weigh days the weight gain was slightly lower than the controls. At the end of the 104 week treatment period the mean body weight was of the control weight. The slight reduction in weight gain was 97% also accompanied by a minimal decrease in food consumption. The female body weight was comparable to the control over the first 40 weeks of the study. From week 40 to 95 the weight gain was 9% lower than the control. However at the end of the study the mean weight was 97% of the control weight. Fluctuations in body weight can occur during long term studies and no toxicological is attached to the finding. No treatment-related changes ware seen in ophthalmoscopy or in any of the haematology or urinary parameters measured. At the week 78 and 102 laboratory examinations the mean GOT and GPT activities for the males were increased when compared to the control means. The increases were statistically significant (p<.05 for all except GPT in week 78 (p<.01). The mean alkaline phosphatase activity was increased at the week 25. 51, 78 and 102 examinations when compared with the control. The increase was statistically significant in the males in weeks 25 (p<0.01), 51 (p<0.001), 78 (p<0.01) and week 12 (p<0.001). In females these differences were statistically significant in week 25 (p<0.05). 51 (p<0.01) and week 102 (p<0.05). The increases, however. were at the upper limit of the range normally seen in untreated rats. No other changes in clinical chemistry were observed. At the interim kill the mean relative liver weight for the males was 13% higher than the controls and the liver weight adjusted to overall necropsy body weight for females was 26% higher. The liver weight for females was statistically significant (p<0.01). At the terminal kill the liver weight adjusted to overall necropsy body weight for the males was 6% higher than the controls and the female weight was 28% higher and was only statistically significant in the females (p<0.01). Histologically at the interim kill 5 out of 10 males and 2 out of 10 females showed minimal to slight centrilobular hypertrophy of the liver, usually associated with moderate focal necrosis and foci of pigmented histiocytes in the males. Minimal to slight focal fibrosis was also seen in the livers of 3 males. At the terminal kill the males showed a moderate incidence of centrilobular hypertrophy of the liver (16 out of 37 animals) and an increased. Incidence of focal necrosis (14 out of 37) and focal fibrosis (9 out of 37) of the liver compared with the controls. Eight out of 32 females showed centrilobular hypertrophy only. The incidence of all other lesions was considered to be comparable to the control. There was no effect of treatment on tumour incidence. The incidence of pituitary carcinoma in males has been changed compared to the original study report, based on a re-examination. This re-read established that several of the pituitary tumours previously diagnosed as carcinoma should more accurately be diagnosed as adenoma. The results of this re-read show that there is no effect of treatment on pituitary adenoma or carcinoma incidence in males.

In conclusion, after 104 weeks of treatment no toxicologically significant changes were seen in group 2 (200 ppm) and this was considered to be a no effect level. In addition, treatment for 104 weeks at a dose of 1800 ppm (group 4) had no on the incidence of morphology of tumours. The NOAEL for systemic toxicity was set at 200 ppm (dietary equivalent to 24.7 ± 5.4 mg/kg bw/day) since no toxicological changes were seen at this dose level. The NOAEL for carcinogenicity was set at 1800 ppm (dietary equivalent to 217.2± 52.6 mg/kg bw/day) because there was no incidence of morphology of tumours.

80-week dietary carcinogenicity study in mice, Everett 1987 - Supporting

The purpose of the study is to determine the toxicity of the test substance and its effects on the incidence and morphology of tumours according to OECD TG 453 and GLP principles. Groups of 50 male and 50 female CD-1 mice were dosed with the test substance for at least 80 weeks at dose levels of 0, 30, 110 or 420 ppm for males and 0, 20, 80 or 320 ppm for females. These animals comprised the carcinogenicity study. Groups of 10 male and 10 female CD-1 mice were dosed for 26 weeks and groups of 10 male and 10 female CD-1 mice were dosed for 52 weeks. A further 10 males and 10 females (control and high dose groups only) were dosed for 52 weeks and allowed to survive for at least another 6 weeks to provide recovery data. These animals comprised the toxicity study; all were dosed at the same dose levels as mentioned previously. Haematology, clinical chemistry and urinalysis samples were taken before dosing commenced and during weeks 26, 52 and 80. Post-mortem examination comprised full autopsy with organ weights, tissue preservation and histopathological evaluation.

Results showed that there were no intergroup differences in mortality, clinical signs, body weight performance, food and water consumption, haematology and both macroscopic and microscopic pathology. At the high dose level, males only showed a small increase in AST after 26 weeks of treatment and a small increase in alkaline phosphatase value after 52 weeks of dosing, the latter not being evident after the recovery period. Urine specific gravity was reduced in males after 52 weeks. In both males and females liver weight was increased after 52 weeks. This increase was not evident after 6 weeks recovery. At the intermediate dose level, only 2 males showed a small increase in AST levels after 26 weeks and there were no clinical chemistry differences in intermediate dose males after 52 weeks of dosing. There was a reduction in urine specific gravity in males only after 52 weeks. Liver weights were marginally increased in males only after 52 weeks. Intermediate dose males and females showed increases and decreases in various other clinical chemistry and organ weight parameters after 26 and 52 weeks of dosing but were disregarded due to lack of consistency and dose relationship of these findings. At the low dose level, the only findings were occasional clinical chemistry, urinalysis and organ weight parameters which were different from controls. These were disregarded due to the lack of consistency and dose relationship. In the carcinogenicity study, there were no intergroup differences in mortality, clinical signs, body weight performance, food and water consumption, haematology and urinalysis. At the high dose level. lactate dehydrogenase levels were increased in males after 80 weeks of dosing. Various other clinical chemistry parameters were different from control values but were disregarded because of a lack of dose relationship and small magnitude. There was an increased incidence of alveolar/bronchiolar tumours and increased liver weight in males only. At the intermediate dose level, the only clinical chemistry and organ weight findings were occasional differences from control values. These were disregarded due to the lack of consistency and dose relationship. There was an increased incidence of alveolar bronchiolar tumours in-males only. At the low dose level, the only clinical chemistry and organ weight changes were occasional differences from control values. These were disregarded due to the lack of consistency and dose relationship. There was an increased incidence of alveolar/bronchiolar tumours in males only.

In conclusion, the NOAEL for carcinogenicity could not be established since an increased incidence of lung tumours in males in all dose groups. A LOAEL of ≤ 30 and ≤ 20 ppm food for males and females, equivalent to ≤ 5.3 and ≤ 4.8 mg/kg bw/day was set for males and females respectively.