Registration Dossier

Toxicological information

Carcinogenicity

Currently viewing:

Administrative data

Description of key information

Carcinogenesis bioassay, male/female mice, National Toxicology Program, 1982

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
April 1977 to May 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: A non-GLP study conducted to sound scientific principles with the read across substance, bis(2-ethylhexyl) adipate.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Deviations:
yes
Remarks:
(environmental paprmeters were outside guideline ranges, the chronic toxicity studies were not long enough in duration, only two dose levels were used in the main study and heamotology and blood parameters were not analysed)
GLP compliance:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: NCI Frederick Cancer Research Center, Frederick, Maryland, USA
- Age at study initiation: 4 weeks
- Housing: 5 animals per cage in solid bottom suspended polycarbonate cages
- Diet: powdered Wayne® Lab Blox diet, ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 31 °C
- Humidity (%): 10 - 88 %
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
Test diets were prepared by mixing the test material with an aliquot of diet, the mixture was then placed into a Patterson-Kelly® twin-shell intennsifier bar V-blender with the remainder of the feed, and mixed for 10 minutes.
- Storage temperature of food: Test diets were dealed in labelled plastic bags and stored at 4 °C for no longer than 14 days.

Stability of test material in feed was determined by analysing sample diet mixtures containing 100,000 ppm test material that had been stored at -20, 5, 25 or 45 °C for 2 weeks. The amounts of test material found to be present by vapour-phase chromatography indicate that the test material was stable in feed for 2 weeks at temperatures as high as 45 °C.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The amounts of test material in selected batches of feed were measured by vapour-phase chromatography. 2 g samples of feed were extracted with 50 mL portions of methanol. The supernatant solutions were combined and diluted to a volume of 100 mL and analysed by vapour-phase chromatography using the following system:
- Instrument: Bendix 2500
- Column: 3 % OV-17 on 80/100 Supelcoport, glass, 1.8 m x 4 mm ID
- Oven temperature: 250 °C
- Retention time of test material: 1.9 minutes
- Inlet temperature: 235 °C
- Detctor temerature: 250 °C
Duration of treatment / exposure:
Single administration followed by 14 days observation (acute study)
14 days (14-day repeated dose toxicity study)
13 weeks (sub-chronic toxicity study)
103 weeks (chronic toxicity/ carcinogenicity study)
Frequency of treatment:
Single administration (acute study)
Daily treatment (repeated dose toxicity studies/carcinogenicity study)
Post exposure period:
14 days (acute toxicity study)
1 day (14 day repeated dose toxicity study)
No post exposure period (13 week repeated dose toxicity study)
1 - 3 weeks (103 week repeated dose toxicity study/carcinogenicity study)
Remarks:
Doses / Concentrations:
0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5.0, 10, 20 g/kg (acute toxicity study)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 3100, 6300, 12500, 25000, 50000 ppm (14 day repeated dose toxicity study)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 1600, 3100, 6300, 12500, 25000 ppm (13 week repeated dose toxicity study)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 12000, 25000 ppm (103 week repeated dose toxicity study/carcinogenicity study)
Basis:
nominal in diet
No. of animals per sex per dose:
5 males and 5 females per dose (acute toxicity study)
5 males and 5 females per dose (14 day repeated dose toxicity study)
10 males and 10 females per dose (13 week repeated dose toxicity study)
50 males and 50 females (103 week repeated dose toxicity/carcinogenicity study)
Control animals:
other: no (acute toxicity study)... (see attached file)
Details on study design:
- Dose selection rationale: dose selection for the 103 week repeated dose toxicity/carcinogenicity study was based on findings of the preceding acute, 14 day repeated dose, and 13 week repeated dose toxicity studies (see below)

ACUTE TOXICITY STUDY
The estimated LD50 for male and female rats was 15.0 and 24.6 g/kg, respectively.

14 DAY REPEATED DOSE TOXICITY STUDY
All female mice fed 100,000 ppm died. Weight loss occurred among male mice fed 50,000 ppm and females fed 25,000 ppm or more. Feed consumption was reduced in females fed 100,000 ppm.

13 WEEK REPEATED DOSE TOXICITY STUDY
One female mouse died as a result of an accident. Weight gain depression was 10 % or more for male mice fed 3,100 ppm or more. Weight gain depression was 10 % or more for females fed 6,000 or 25,000 ppm. No treatement-related histopathologic effects or reduction in feed consumption were observed. High and low doses selected for the chronic (103 week) study with rats were 12,000 ppm and 25,000 ppm test material in feed.

- Rationale for animal assignment (if not random): animals were assigned to control or dose groups so that average cage weights were approximately equal for all animals of the same sex.
Observations and examinations performed and frequency:
103 Week Study

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were inspected twice daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were recorded every 4 weeks

FOOD CONSUMPTION AND COMPOUND INTAKE: No data

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:
103 Week Study

SACRIFICE
Animals that were moribund and those that survived to the end of the study were killed using CO2 inhalation and necropsied.

GROSS PATHOLOGY: Yes
A gross pathological examination was performed on major tissues, major organs, and all gross lesions.

HISTOPATHOLOGY: Yes
Tissues were preserved in 10 % neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The following tissues were examined microscopically: skin, lungs and bronchi, trachea, bone and bone marrow, spleen, lymph nodes, heart, salivary gland, liver, pancreas, stomach, thyroid, parathyroid, mammary gland, prostate and seminal vesicles or uterus, testis or ovary, brain, thymus, larynx, and esophagus.
Necropsies were performed on all animals found dead unless precluded in whole or in part by autolysis or cannibalisation. Thus, the number of animals from which particular organs or tissues were examined microscopically varied.
Statistics:
Statistical analyses for a possible dose-related effect on survival used the method of Cox. The incidence of neoplastic or non-neoplastic lesions was given as the ratio of the number of animals bearing such lesions at a specific anatomic site to the number of animals in which that site was examined. A one-tailed Fisher exact test was used to compare the tumour incidence of a control group with that of a group of dosed animals at each dose level. Life table methods were used to analyse the incidence of tumours. Curved of the proportions surviving without an observed tumour were computed. the week during which an animal died naturally or was killed was entered as the time point of tumour observation. the methods of Cox and Tarone were used for the statistical tests of the groups. The statistical tests were one-tailed.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See "Details on results" for information
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See "Details on results" for information
Details on results:
CLINICAL SIGNS AND MORTALITY
In male mice, 36/50 (72 %) of the control group, 32/50 (64 %) of the low-dose group, and 41/50 (82 %) of the high-dose group survived until the end of the study at 105 - 107 weeks. In females, 42/580 (84 %) of the control group, 39/50 (78 %) of the low-dose group, and 36/49 (73 %) of the high-dose group survived to the end of the study at 105 - 107 weeks.
No treatment-related clinical signs were observed.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of the dosed mice of either sex were lower than those of the corresponding controls throughout the study, and the decrease in weight gain was dose-related.

HISTOPATHOLOGY: NON-NEOPLASTIC
A variety of non-neoplastic lesions were seen in control and dosed mice. None appeared to be related to test material administration. No toxic lesions were seen in livers of dosed mice.

HISTOPATHOLOGY: NEOPLASTIC
The incidence of neoplasms of the liver appeared to be related to treatment with the test material. Hepatocellular adenoma compressed the adjacent liver tissue. Cells in the adenoma were large. Cytoplasm of the cells was acidophillic or vacuolated and nuclei were hyperchromatic. Hepatocellular carcinoma involved a part or an entire lobe of the liver. The lobular architecture was distorted and cell plates were two or more cells thick, forming trabeculae. A pleomorphism in the size of cells was apparent. The nuclei had coarse chromatin, and the nuclei were prominant. Both normal and abnormal mitotic figures were numerous. Areas of necrosis and mineralisation were common in the large tumours.
The hepatocellular carcinoma metastasized to the lung in 14 male mice (control - 5; low-dose -4; high-dose - 5) and in 11 female mice (low-dose - 6; high-dose - 5). In all cases, the primary liver tumours were of the trabecular type.
The incidence of hepatocellular adenomas in male mice was dose related and statistically significant in the high-dose group. The incidence of male mice with either hepatocelular adenomas or carcinomas was dose related, and the high-dose group incidence was significantly higher than that in the controls. In female mmice, there was a significant dose-related trend and significantly higher incidence of animals with hepatocellular adenoma or carcinomas in each of the dosed groups than in the control group.
The other sites of metaphases were the kidney, adrenal, and lymph nodes in dosed female mice.

Relevance of carcinogenic effects / potential:
The carcinogenic mechanism observed in the mouse study was characteristic of rodent-specific peroxisome proliferation and hence is not relevant to humans. On this basis there is no evidence to suggest a carcinogenic effect in humans and hence classification is not necessary.
Dose descriptor:
LOAEL
Effect level:
12 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: (equivalent to 1715 mg/kg bw)
Remarks on result:
other: Effect type: carcinogenicity (migrated information)

Table 1: Incidence of Primary Tumours in Male Mice

Topography: Morphology

Control

Low Dose

High Dose

subcutaneous tissue: fibroma or fibrosarcoma

3/50

2/50

0/49

lung: alveolar/bronchiolar adenoma

8/50

9/49

3/49

hematopoietic system: malignant lymphoma, NOS

16/50

5/50

4/49

hematopoietic system: all lymphomas

16/50

7/50

6/49

hematopoietic system: leukemia or lymphoma

16/50

7/50

6/49

liver: hepatocellular adonema

6/50

8/49

15/49

liver: hepatocellular carcinoma

7/50

12/49

12/49

liver: hepatocellular adonema or carcinoma

13/50

20/49

27/49

 

Table 2: Incidence of Primary Tumours in Female Mice

Topography: Morphology

Control

Low Dose

High Dose

lung: alveolar/bronchiolar adenoma

5/49

1/49

3/48

lung: alveolar/bronchiolar adenoma or carcinoma

6/49

1/49

3/48

hematopoietic system: malignant lymphoma, NOS

23/50

14/50

7/49

hematopoietic system: all lymphomas

23/50

14/50

7/49

hematopoietic system: leukemia or lymphoma

23/50

14/50

7/49

circulatory system: angiosarcoma

3/50

1/50

1/49

liver: hepatocellular adonema

2/50

5/50

6/49

liver: hepatocellular carcinoma

1/50

14/50

12/49

liver: hepatocellular adonema or carcinoma

3/50

19/50

18/49

pituitary: adenoma, NOS

8/39

6/37

0/39

Table 3: Hepatocelular Neoplasms and Sites of Metastases

Males

Females

Control

Low Dose

High Dose

Control

Low Dose

High Dose

Number of livers evaluated

50

49

49

50

50

49

Hepatocellular

adenoma

6

8

15*

2

5

6

carcinoma

7

12

12

1

14#

12~

Neoplasms, NOS

1

Percent mice with liver tumours

26

41

56

6

38

37

Metastasis

lung

5

4

5

6

5

kidney

1

adrenal

1

lymph node

1

* P < 0.025

# P < 0.001

~ P = 0.001

Conclusions:
Under the conditions of the study, the test material was found to be carcinogenic for female mice, causing increased incidences of hepatocellular carcinomas, and was probably carcinogenic for male mice, causing hepatocellular adenomas.
Executive summary:

The carcinogenicity of the test material was determined in a non-GLP study which was conducted to a methodology which was similar to that which is outlined in the standardised guideline OECD 453. Results from an acute toxicity study, a 14 day repeated dose toxicity study and a 13 week repeated dose toxicity study were used to inform on suitable dose level for the definitive carcinogenesis bioassay. The study was conducted by feeding diets containing 12,000 or 25,000 ppm of test material to groups of 50 male and 50 female mice for 103 weeks. Groups of 50 undosed mice of each sex served as controls. During the study animals were inspected twice daily and bodyweights were recorded every 4 weeks. Animals that were moribund and those that survived to the end of the study were killed at 104 to 107 weeks. Gross and microscopic examinations were performed on major tissues, major organs and all gross lesions.

During the study no treatment-related clinical signs were observed. Mean body weights of the dosed mice of either sex were lower than those of the corresponding controls throughout the study, and the decrease in weight gain was dose-related. A variety of non-neoplastic lesions were seen in control and dosed mice. None appeared to be related to test material administration. No toxic lesions were seen in livers of dosed mice.

Hepatocellular adenomas or carcinomas occurred in high-dose mice of either sex and in low-dose female mice at incidences that were dose related and significantly higher than those in the controls. The time to observation of hepatocellular adenomas or carcinomas in the dosed female mice, but not in the dosed male mice, was significantly shorter than the time to observation of these tumours in the controls. Because the incidence of hepatocellular adenomas or carcinomas in the male high-dose greoup was not greatly increased over that in the male historical control mice in the same laboratory and because the time to observation of tumours in the dosed groups as compared with the control group was not significantly diferent, the association of liver tumours in the males with administration of the test material was not considered conclusive. Therefore, under the conditions of the study, the test material was found to be carcinogenic for female mice, causing increased incidences of hepatocellular carcinomas, and was probably carcinogenic for male mice, causing hepatocellular adenomas.

The carcinogenic mechanism observed in the mouse study was characteristic of rodent-specific peroxisome proliferation and hence is not relevant to humans. On this basis there is no evidence to suggest a carcinogenic effect in humans and hence classification is not necessary.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
1 715 mg/kg bw/day
Study duration:
chronic
Species:
mouse
Quality of whole database:
Available data are taken from a non-GLP study conducted to sound scientific principles with the read across substance, bis(2-ethylhexyl) adipate. The study was assigned a reliability score of 2 in accordance with the criteria of Klimisch (1997).

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The carcinogenicity of the test material was determined in a non-GLP study which was conducted to a methodology which was similar to that which is outlined in the standardised guideline OECD 453. Results from an acute toxicity study, a 14 day repeated dose toxicity study and a 13 week repeated dose toxicity study were used to inform on suitable dose level for the definitive carcinogenesis bioassay. The study was conducted by feeding diets containing 12,000 or 25,000 ppm of test material to groups of 50 rats of each sex, and 50 mice of each sex, for 103 weeks. 12,000 ppm was considered to be equivalent of 1715 mg/kg/day bw. Groups of 50 undosed rats and mice of each sex served as controls. During the study animals were inspected twice daily and bodyweights were recorded every 4 weeks. Animals that were moribund and those that survived to the end of the study were killed at 104 to 107 weeks. Gross and microscopic examinations were performed on major tissues, major organs and all gross lesions.

 

In rats, mean body weights of high-dose rats of either sex were lower than those of the controls throughout the study. Several non-neoplastic lesions were seen in control and dosed rats. None appeared to be related to treatment with the test material. A variety of neoplasms were seen in both control and dosed rats. Tumours noted were those seen routinely in this strain of rat, and they occurred in comparable numbers in control and dosed rats. Therefore, under the conditions of the study the test material was determined to be non-carcinogenic for F344 rats.

 

In mice, no treatment-related clinical signs were observed. Mean body weights of the dosed mice of either sex were lower than those of the corresponding controls throughout the study, and the decrease in weight gain was dose-related. A variety of non-neoplastic lesions were seen in control and dosed mice. None appeared to be related to test material administration. No toxic lesions were seen in livers of dosed mice.

 

Hepatocellular adenomas or carcinomas occurred in high-dose mice of either sex and in low-dose female mice at incidences that were dose related and significantly higher than those in the controls. The time to observation of hepatocellular adenomas or carcinomas in the dosed female mice, but not in the dosed male mice, was significantly shorter than the time to observation of these tumours in the controls. Because the incidence of hepatocellular adenomas or carcinomas in the male high-dose group was not greatly increased over that in the male historical control mice in the same laboratory and because the time to observation of tumours in the dosed groups as compared with the control group was not significantly diferent, the association of liver tumours in the males with administration of the test material was not considered conclusive. Therefore, under the conditions of the study, the test material was found to be carcinogenic for female mice, causing increased incidences of hepatocellular carcinomas, and was probably carcinogenic for male mice, causing hepatocellular adenomas.


Justification for selection of carcinogenicity via oral route endpoint:
This study was selected since adverse effects were observed in the study with mice whereas no adverse effects were observed in the study conducted with rats. The use of this study is therefore considered to be the most conservative approach.

Carcinogenicity: via oral route (target organ): digestive: liver

Justification for classification or non-classification

The carcinogenic mechanism observed in the mouse study was characteristic of rodent-specific peroxisome proliferation and hence is not relevant to humans. On this basis there is no evidence to suggest a carcinogenic effect in humans and hence classification is not necessary.