1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole

Carcinogenicity

Description of key information

NOAEL for systemic toxicity = 100 ppm (equivalent to 11 mg/kg bw/day), NOAEL for carcinogenicity > 850 ppm (equivalent to 107.6 mg/kg bw/day), OECD TG 451, GLP, Gerspach 1999

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:

19 Dec 1994 to 21 Jun 1996

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:

other: FIFRA 83-2

Version / remarks:

1982

Qualifier:

according to guideline

Guideline:

other: JAP MAFF Noh San No. 4200

Version / remarks:

1985

GLP compliance:

yes (incl. QA statement)

Species:

mouse

Strain:

CD-1

Remarks:

CrI: CD-1r (ICR) BR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age and weight at arrival: 4 weeks old, 26.59-39.52 g in males
- Housing: All males and the females of the 3-month interim sacrifice group were housed individually in macrolon cages type 2. All other females were housed in groups of 5 in macrolon cages type 3.
- Diet: Standard diet: ad libitium
- Water: Tap water, ad libitum
- Acclimation period: 26 days

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

IN-LIFE DATES: From: 19 Dec 1994 To: 21 Jun 1996

Route of administration:

oral: feed

Vehicle:

acetone

Remarks:

(evaporised after premix)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: 4 weeks
- Mixing appropriate amounts with: Pelleted diet
- Storage temperature of food: Room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Pellet samples (about 200 g each) were collected during the pelleting process for homogeneity analyses at beginning, middle, and end of pelleting process. Diet mixtures were measured at the beginning of the experiment. Samples should not differ from the mean value by > 15 %.
- Diet mixtures were analysed for test article stability at the beginning of the experiment and the test article in diet is considered stable, when the content does not decline > 10 % during the dosing period.
- Periodically during the study, diet mixtures were analysed for test article concentration. An analytical result of test article content in the range between 75-125 % is considered acceptable.

Duration of treatment / exposure:

18 months

Frequency of treatment:

Continuously

Dose / conc.:

100 ppm

Remarks:

Equivalent to 11 mg/kg bw/day

Dose / conc.:

500 ppm

Remarks:

Equivalent to 59 mg/kg bw/day

Dose / conc.:

850 ppm

Remarks:

Equivalent to 107.6 mg/kg bw/day

No. of animals per sex per dose:

80 (of which 50 animals/group for evaluation of the carcinogenic potential; 10 animals/group for blood chemistry investigations, scheduled at weeks 1, 9, 53 and 79; 10 animals/group for interim sacrifice at week 53; 10 animals/group for interim sacrifice at week 9)

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

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

MORTALITY:
- Time schedule: daily (a.m. and p.m. on working days, a.m. on weekends and holidays).

BODY WEIGHT:
- Time schedule for examinations: weekly (midweek) for the first 3 months once 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

BLOOD CHEMISTRY: (10 animals)
- Investigations were carried out on 10 males per group (experimental group II). For terminal laboratory investigation at week 79, the number of animals subjected to examination was supplemented by animals of the carcinogenicity group (I) to yield 10 samples per group.
- Laboratory investigations were performed at: pretest, week 9, 14, 53 and 79.
- With respect to biological variability due to circadian rhythms, blood was consistently sampled in the Ether was used to anesthetize the animals. Blood was withdrawn from the orbital sinus using glass capillary tubes. Heparin was used as anticoagulant. Food was withheld overnight prior to blood collection.
- Parameters: Cholesterol, Asparate amino transferase, Alanine amino-transferase, Alkaline phosphatase, Sorbitol dehydrogenase

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- At scheduled sacrifices at weeks 9, 53 and 79 all surviving control and treated animals were bled under ether anesthesia and subjected to detailed necropsy. At necropsy the following weights were recorded from all animals: body, brain, heart, liver, kidneys, testes (without epididymides).
- The following organs and were preserved in neutral buffered formalin: skin, mammary area, spleen, mesenteric lymph node, axillary 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 (duodenum, ileum, jejunum), large intestine (cecum, colon, rectum), kidney, both, urinary bladder, prostate, seminal vesicle, testis, both, epididymis, both, pituitary gland, adrenal gland, both, thyroid with parathyroid gland, thymusperipheral nerve, brain, spinal cord, eye with optic nerve, both, orbital gland, both, extraorbital lacrimal gland, both, Zymbal gland, both, muzzle, tongue, any tissue with gross lesions and masses.
- A complete necropsy with tissue preservation was performed also on all animals of all groups which died during the test period or which had to be sacrificed in moribund condition.

HISTOPATHOLOGY: Yes
- Two samples of the liver were taken, embedded in paraplast, sectioned at 3-5 microns, stained with hematoxylin and eosin, and subjected to a microscopical examination. Since no other target organ was revealed by clinical observations or at necropsy, no supplementary organ/tissue was processed for histopathology according to the protocol.
- All livers of all animals of the oncogenicity group and interim sacrifice groups were re-evaluated by the reviewing pathologist. An external peer review was also performed for the 9-week interim slides.
- The histopathological lesions observed were graded as to degree of severity according to the following criteria: Grade "+" : Minimal (slight). Includes histopathological change that is a noticeable but not prominent feature of the tissue. Grade "++" : Moderate. Includes histopathological change that is a prominent but not dominant feature of the tissue. Grade "+++" : Marked. Includes histopathological change that is a dominant feature of the tissue.

Statistics:

For each time point and parameter an uni-variate statistical analysis was performed. Non-parametric methods (Lehmann, 1975) were applied, to allow for non normal as well as normal data distribution. Each treated group was compared to the control group Lepage's 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. Two-sided asymptotic p-values are reported. Survival analysis was performed by the regression model (partial likelihood) (Cox, 1972) in order to compare survival time of treated animals (experimental group 1) with control animals. To establish possible relationships between pathological changes and treatment, all microscopical findings occurring in animals of the oncogenicity group (experimental group 1) were subjected to a statistical analysis according to the method described by Pete et al. (1980) for neoplastic lesions and Gart et al. (1986) for non-neoplastic changes with a program described by Seewald et al. (1994).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were neither clinical signs nor behavioral changes indicative of a treatment-related effect.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No statistically significant effects on survival were observed in this study. The survival % was 32, 34, 40 and 36 % in the control, 100 ppm test substance, 500 ppm test substance, and 850 ppm test substance groups, respectively.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At week 14, the cumulative body weight gain was lower than that of the controls by up to 19 % in the 850 ppm group and by up to 10 % in the 500 ppm group. The mean body weight gain of the 100 ppm group was comparable to that of the control group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Throughout the acclimatization and treatment periods the mean food consumptions and the overall food consumptions were comparable in all groups. The high values in the 850 ppm group during the first 4 weeks of treatment were considered due to food spillage of individual animals.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related decreases in plasma cholesterol levels were recorded throughout the treatment period in mice treated at 500 ppm and greater; at weeks 9 and 14, the decreases in the 850 ppm group were statistically significant. Furthermore, increased sorbitol dehydrogenase activities were seen in mice treated at 850 ppm at week 9 and 14 while subsequent values were similar to those of controls.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the two interim sacrifices as well as at the final sacrifice, the mean liver weights and the mean liver to body weight ratios were slightly to markedly increased in the 500 and 850 ppm groups, compared to the control group. Weights of other organs were not affected by treatment. The lower mean carcass weight of the 850 ppm group (in comparison with the other groups) at the 53 weeks interim sacrifice was due to the general light weight of the animals randomly assigned to this group and considered incidental.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No dose-related increase of macroscopical findings were observed in the liver at sacrifices of week 9 and 53 including the 2 animals which died intercurrently. At terminal sacrifice (79 weeks), enlarged livers were observed in higher numbers of animals of the 850 ppm group than in other groups, confirming the increased liver weights recorded at necropsy. Increased numbers of animals having, except 1, single masses in the liver up to 5 cm in size were observed in the 100, 500 and 850 ppm groups, and several nodules were found in the liver of 1/50 mice of the 850 ppm group. All other findings occurred in comparable numbers in all dose groups and were similar to those occurring spontaneously in this strain of mice.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 9 weeks sacrifice, increased numbers of mice with hepatocellular hypertrophy of minimal to marked degree of severity were present in the 500 and 850 ppm groups. The centrilobular hepatocytes were typically enlarged and had a very pale eosinophilic cytoplasm which was considered treatment related. Increased numbers of mice of the 850 ppm group had fatty change of the liver, consisting generally of minimal amounts of small-sized fatty vacuoles in the cytoplasm of the centrilobular hepatocytes. Furthermore, mice of the 850 ppm group compared with the controls, showed a higher incidence of necrosis of the liver, including monocellular necrosis of hepatocytes in 3/10 males and necrosis of small groups of hepatocytes in 5/10 males. Overall, there were 8/10 high-dose males with necrosis in the liver versus only 2/10 in the controls. Slightly increased numbers of mice with generally minimal lympho-histiocytic infiltration of the liver were observed in the 850 ppm group. All of these findings were considered treatment related. At week 53 sacrifice, higher numbers of mice having hepatocellular hypertrophy were observed in the 500 and 850 ppm groups. The typically enlarged hepatocytes with centrilobular distribution had eosinophilic cytoplasm and large nuclei with multiple prominent nucleoli. The severity of this finding was minimal in the 500 ppm group and moderate in the 850 ppm group. All other findings were considered non-treatment related. At terminal sacrifice (79 weeks), significantly higher numbers of animals with hypertrophy of hepatocytes were observed in the 500 and 850 ppm groups and were considered treatment related. Hepatocellular hypertrophy corresponded in majority with the enlarged livers observed at necropsy. The enlarged centrilobular hepatocytes usually had an eosinophilic cytoplasm and enlarged nuclei with prominent nucleoli. The occurrence of animals having pigmentation of Kupffer cells of the liver was increased in the 850 ppm group compared with the controls and was considered related to treatment. The occurrence of foci of cellular change of minimal to moderate size appeared to be significantly increased in animals of the 850 ppm group and were considered associated with treatment. All other findings were considered non-treatment related.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the 850 ppm treatment group, 10/50 mice (20 %) were affected by hepatocellular adenoma. These benign tumors showed eosinophilic features, except for one animal, where an eosinophilic and a basophilic variant of the tumor were present. In the original study report, these effects were considered treatment related when compared to the incidence of hepatocellular tumors in the control group of this study (1/50 or 2 %) and historical control data (up to 18.4 %). However, in a review of the evidence, additional control animal data from a reference study and literature were used to critically compare the results. The exact incidences of hepatocellular tumor bearing animals in the reference study were 14, 14, 24, 26, and 30 %. Similarly variable incidences were also reported in the literature (Vandenberghe, 1993; Lang, 1995; Harada et al., 1996; RITA, 1997). When considering this control data, the findings of hepatocellular neoplasia in the high dose group are no longer considered of toxicological relevance. The hepatic masses in the 100 and 500 ppm groups corresponding with adenomas were considered devoid of toxicological relevance.

Relevance of carcinogenic effects / potential:

An increased incidence of hepatocellular adenomas was observed in the high dose group following treatment with 850 ppm of the test substance. However, the incidences were not considered of toxicological relevance as similar levels have been reported in control mice of the same strain in a reference study and from literature (up to 30 %). Therefore, the carcinogenic potential of the test substance is doubtful.

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

100 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Remarks:

Carcinogenicity

Effect level:

> 850 ppm

Based on:

test mat.

Sex:

male

Critical effects observed:

yes

Lowest effective dose / conc.:

500 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

ANALYTICAL VERIFICATION OF DIET

The test article in diet is considered stable, when the content does not decline more than 10 during the dosing period.

The methods and procedure yielded diets with a homogeneous distribution of test article.

The test article mixed with rodent diet was stable for at least 5 weeks at room temperature.

The amount of test article in the diet was determined analytically during the study. The results of these analyses revealed concentrations of 80.9-116 % of the nominal values.

 

ADDITIONAL INFORMATION

This is an amendment to the final report which contains data concerning the spontaneous occurrence of liver tumors in CD-1 mice (microscopical examination). These data were collected from a separately conducted reference study with CD-1 mice at the performing laboratory. In five control groups with 50 untreated CD-1 mice per group/sex the following primary tumors and foci were observed in the liver: in male mice designated for terminal sacrifice, 2 % to foci of cellular alteration, 6 % to hepatocellular adenomas and to hepatocellular adenocarcinomas corresponding to to male CD-1 mice bearing at least one hepatocellular neoplasia, 2 % hemangiomas and hemangiocarcinomas were seen per control group. Female mice designated for terminal sacrifice presented neither primary tumors nor foci of cellular alteration.

In the initial interpretation of the results of the present study, when in-house reference control data were not available, it was concluded in the final report that the liver tumor incidence at the top dose (850 ppm) is treatment-related. This assessment was driven by the fact that the incidence of hepatocellular neoplasm was substantially higher (20 % hepatocellular adenoma and 4 % hepatocellular carcinoma corresponding to 24 % male CD-1 mice bearing hepatocellular neoplasia) in high dose males than the concurrent controls (2 % adenoma and 2 % carcinoma) and that there was not other in-house control data for CD-1 mice.

Additional historical control data for CD-1 mice generated under the same experimental and environmental conditions including temperature, humidity, light-cycle, food, water, type of cage, and animal facility location, as in the present study are now available. These new reference control data are appropriate for the re-evaluation of the toxicological relevance of the hepatocellular neoplasms which were observed in animals treated at 850 ppm in the present study. While the source of the CD-1 mice is not exactly the same stock used in this study, these are comparable animals since the CD-1 mice are an outbred strain. As confirmed by the data shown below, CD-1 mice supplied by different breeders have similar ranges for hepatocellular tumor incidence.

There is a great intergroup variance in the stock used for the new reference control study, with the incidences ranging from 6 to 18 % for hepatocellular adenoma and from 8 to 16 % of hepatocellular adenocarcinoma. The exact incidences of hepatocellular tumor bearing animals in this reference study are 14, 14, 24, 26, and 30 %. Similarly variable incidences were also reported in the literature (Vandenberghe, 1993; Lang, 1995; Harada et al., 1996; RITA, 1997).

In considering these various historical control data in CD-1 mice, it can be observed that the incidence of hepatocellular adenoma alone at 850 ppm of the present study (20 %) is at the top end of this range. However, because of the great variance in the relationship between benign and malignant tumors observed in these different control data, and of the diagnostic difficulty in determining the exact identity of a tumor in the continuum of adenoma and carcinoma in borderline lesions, an evaluation based on the combined incidence of these two tumor types is appropriate. So, then counting the animals bearing at least one hepatocellular neoplasia regardless of type, high dose male mice in the present study have a combined incidence of 24 % which is right in the middle of in-house historical control data for hepatocellular neoplasia.

In conclusion, a great spontaneous variability in incidences of hepatocellular neoplasia and in the relationship between benign and malignant tumors in CD-1 mouse populations can be observed. The occurrence of hepatocellular tumors is abnormally low in controls of the present study and therefore do not represent a reliable point for comparison. In the context of these historical control data, from within the laboratory and from outside, it is concluded that the finding of hepatocellular neoplasia in the present study is of doubtful toxicological relevance.

Conclusions:

Dietary administration of the test substance to male albino mice at concentrations of 100, 500 and 850 ppm was tolerated without occurrence of overt signs of toxicity. The MTD (Maximum Tolerated Dose) was exceeded at the high dose level of 850 ppm, based on evidence of liver damage at early stage of treatment and marked effects on body weight gain. The NOAEL oncogenicity was 800 ppm. Based on the above findings, the NOAEL for systemic effects was 100 ppm, equivalent to an average intake of 11 mg/kg bw/day.

Executive summary:

This study was conducted to assess the oncogenic potential of the test substance when administered in the diet to male mice. The test article was administered in the diet at concentrations of 0, 100, 500, and 850 ppm (mg/kg food) for 18 months to a total of 320 albino mice, 80 animals per dose group. This study was conducted according to OECD TG 451 and followed GLP. Clinical signs, body weight, food consumption and mortality were monitored throughout the study for all animals. Blood chemistry investigation was performed at weeks -1, 9, 14, 53 and 79. At sacrifice, animals were examined macroscopically and organ weights were recorded. Organs and tissues were collected and the liver was prepared for histopathological evaluation. The liver from all animals of the 9-week and 53-week interim sacrifices and of the oncogenicity group was examined microscopically.

Survival in the treated groups was comparable to that in the control group. The appearance and behaviour of the animals were not influenced by the treatment. During acclimatisation period and the first 3 months of treatment the mean body weights of 850 ppm group were comparable to those of the control group. Statistically significant lower mean body weights were measured during weeks 18-50. No differences in body weight gains were obvious during the first 3 months of treatment. However, significantly increased liver weights masked body weight gain decrement during this period of treatment at feeding levels of 500 and 850 ppm. For the remaining period of the first year, the body weight gains of the top dose group were up to 19 % below those of the control group. During the same time period, slightly lower body weight gains (up to 10 %) were measured for the 500 ppm group. The mean food consumptions and the overall food consumptions were
comparable in all groups. Treatment-related decreases in plasma cholesterol levels were recorded throughout the treatment period in mice treated at greater than 500 ppm. At weeks 9 and 14, the decreases in the 850 ppm group were statistically significant. Furthermore, increased sorbitol dehydrogenase activities were seen in mice treated at 850 ppm at week 9 and 14 investigations. At the two interim sacrifices and the final sacrifice as well, the mean liver weights and the mean liver to body weight ratios were slightly to markedly increased in treatment 500 and 850 ppm groups, compared to the control group's values. Macroscopical examination revealed a treatment-related increase in incidence of enlarged livers and of masses and nodules in the liver of male mice of the 850 ppm group designated for terminal sacrifice. In relation to microscopical examination, hepatocellular hypertrophy, fatty change, and single and multicellular necrosis were evident in the livers of mice fed 850 ppm as early as 9 weeks of treatment. Hepatocellular hypertrophy was also seen at 500 ppm at this time interval, and at 500 and 850 ppm at both the week 53 and terminal sacrifices. Additionally at study end, in the high dose group (850 ppm) an increased incidence of foci of cellular change and of hepatocellular adenomas was noted. Such effects to the liver were considered treatment related except for the increased incidence of hepatocellular adenomas, which on review were similar to control mice data. 

Dietary administration of the test substance to male albino mice at concentrations of 100, 500 and 850 ppm was tolerated without occurrence of overt signs of toxicity. The MTD (Maximum Tolerated Dose) was exceeded at the high dose level of 850 ppm, based on evidence of liver damage at early stage of treatment and marked effects on body weight gain. The NOAEL oncogenicity was 850 ppm. Based on the above findings, the NOAEL for systemic effects was 100 ppm, equivalent to an average intake of 11 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

107.6 mg/kg bw/day

Study duration:

chronic

Species:

mouse

Quality of whole database:

OECD TG 451 and GLP complaint study

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The following considerations were taken into account for the carcinogenicity classification:

 

• Significantly increased liver tumour incidences were observed in one species (mice) and in males only;


• Only at dose levels clearly exceeding the maximum tolerated dose and at the end of normal lifespan of mice;


• The increased incidence of live adenomas and carcinomas at the highest dose level were only slightly above the contemporary historical control range. The contemporary historical control studies show relatively high spontaneous variability in incidence of hepatocellular adenomas (6-18%) and carcinomas (8-16%);


• Based on histopathological findings and mechanistic studies, the test substance induces liver tumours by a non-genotoxic mechanism which involves CAR activation, induction of mitogenic hepatocyte proliferation and enlargement, hepatomegaly, and increase of xenobiotic metabolism.

 

Based on the available data classification for carcinogenicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.

Additional information

All available data was assessed and the study representing the worst-case effects was included as key study. The test substance did not elicit an oncogenic response in mice and rats.

 

In the key study (Gerspach 1999), the test article was administered in the diet at concentrations of 0, 100, 500, and 850 ppm (mg/kg food) for 18 months to albino mice. This study was conducted according to OECD TG 451 and followed GLP. Clinical signs, body weight, food consumption and mortality were monitored throughout the study for all animals. Blood chemistry investigation was performed at weeks -1, 9, 14, 53 and 79. At sacrifice, animals were examined macroscopically and organ weights were recorded. Organs and tissues were collected and the liver was prepared for histopathological evaluation. The liver from all animals of the 9-week and 53-week interim sacrifices and of the oncogenicity group was examined microscopically.

Survival in the treated groups was comparable to that in the control group. The appearance and behaviour of the animals were not influenced by the treatment. During acclimatisation period and the first 3 months of treatment the mean body weights of 850 ppm group were comparable to those of the control group. Statistically significant lower mean body weights were measured during weeks 18-50. No differences in body weight gains were obvious during the first 3 months of treatment. However, significantly increased liver weights masked body weight gain decrement during this period of treatment at feeding levels of 500 and 850 ppm. For the remaining period of the first year, the body weight gains of the top dose group were up to 19 % below those of the control group. During the same time period, slightly lower body weight gains (up to 10 %) were measured for the 500 ppm group. The mean food consumptions and the overall food consumptions were comparable in all groups. Treatment-related decreases in plasma cholesterol levels were recorded throughout the treatment period in mice treated at greater than 500 ppm. At weeks 9 and 14, the decreases in the 850 ppm group were statistically significant. Increased sorbitol dehydrogenase activities were seen in mice treated at 850 ppm at week 9 and 14 investigations. At the two interim sacrifices and the final sacrifice as well, the mean liver weights and the mean liver to body weight ratios were slightly to markedly increased in treatment 500 and 850 ppm groups, compared to the control group's values. Macroscopical examination revealed a treatment-related increase in incidence of enlarged livers and of masses and nodules in the liver of male mice of the 850 ppm group designated for terminal sacrifice. In relation to microscopical examination, hepatocellular hypertrophy, fatty change, and single and multicellular necrosis were evident in the livers of mice fed 850 ppm as early as 9 weeks of treatment. Hepatocellular hypertrophy was also seen at 500 ppm at this time interval, and at 500 and 850 ppm at both the week 53 and terminal sacrifices. Additionally at study end, in the high dose group (850 ppm) an increased incidence of foci of cellular change and of hepatocellular adenomas was noted. Such effects to the liver were considered treatment related except for the increased incidence of hepatocellular adenomas, which on review were similar to control mice data. 

Dietary administration of the test substance to male albino mice at concentrations of 100, 500 and 850 ppm was tolerated without occurrence of overt signs of toxicity. The MTD (Maximum Tolerated Dose) was exceeded at the high dose level of 850 ppm, based on evidence of liver damage at early stage of treatment and marked effects on body weight gain. The NOAEL oncogenicity was 850 ppm. Based on the above findings, the NOAEL for systemic effects was 100 ppm, equivalent to an average intake of 11 mg/kg bw/day.

 

Two additional studies for this endpoint are available as supporting information. The first study (Froehlich 1984) was not conducted according to a particular test guideline and did not follow GLP. Rats were administered a dose of 2000 ppm of the test substance for 8 weeks. The results presented in this study support the hypothesis that 2000 ppm of the test substance acts as a promotor of non-neoplastic and neoplastic proliferation changes in the rat liver. In the second study (Hunter 1985), rats were administered a dose of either 0, 100, 500, 2500 ppm test substance for up to 109 weeks via the diet. The study was not conducted according to a particular test guideline and did follow GLP. Treatment at 2500 ppm of the test substance produced a significant effect on body weight gain to a level greater than expected by the small reduction in food intake. Minor changes in a few blood chemistry parameters and changes in liver and adrenal weights were not associated with morphological change. Treatment with the test substance produced no change in any tissues examined, and in particular there was no indication of an effect on the spontaneous tumor profile of the SD rat. The NOAEL in this study is 100 ppm, equivalent to 3.6 mg/kg bw/day for males and 4.57 mg/kg bw/day for females.