pymetrozine (ISO); (E)-4,5-dihydro-6-methyl-4-(3-pyridylmethyleneamino)-1,2,4-triazin-3(2H)-one

Carcinogenicity

Description of key information

- Oral, NOAEL = 100 ppm (both sexes), equivalent to dose levels of 3.7 mg/kg bw/day for males and 4.45 mg/kg bw/day for females, rats (Tif: RAIf (SPF)), chronic, 2 -years, feeding, OECD 453, Gerspach 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:

4 May 1992 to 11 May 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

Adopted May 1981

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: FIFRA, Pesticide Assessment Guidelines, subdivision F: Hazard Evaluation, Human and Domestic Animals; section 83-5: Combined chronic toxicity/oncogenicity studies.

Version / remarks:

November 1984

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: Japanese MAFF 59 NoSan No. 4200

Version / remarks:

January 1985

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

other: Tif: RAIf (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 4 to 5 weeks
- Weight at study initiation: 134.6 to 183.3 g (males) and 128.1 to 209.8 g (females)
- Fasting period before study: No
- Housing: The experiment was carried out under specified pathogen free (SPF) standard laboratory conditions. The animals were housed in groups of 5 in Macrolon cages type 4 (area: 1800 square centimeters) with standardized granulated soft wood bedding. Cages were allocated to racks by sex and group. Cages and feeders were changed at 1-week and 5-week intervals, respectively.
- Diet: ad libitum. Pelleted, certified standard diet, containing the appropriate concentrations of the test article was provided ad libitum except for fasting prior to blood collection.
- Water: ad libitum, tap water
- Acclimation period: 10 days
- Additional information on strain: Hybrids of RII/1 X RII/2 (Sprague-Dawley derived)

DETAILS OF FOOD AND WATER QUALITY:
The drinking water quality fulfilled the critical parameters in the specifications of the "Schweizerisches Lebensmittelbuch" (Ed. 1972). There were no contaminants present at concentrations considered likely to have affected the conduct or purpose of the study.
All batches of diet were assayed for composition and contaminant levels by the manufacturer. None of the contaminants reported in the analysis profile were considered likely to have affected the conduct or purpose of the study.

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

IN-LIFE DATES:
4 May 1992 to 11 May 1994

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed on a calibrated Mettler balance. The pulverized food was then homogeneously mixed with the appropriate concentrations of the test article and about 25% water was added before pelleting to ensure the necessary pellet quality. The pellets were subsequently airdried. The test substance was used without adjustment for purity.

DIET PREPARATION
- Rate of preparation of diet: about monthly intervals
- Mixing appropriate amounts with: certified standard diet
- Storage temperature of food: stored in stainless steel containers at room temperature

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Initially, pellet diets were analysed for test article homogeneity and stability. Periodically during the study, diets were analysed for test article concentration.
Pellet samples (about 200 g) were taken during the pelleting process (for homogeneity analysis at beginning, middle and end of pelleting process) and dispatched deep-frozen for analysis. For stability analysis, the sample was stored five weeks at room temperature before analysis.

Duration of treatment / exposure:

24 months

Frequency of treatment:

Daily; the test substance is available in the diet throughout the study. The diet was ad libitum except as noted under laboratory investigations.

Post exposure period:

None

Dose / conc.:

10 ppm (nominal)

Remarks:

Equivalent to 0.4 and 0.4 mg/kg bw/day for males and females respectively.

Dose / conc.:

100 ppm (nominal)

Remarks:

Equivalent to 3.7 and 4.5 mg/kg bw/day for males and females respectively.

Dose / conc.:

1 000 ppm (nominal)

Remarks:

Equivalent to 39.3 and 47.1 mg/kg bw/day for males and females respectively.

Dose / conc.:

3 000 ppm (nominal)

Remarks:

Equivalent to 128 and 154 mg/kg bw/day for males and females respectively.

No. of animals per sex per dose:

80

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The selection was based on the following previously conducted studies: acute oral toxicity in rats (LD50: 5820 mg/kg), 28-days range finding study in rats (NOEL: 500 ppm, corresponding to a daily intake of 55 mg/kg bw/day), 3-months toxicity study in rats (NOEL: 500 ppm, corresponding to a daily intake of 33 mg/kg bw/day in females).
Based on these findings, the 3000 ppm dose level selected for the chronic study was expected to result in overt toxicity without significantly reducing survival. The 1000 ppm level was selected in order to evaluate dose-response relationships. The 100 ppm levels was expected to cause no or minimal toxic effects and the 10 ppm level was projected to be a no observed effect level.

In total 80 animals/sex/group, of which:
- 50 animals/sex/group for evaluation of the carcinogenic potential
- 10 animals/sex/group for evaluation of haematological, biochemical and urine analysis
- 10 animals/sex/group for evaluation of haematological investigations
- 10 animals/sex/group for interim sacrifice at 12 months

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, clinical signs present were recorded. Mortality: daily (a.m. and p.m. on working days, a.m. on weekends and holidays).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, clinical signs present were recorded

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

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, mean of individual calculations according to the following formula: (weekly food consumption in g) / (midweek body weight in g) X (1000/7).
The unit of the food consumption ratio is g food/kg body weight/day.
- Time schedule: Weekly for the first 3 months and monthly thereafter.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: calculated according to the following formula and rounded to 3 meaningful digits: (food consumption ratio X nominal dose (ppm)) / 1000. The unit of the test article intage is mg test article/kg body weight/day.
An overall mean value (TWA1, time weighted average) was calculated based on the nominal content of the test article in the food. Additionally, this value was corrected for the analytically determined content (TWA2). The formula can be found in the section ‘Any other information on materials and methods incl. tables’.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the beginning of the treatment, and at 6, 12, 18 and 24 months.
- Dose groups that were examined: male and female animals of the highest dose level and of the control group were examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Wk 13, 27, 53, 78 and 105. To reduce the biological variability due to circadian rhythms, blood sampling was performed in the morning.
- Anaesthetic used for blood collection: Yes, ether anaesthesia
- Animals fasted: Yes, food was withheld overnight prior to blood removal
- How many animals: haematology investigations were carried out on surviving animals of experimental groups II and III (comprising initially 20 animals per sex and dose group). For terminal laboratory investigations at week 105, the number of animals subjected to examination was supplemented by animals of the carcinogenicity group (I) and/or experimental group III to yield 20 samples/sex/group for haematology.
- The following parameters were examined:
Red blood cell parameters: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width.
White blood cell parameters: leukocyte count, differential leukocyte count, neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unstained cells.
Blood platelets: Trombocyte count.
Prothrombin time: photometric assay using chromogenic substrate on a Cobas Bio centrifugal analyser.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Wk 13, 27, 53, 78 and 105. To reduce the biological variability due to circadian rhythms, blood sampling was performed in the morning.
- Animals fasted: Yes, food was withheld overnight prior to blood removal.
- How many animals: Blood chemistry investigations and urine analysis were performed on surviving animals of experimental group II (comprising initially 10 animals per sex and dose group). For terminal laboratory investigations at week 105, the number of animals subjected to examination was supplemented by animals of the carcinogenicity group (I) and/or experimental group III to yield 10 samples/sex/group for blood chemistry and urinalysis.
- The following parameters were examined: glucose, urea, creatinine, total bilirubin, total protein, albumin, globulin, A/G ratio, cholesterol, triglycerides, phospholipids, sodium, potassium, calcium, chloride, phosphorus inorganic, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, Gamma-glutamyl transpeptidase.

URINALYSIS: Yes
- Time schedule for collection of urine: Wk 13, 27, 53, 78 and 105.
- Metabolism cages used for collection of urine: Yes, urine for analysis was collected overnight.
- Animals fasted: Yes, food and water were withheld during the time of urine collection.
- The following parameters were examined:
Physical and chemical examination: urine volume, relative density, pH value, urine colour, protein, glucose, ketones, bilirubin, blood, urobilinogen.
Microscopic examination: cells (erythrocytes, leukocytes, squamous epithelial cells, transitional epithelial cells, renal epithelial cells), casts (hyaline casts, granular casts, waxy casts, erythrocyte casts, leukocyte casts, epithelial cell casts), crystals (triple phosphate crystals, calcium oxalate crystals, uric acid crystals, amorphous urate crystals, tyrosine crystals, cystine crystals, di-calcium phosphate crystals, unidentified crystals).

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
After one year of treatment and at the end of the 2-year treatment period, the surviving scheduled control and treated animals were bled under ether anaesthesia and subjected to a detailed necropsy.
At necropsy, the following weights were recorded from all animals: body (exsanguinated), brain, liver, kidneys, adrenals, ovaries/testes, spleen.
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, pancreas, oesophagus, 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 tissue with gross lesions.
Unless prevented by cannibalism or advanced autolysis a complete necropsy with tissue preservation was performed also on all animals which died during the test period or which had to be sacrificed in moribund condition.

HISTOPATHOLOGY: Yes
After the fixation, organ samples listed below were taken, embedded in paraplast, sectioned at 3-5 microns, stained with hematoxylin 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, pancreas, oesophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), 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 (sciatic nerve), brain (including medulla, pons, cerebral cortex, cerebellar cortex), spinal cord (cervical, thoracolumbar), eye with optic nerve (both), Harderian gland (both), extraorbital lacrimal gland (both), and any tissue with gross lesions.
Twenty satellite animals per sex and group, used for laboratory investigations, were excluded from microscopical examination according to the protocol.
All organs from 10% of animals randomised from control and all treated groups, all tumours, all livers, all thyroid with parathyroid glands, all adrenal glands and testes in males, and the uterus in control and high dose females were re-evaluated by the reviewing pathologist.

Statistics:

IN-LIFE OBSERVATIONS AND NECROPSY
For each time point and parameter an univariate statistical analysis was performed. Nonparametric methods were applied, to allow for non-normal as well as normal data distribution.
Lepage test and Jonckheere test was performed on the following data: Body weight, food consumption, haematology parameters, organ weights, organ to body weight ratio.
Cox regression model was performed on the following data: Survival.
Each treated group was compared to the control group by 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. 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.
Survival analysis was performed by the regression model (partial likelihood) introduced by Cox in order to compare survival time of treated animals with control animals.

PATHOLOGY DATA
To establish possible relationships between pathological changes and treatment, all microscopical findings were subjected to a statistical analysis according to the method described by Peto et al. (1980) for neoplastic lesions and Gart et al. (1985) for non-neoplastic changes with a program described by Seewald et al. (1994).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence and distribution of clinical signs recorded during the study gave no indication of a treatment-related effect on the appearance or behavior of the rats.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

A significantly higher number of males treated with 3000 ppm survived the treatment period. The survival of other treated groups was similar to that of the control group. An overview of the group mortality data can be found table 1 in 'Any other information on results incl. tables'.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

From the first week of treatment a lower rate of body weight gain was recorded for males and females of group 5 (3000 ppm) which at termination of the study resulted in mean body weights 15% (males) and 24% (females) lower than control values. A similar effect, to a lesser degree, was recorded for rats of group 4 (1000 ppm) with preterminal mean body weights 4% (males) and 7% (females) lower than control values. Correspondingly, the reduction in mean body weight gains for males and females of groups 4 and 5 (1000 and 3000 ppm) attained values of 6% and 19% (males), respectively, and 9% and 34% (females), respectively, below the values recorded for the control group.
The body weight development of males and females of groups 2 and 3 (10 and 100 ppm) was not disturbed by treatment.
An overview of the body weight data can be found table 2 in 'Any other information on results incl. tables'.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Markedly lower food intakes were recorded for males and females of group 5 (3000 ppm) during the first week of the study. Thereafter, lower intakes continued to be recorded for this group with overall mean values 6% (males) and 13% (females) lower than those of the respective control group. Slightly lower food intakes were also recorded for males and females of group 4 (1000 ppm) during the first week of treatment. Thereafter, the intakes for group 4 females remained marginally lower than those of the control group.
Food intakes for males and females of groups 2 and 3 (10 and 100 ppm) were not disturbed by treatment.
Lower food consumption ratios were obtained for groups 4 and 5 (1000 and 3000 ppm) for the first week of treatment. Thereafter, ratios for males and females of group 5 (3000 ppm) tended to be slightly higher whereas ratios for group 4 (1000 ppm) were essentially similar to those of the control group.
Food consumption ratios for males and females of groups 2 and 3 were similar to the control values.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No trends of toxicological relevance developed. Therefore, measurement of water intake was discontinued according to the proviso in the study protocol.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Examinations included inspection of the surroundings of the eyes, of sclera, cornea, iris and adaptation of the pupil to the ophthalmoscopic light beam. Animals of control and highest treatment level were examined at day -6 (acclimation period), during days 179, 359, 536, and towards the end (day 725) of the treatment period.
The examinations revealed no evidence of a treatment-related effect on the eyes of the rats.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A marginally lower red blood cell count was recorded at week 13 for males of group 5 (3000 ppm) and was associated with a higher mean cell volume and higher mean cell haemoglobin. However, subsequent values were similar to those of the control group.
Throughout the study the mean platelet count for females of group 5 (3000 ppm) was significantly higher than that of the control group although the differences from control values were of doubtful biological relevance.
Other differences from control values which attained a level of statistical significance were of insufficient magnitude to be of toxicological relevance.
At the end of the treatment period, one male of the control group had a myeloid leukaemia, and one male each of group 4 and of group 5 had a lymphatic leukaemia. No relevance is attributed to these findings as this pathology occurs spontaneously in this colony of rats.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At each investigation, slightly higher values were recorded for plasma bilirubin and albumin in males of group 5 (3000 ppm) and for plasma cholesterol and inorganic phosphorus in females of group 5 (3000 ppm). In addition, in group 5 males, slightly lower values were obtained throughout for chloride levels and during the first 18 months for glucose levels.
For both males and females of group 5 (3000 ppm), mean activities for alanine aminotransferase remained lower than control values throughout the treatment period.
Other differences which attained a level of statistical significance were of insufficient magnitude to be of toxicological relevance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on any urine parameters in males or females at any dose level tested.

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):

Compared to the respective control values, the mean carcass weights were decreased in groups 4 and 5 (1000 and 3000 ppm) both after one year of treatment (-21% in males / -29% in females) and at termination of the study (-14% in males / -23% in females). Therefore, for these groups organ to body weight ratios were especially considered.
After one year of treatment, significantly higher organ to body weight ratios were obtained for liver, kidneys and spleen of males and females of group 5 (3000 ppm) and for males of group 4 (1000 ppm).
At termination of the study, significantly higher organ to body weight ratios were obtained for liver and spleen of males and females of group 5 (3000 ppm) and for kidneys of group 5 females. However, the absence of morphological change in the spleen and kidneys suggests the differences were a reflection of the marked reduction of body weight in this group.
The mean adrenals weight for males of group 3 (100 ppm) was unduly influenced by the high value recorded for one rat. Similarly, the high mean adrenals weight for group 5 (3000 ppm) was due to the gross values recorded for three rats. Excluding the above values and recalculating the means resulted in similar mean values for control and treated groups. Therefore, a treatment-related effect on these organs was not apparent.
Other differences between the means which attained a level of statistical significance were of insufficient magnitude to be of toxicological relevance or, as with ratios for brain and gonads, reflected the difference in group mean body weights.
Organ weights and ratios for animals of groups 2 and 3 (10 and 100 ppm) were not influenced by treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

A treatment-related increased incidence of female rats with liver cysts was noted in the 3000 ppm group. In females, a slight increase in the number of animals with liver masses and in males, a slight increase in the number of animals with mottled livers were also noted at 3000 ppm. Correlative microscopical pathology is discussed in the section "Microscopical findings".
Changes in the incidences of nodules in the uterus and ovaries in females at 3000 ppm appeared to be of no toxicological relevance and are considered incidental.
All other findings occurred in comparable numbers in all experimental groups and were similar to those occurring spontaneously in our colony of rat. Thus, no experimental relevance was attributed to these findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

LIVER – EFFECTS OBSERVED AND TREATMENT RELATED
The liver was clearly a target organ as indicated by a significantly increased incidence of hepatocellular hypertrophy in both males and females at feeding levels of 1000 and 3000 ppm, increased incidence of foci of cellular change in males and females at 3000 ppm, and increased incidences of biliary cysts in females at 3000 ppm.
Four out of five males at 100 ppm revealed the liver cell hypertrophy at the 12 month interim sacrifice and only one male showed this finding after 24 month of treatment. Hepatocellular hypertrophy was mainly confined to the centrilobular and midzonal region of the liver.
The cellular morphology and tinctorial appearance of foci of cellular change in males and females at 3000 ppm varied from clear or eosinophilic to basophilic cells. Slight increases in incidence of foci of cellular change in females at 100 and 1000 ppm are within the historical control range for this strain of rats used in this laboratory and are therefore considered of no toxicological relevance. The mottled appearance of liver seen in the male 3000 ppm group was mainly consequent to foci of cellular change observed at microscopical level.
Biliary cysts in females in the 3000 ppm group were characterized morphologically by unilocular or multilocular cysts lined by an unilayered flattened or cuboidal epithelium. These biliary cysts accounted for most of the liver cysts observed at necropsy. In males at 3000 ppm, the liver cysts correlated microscopically also mainly with biliary cysts but there was no statistical significance, and therefore no toxicological relevance was attributed to this finding.

THYROID GLAND – EFFECTS OBSERVED, BUT NOT TREATMENT RELATED
Hyperplasia of thyroid follicular epithelium was observed in a higher number of males at 1000 and 3000 ppm and females at 3000 ppm. However, this lesion was considered to be secondary to the liver changes.

SPLEEN – EFFECTS OBSERVED, BUT NOT TREATMENT RELATED
The minimal congestion detected in males and females of the 3000 ppm group was observed at the 12 month interim sacrifice but was no longer present at the end of 2 years of treatment, therefore no toxicological relevance was attributed to this finding.

UTERUS – EFFECTS OBSERVED, BUT NOT TREATMENT RELATED
The statistically significant increase in dilatation of the uterus in females at 3000 ppm is considered of no toxicological relevance since the reported incidence is within the historical control range for this strain of rats used in this laboratory. These dilatations correlated with the uterine nodules reported at necropsy.

VARIOUS TISSUES – EFFECTS OBSERVED, BUT NOT TREATMENT RELATED
The slightly higher incidence of hypertrophy of exocrine pancreas in males at 3000 ppm, the pigmentation of Kupffer cells of the liver and the ovarian atrophy in females at 3000 ppm, and the sinusoid cystic dilatation of mesenteric lymph node in females of the 3000 ppm group were considered to reflect the biological variation occurring in rats at this age and, therefore, are regarded to be of no toxicological relevance.
The ovarian nodules observed at necropsy correlated mainly with stromal hyperplasias of the ovaries. There were no differences in the incidence or severity of this finding among the groups, and, therefore, no toxicological relevance was attributed to it.
Additionally, a variety of other changes was found in this study. They commonly occur in our colony of rats, and, neither their incidences nor their distribution and morphological appearance gave any indication of a treatment-related association.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

An overview of the group incidences of neoplastic microscopic lesions in rats can be found table 3 in 'Any other information on results incl. tables'.

LIVER – EFFECTS OBSERVED AND TREATMENT RELATED
The significantly increased incidence of hepatocellular hypertrophy noted in males and females at the 3000 ppm feeding level was accompanied by an increased incidence of benign liver cell tumours in females at 3000 but not in males. The incidence of hepatocellular hypertrophy which was also elevated in males and females at 1000 ppm was not associated with an increased incidence of liver tumours at this dose. The few hepatomas in females at 1000 ppm are slightly above the historical control range for this strain of rats used in this laboratory but still within the historical control range for aged female Sprague-Dawley-derived rats (the incidence of spontaneously occurring hepatomas in female Sprague-Dawley-derived rats from 24-month studies varies up to 8%). Therefore, no toxicological relevance was attributed to the hepatomas in females at 1000 ppm. The benign hepatomas in females at 3000 ppm correlated with the macroscopically reported liver masses.

ADRENAL GLAND – EFFECTS OBSERVED, BUT NOT TREATMENT RELATED
Malignant medullary tumours were noticed in a slightly increased incidence in males at 3000 ppm. However, on pooling both the benign and malignant medullary tumours for statistical analysis, there was no longer a significance and the combined tumour incidence is within the historical control range for this strain of rats used in this laboratory. Therefore, no toxicological relevance was attributed to these tumours.

CEREBRAL MENINGES – EFFECTS OBSERVED, BUT NOT TREATMENT RELATED
The slightly higher incidence of benign granular cell tumours seen in the cerebral meninges of males at 3000 ppm is within the historical control range for this strain of rats used in this laboratory and, therefore, was considered of no toxicological relevance.

VARIOUS TISSUES – EFFECTS OBSERVED, BUT NOT TREATMENT RELATED
Primary malignant lymphomas were slightly significant by the statistical analysis in the lymphoreticular tissue of males at 3000 ppm as well as the systemic infiltration in organs such as mesenteric lymph node, lung, heart, kidney, urinary bladder, brain and eye. However, due to the high biological variation of this generalised neoplasm and the low incidence observed, no toxicological relevance was attributed to it.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

100 ppm (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: equivalent to 3.73 mg/kg bw/day

Dose descriptor:

NOAEL

Effect level:

100 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Remarks on result:

other: equivalent to 4.45 mg/kg bw/day

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 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.:

1 000 ppm

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Table 1. Group mortality data, number surviving to terminal necropsy

	Males	Females
Control	20	30
10 ppm	25	35
100 ppm	23	30
1000 ppm	28	34
3000 ppm	35*	37

* p= 0.0056 when compared with the survival in the control group.

Table 2. Body weights (means)

Feeding level [ppm]	0	10 ppm	100 ppm	1000 ppm	3000 ppm
Parameter	Males
Week -1	159	160	161	163*+	162+
Week 50	733	727	731	689*-	609*-
Week 104	725	748	718	695	619*-
Parameter	Females
Week -1	146	146	146	144	143-
Week 50	392	391	399	375*	305*-
Week 104	467	471	458	436	356*-

Lepage: * if p <0.01; Jonckheere: +/- if p <0.01

 

Table 3. Increased incidences of neoplastic microscopic lesions in rats

Sex	Males					Females
Feeding level, ppm	0	10	100	1000	3000	0	10	100	1000	3000
Liver / Total examined	60	60	60	60	60	60	60	60	59	60
Benign hepatoma +)	2 (3 %)	0	2 (3 %)	0	2 (3 %)	0	0	0	2(3 %)	7 (12 %)**
Adrenal medulla / Total examined	60	60	60	60	60	60	60	60	60	60
Benign medullary tumor	2	0	3	2	1	0	1	0	0	1
Malignant medullary tumor ++)	0	0	1	0	3 (5 %)	0	0	0	0	0
Cereb. meninges / Total examined	60	60	60	60	60	60	60	60	60	60
Benign gran. cell tumor +++)	0	0	0	1	2*	1	0	1	0	1

Peto-Test: ** = p<0.05; ** = p<0.0001

+) Historical control incidence in females: 0 - 3 % in the conducting laboratory between 1989 & 1993, up to 8 % according to Registry of Industrial Toxicology Animal-data, Hannover, Germany

++) Historical control incidence in males: 0 - 3 % for malignant medullary tumour and 4 - 12 % for benign & malignant medullary tumour in the conducting laboratory between 1989 & 1993.

+++) Historical control incidence in males: 0 - 5 % in the conducting laboratory between 1989 & 1993.

Conclusions:

In this GLP compliant OECD 453 study, a NOAEL of 100 ppm, equivalent to 3.7 or 4.45 mg/kg bw/day for males and females, respectively, was derived based on liver toxicity evidenced by hypertrophy of hepatocytes in both sexes at 1000 ppm.

Executive summary:

This GPL compliant OECD 453 study was conducted to assess the carcinogenicity and chronic toxicity of the test substance when administered in the diet to rats (strain: Tif: RAIf (SPF), at dietary concentrations of 0, 10, 100, 1000 and 3000 ppm (= mg/kg food) for 24 months (for females equivalent to 0, 0.357, 3.73, 39.3 and 128 mg/kg body weight/day; for males equivalent to 0, 0.430, 4.45, 47.1 and 154 mg/kg body weight/day). Fifty animals per sex and group were utilised for evaluation of carcinogenic potential, 10 animals per sex and group were utilised for haematological, biochemical and urine parameters. Additionally, 10 animals per sex and group were utilised for haematological parameters, and 10 animals per sex and group were used for interim sacrifice at 12 months. Clinical signs, body weight, food consumption and mortality were monitored throughout the study for all animals. Haematological, blood chemistry and urine analyses were performed at 13, 27, 53, 78 and 105 weeks. At sacrifice, animals were examined macroscopically and organ weights were recorded. Organs and tissues were collected and prepared for histopathological evaluation. Organs and tissues from all animals in the carcinogenicity group were examined microscopically.

Dietary levels of 1000 and 3000 ppm of the test substance caused inappetence with the resulting reduction of body weight gain, especially marked in males and females in the highest treatment group. Changes to the haematology and blood chemistry profiles may have been a reflection of the nutritional status of the animals these groups. Higher liver, kidney and spleen to body weight ratios were noted in males and females of the 3000 ppm and in males of the 1000 ppm exposed groups. The marginal increased incidences of neoplasms in the adrenal medulla, cerebral meninges and lymphoreticular tissue were considered of no toxicological relevance, especially with regard to the historical control data in the conducting laboratory. Microscopical examination revealed the liver to be the target organ as indicated by a significantly increased incidence of hepatocellular hypertrophy in both sexes at 1000 and 3000 ppm, an increased incidence of foci of cellular change in both sexes at 3000 ppm, and increased incidences of benign hepatomas and biliary cysts in females at 3000 ppm. Mainly based on liver toxicity evidenced by hypertrophy of hepatocytes in both sexes at 1000 ppm, the No Observed Adverse Effect Level (NOAEL) is 100 ppm, equivalent to 3.7 or 4.45 mg/kg bw/day for males and females, respectively.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

3.7 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

GLP compliant OECD 453 study.

System:

hepatobiliary

Organ:

liver

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

Based on the available information, the test substance is classified as carcinogen category 2 in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.

Additional information

Carcinogenicity - rats

This GPL compliant OECD 453 study was conducted to assess the carcinogenicity and chronic toxicity of the test substance when administered in the diet to rats (strain: Tif: RAIf (SPF), at dietary concentrations of 0, 10, 100, 1000 and 3000 ppm (= mg/kg food) for 24 months (for females equivalent to 0.357, 3.73, 39.3 and 128 mg/kg body weight/day; for males equivalent to 0.430, 4.45, 47.1 and 154 mg/kg body weight/day) (Gerspach 1995). Fifty animals per sex and group were utilised for evaluation of carcinogenic potential, 10 animals per sex and group were utilised for haematological, biochemical and urine parameters. Additionally, 10 animals per sex and group were utilised for haematological parameters, and 10 animals per sex and group were used for interim sacrifice at 12 months. Clinical signs, body weight, food consumption and mortality were monitored throughout the study for all animals. Haematological, blood chemistry and urine analyses were performed at 13, 27, 53, 78 and 105 weeks. At sacrifice, animals were examined macroscopically and organ weights were recorded. Organs and tissues were collected and prepared for histopathological evaluation. Organs and tissues from all animals in the carcinogenicity group were examined microscopically.

Dietary levels of 1000 and 3000 ppm of the test substance caused inappetence with the resulting reduction of body weight gain, especially marked in males and females in the highest treatment group. Changes to the haematology and blood chemistry profiles may have been a reflection of the nutritional status of the animals these groups. Higher liver, kidney and spleen to body weight ratios were noted in all 3000 ppm and for males of 1000 ppm exposed groups. The marginal increased incidences of neoplasms in the adrenal medulla, cerebral meninges and lymphoreticular tissue were considered of no toxicological relevance, especially with regard to the historical control data in the conducting laboratory. Microscopical examination revealed the liver to be the target organ as indicated by a significantly increased incidence of hepatocellular hypertrophy in both sexes at 1000 and 3000 ppm, an increased incidence of foci of cellular change in both sexes at 3000 ppm, and increased incidences of benign hepatomas and biliary cysts in females at 3000 ppm. Mainly based on liver toxicity evidenced by hypertrophy of hepatocytes in both sexes at 1000 ppm, the No Observed Adverse Effect Level (NOAEL) is 100 ppm, equivalent to 3.7 or 4.45 mg/kg bw/day for males and females, respectively.

 

Carcinogenicity - mice

In this GLP compliant, OECD 451 study, groups of 60 male and 60 female mice (strain Tif:MAGf) were administered with the test substance orally for 18 months, by admixture in the diet, at concentrations of 0, 10, 100, 2000 or 5000 ppm (dietary equivalent of 1.24, 12.0, 254 and 678 mg/kg bw/day for males and 1.17, 11.4, 243 and 673 mg/kg bw/day for females) (Gerspach 1995). Per dose 50 animals per sex were designated for evaluation of the carcinogenic potential and 10 animals per sex for evaluation of haematological parameters. Clinical observations were made daily, body weights and food consumption recorded weekly for 3 months and monthly thereafter, and water consumption recorded monthly. Laboratory investigations were performed at weeks 53 and 78 on the animals designated for the evaluation of haematological parameters. At the end of the test period, all surviving animals were subjected to detailed necropsy and post mortem examination. Organ weights of all animals which were recorded, tissue/organ samples were preserved. Microscopic examination of tissues was performed on all animals, of all treatment and control groups, with the exception of the 10 animals/sex/group designated for evaluation of haematological parameters.

The treatment did not adversely affect survival or appearance or behaviour of the animals. Compared with the controls, a significantly higher number of group 5 males (5000 ppm) survived the treatment period. At the end of the study, mean body weight were decreased relatively to the control for animals in the groups receiving 2000 and 5000 ppm (group 4 and 5), for males -7% and -23% and for females -15% and -44%, respectively. Food consumption was not affected by treatment at any dose level except in the high dose (5000 ppm) females. For the majority of the treatment period, higher food consumption ratios were obtained for males and females of group 5 (5000 ppm). The treatment did not influence the haematological profile of the mice. Significantly higher organ weights and organ to body weight ratios were obtained for liver of males and females of groups 4 and 5 (2000 and 5000 ppm) and for adrenals of males of groups 4 and 5. Significantly higher ratios were also obtained for spleen of males of group 5 (5000 ppm) and for kidneys of females of group 5. Necropsy revealed a higher incidence of liver masses in male groups 4 and 5 (2000 and 5000 ppm) and female group 5. An increased incidence of animals showed enlarged livers in groups 4 and 5 and mottled livers in group 5. Splenic enlargement was noted in male groups 4 and 5. Microscopical examination revealed a significantly increased incidence and severity of liver cell hypertrophy in male and female groups 4 and 5. Furthermore, an increased number of liver nodules in animals of group 5 was observed. In addition, higher incidences were recorded for chronic inflammation of the glandular stomach and hyperplasia of the gastric mucosa in male group 5, extramedullary haematopoiesis in the spleen in male and female groups 4 and 5, hypercellularity of the bone marrow in male groups 4 and 5 and female group 5, and haemosiderosis of the spleen in male group 5 and female groups 4 and 5. A significantly increased incidence of benign and malignant liver tumours in both male and female groups 5 and of malignant tumours also in male group 4 were observed.

Mainly based on toxicity to the liver evidence by increased tumour formation in males, the No Observed Adverse Effect Level (NOAEL) is 100 ppm, equivalent to a lifetime daily dose of 11.4 mg/kg bw/d.