mecoprop-P (ISO); (R)-2-(4-chloro-2-methylphenoxy)propionic acid

Carcinogenicity

Description of key information

Milburn (2008)

Under the conditions of the study, the test material is concluded not to be carcinogenic.

Supporting Study: Mellert (1996)

Under the conditions of the study the no observed adverse effect level (NOAEL) for systemic toxicity was 25 ppm (about 4 mg/kg body weight) in females and 250 ppm (43 mg/kg body weight) in males. The test material was not oncogenic under the conditions of this study. The no observed adverse effect level (NOAEL) for carcinogenicity was therefore 250 ppm (43 mg/kg body weight).

Supporting Study: Mellert (1999)

Under the conditions of the study the test material was not oncogenic.

QSAR: Lye (2011)

All substances examined (i.e. the test material and 4 impurities) are predicted to be non-carcinogenic with the restraints of the models.

Read-Across Study: Kuehborth (1988)

Under the conditions of the study the administration of the read-across material over a period of 24 months at a dose level of 20 ppm did not lead to any test material-induced changes. Due to the present test results a carcinogenic potential of the test material is to be ruled out.

A dose-dependent increase in the kidney weights of the male rats of the 100 and 400 ppm groups was observed, while the kidney weights of the female animals remained uninfluenced.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 March 2005 to 17 October 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.4200 (Carcinogenicity)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Annex V to Council Directive 67/548/EEC published in the Ninth Adaptation, Commission Directive 87/302/EEC of 18th November 1987, OJEC, L133, 32-36, 1988 (Carcinogenicity test)

Version / remarks:

1988

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Remarks:

HsdRCCHan:WIST

Details on species / strain selection:

The rat was used because it is one of the species generally recommended for the assessment of toxicity. The HsdRCCHan:WIST strain was used because of the good survival and availability of background data for this strain, in this Laboratory, relating to studies of this type.
The oral route was used as this represents a possible route of human exposure.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 21 to 26 days old. The rats were approximately 5 weeks of age when the experimental diets were first fed.
- Housing: The rats were housed, sexes separately. They were housed in litters initially and in fours after they had been assigned to experimental groups.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: The animals were housed under the experimental conditions for approximately 2 weeks prior to the start of the study.

DETAILS OF FOOD AND WATER QUALITY:
Each batch of diet is routinely analysed for composition and for the presence of contaminants. Water is also periodically analysed for the presence of contaminants. No contaminants were found to be present in the diet or water at levels considered to be capable of interfering with the purpose or outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C. Except for a single day when the minimum temperature recorded was 18 °C, the temperatures were within the specified range.
- Humidity (%): 30 - 70 %. There were a small number of days when the relative humidity was higher than 70 %, the maximum value recorded was 90 %. There was no discernible effect on the animals and no effect on the integrity of the study.
- Air changes (per hr): At least 15 changes/hour.
- Photoperiod (hrs dark / hrs light): Artificial giving 12 hours light, 12 hours dark.

Route of administration:

oral: feed

Vehicle:

other: 2 % amorphous silica as an anti–caking agent.

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): The test and control diets were prepared (using RM1 diet) and transferred to the animal rooms as required. The diets were prepared in 60 kg batches from 1 000 g premixes prepared by triturating the appropriate amount of test material with milled diet. The prepared diets were dispensed into glass jars via an automated system (Autopak Ltd) and the jars fitted with a stainless steel ‘follower’ to prevent excessive wastage of diet. The jars were then stored in labelled plastic trays, which were colour-coded. The trays of diet were stored at room temperature and presented to the animals as required.
- Mixing appropriate amounts with: All diets were based on RM1 diet supplied by Special Diets Services Ltd, Stepfield, Witham, Essex, UK.
- Storage temperature of food: Test diets were stored a room temperature for up to 5 weeks.

VEHICLE
- Justification for use and choice of vehicle: The preparation of the test material as a fine powder necessitated inclusion of 2 % amorphous silica as an anti–caking agent. The control article is Sipernat 505 (amorphous silica supplied by Degussa). This was added to the control diets to a level comparable with that in the high dose diets.
- Lot/batch no.: MC/01/02
- Purity: Assumed 100 %

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples from all dietary levels (including controls) were taken prior to the start of the study at approximately three-monthly intervals throughout the study and analysed quantitatively for the test material.
At the start of the study the homogeneity of the test material in CT1 diet was determined by analysing samples from the low and high dose levels and the chemical stability of the test material in diet at room temperature was determined for these same diets over a period of up to 5 weeks.
The mean concentrations for all batches of diet preparations analysed were within 13 % of the nominal concentration. The overall mean concentrations were within 6 % of nominal.

The homogeneity of the test material in diet preparations at concentrations of 100 ppm and 1 200 ppm, (for a batch size of 60 kg), was determined and considered satisfactory, percentage deviations from the overall mean were within 8 %. Some variation was noted for the 1 200 ppm level when initially analysed, which was not evident to the same extent when repeat analysis was conducted on the same homogeneity samples. As an additional check, on the instruction of the SD, representative 1 200 ppm diet samples were sourced directly from the animal room for analysis. Results from these samples also demonstrated satisfactory homogeneity.

The reanalysis of the test material in diet preparations at concentrations of 100 ppm and 1 200 ppm when stored at room temperature was shown to be acceptable for 36 days, covering the period of dosing. Reanalysis of 1 200 ppm stability day zero samples was repeated on stability day 1, with results confirming that of day zero. Variable results were obtained for recovery diets at the 100 ppm level on stability day 36. Extracted sample solutions were re-analysed, confirming results as being acceptable.

METHOD SUMMARY
Measured amounts of water/trifluoroacetic acid (0.5 % v/v) were added to diet samples, which were allowed to stand at room temperature, before being extracted by the addition of acetonitrile/trifluoroacetic acid (0.5 % v/v). Aliquots of the supernatant were diluted with acetonitrile, as appropriate after filtration, to give sample solution concentrations within the range of the calibration standards used. Samples and standards were analysed by High Performance Liquid Chromatography (HPLC).

CHEMICALS AND REAGENTS
Acetonitrile, HPLC grade
Water, Milli Q+ grade (Millipore)
Acetic acid, Analytical grade
Trifluoroacetic acid, Analytical grade
Water/Trifluoroacetic acid, 0.5 % v/v: A volume of trifluoroacetic acid, 5mL was added to 1 L of water.
Acetonitrile/Trifluoroacetic acid, 0.5 % v/v: A volume of trifluoroacetic acid, 5mL was added to 1 L of acetonitrile.
Acetic acid, 0.1M: A volume of acetic acid, 5.7 mL, was added to 1 L of water.
HPLC mobile phase, 0.1M acetic acid in water/acetonitrile 55/45 v/v
Acetonitrile, 450 mL was added to acetic acid, 0.1M, 550 mL.
Diluting solvent for samples and standards was acetonitrile.

CALIBRATION STANDARDS
Nominally 100 mg of the test material was accurately weighed into a volumetric flask, 100 mL, and diluted to volume with acetonitrile (nominally 1.0 mg/mL). Further appropriate dilutions were made with acetonitrile using volumetric glassware to produce a range of solutions, nominally within 2.5 to 25 μg/mL.
The purity of the test material was not taken into account in the preparation of standard solutions.

PROCEDURE
Sample preparation: Accurately weighed portions of diet samples, 5 g, were added to tared conical flasks, water/trifluoroacetic acid (0.5 % v/v), 20 mL, was added and the flasks stoppered. The contents were left to stand at room temperature for 30 minutes, before the addition of acetonitrile/trifluoroacetic acid (0.5 % v/v), 30 mL followed by a 30-minute extraction with mechanical shaking. The supernatant was filtered and diluted as required with acetonitrile, to a known nominal concentration within the range of the calibration standards. Final sample solutions were filtered a second time prior to transfer to HPLC autosampler vials.

High Performance Liquid Chromatography Conditions
Separations module: Alliance 2695 (Waters)
Mobile phase: 0.1 M acetic acid in water/ acetonitrile, 55/ 45 v/v
Flow rate: 1.5 mL/min
Detector: 2487 dual wavelength UV detector (Waters)
Detector wavelength: 280 nm
Column: 25 cm x 4.6 mm ID Zorbax ODS (Hichrom)
Column temperature: 50 °C
Injection volume: 10 μL
Data handling: Millenium 32 and Empower 2 (Waters)

CALIBRATION
The analysis system was calibrated using a range of standards to determine the linearity of response. An appropriate standard of known concentration was interspersed at intervals throughout the analysis.

CALCULATION OF RESULTS
The analysed concentration was calculated using the equation below, after appropriate calibration and reprocessing.

Analysed concentration (ppm w/w) = (C x Df x 100) / R

Where:
C = Calculated sample concentration from data system (μg/mL)
Df = Dilution factor
R = Recovery (%)

RECOVERY
For each analysis, recovery was determined in triplicate at each level.
1 200 ppm level: An accurately weighed portion of the test material, 60 mg, was quantitatively transferred into an automated pestle and mortar (Fritsch) with 49.94 g of control diet, and ground for 20 minutes. The diet was placed in a suitable container and rotated on a flask rotator (Stuart), for 20 minutes at an appropriate speed.
600 ppm level: An accurately weighed portion of the test material, 30 mg, was quantitatively transferred into an automated pestle and mortar (Fritsch) with 49.97 g of control diet, and ground for 20 minutes. The diet was placed in a suitable container and rotated on a flask rotator (Stuart), for 20 minutes at an appropriate speed.
100 ppm level: An accurately weighed portion of 1 200 ppm recovery diet, 4.17 g, was added to control diet, 45.8g, and processed as previously described.

% Recovery = (Analysed concentration x 100) / Theoretical concentration

LIMIT OF DETECTION
The limit of detection was calculated to be approximately 1 μg/mL test material in the analysed solution, corresponding to a dietary concentration of 10 ppm (v/v).

Duration of treatment / exposure:

Surviving rats were fed for at least 104 weeks.

Frequency of treatment:

Continuous (in diet)

Post exposure period:

None

Dose / conc.:

100 ppm

Remarks:

Corresponding to test material intakes of 5.3 mg/kg bw/day (males) and 6.6 mg/kg bw/day (females)

Dose / conc.:

600 ppm

Remarks:

Corresponding to test material intakes of 32.0 mg/kg bw/day (males) and 39.9 mg/kg bw/day (females)

Dose / conc.:

1 200 ppm

Remarks:

Corresponding to test material intakes of 64.6 mg/kg bw/day (males) and 81.7 mg/kg bw/day (females)

No. of animals per sex per dose:

Treatment groups each contained fifty two males and fifty two females.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The doses were selected by the sponsor by reference to studies with the test material.
A dose of 1 200 ppm in diet was considered to be sufficient to establish an MTD. This dose was expected to be sustainable throughout the study but was anticipated to cause peroxisome proliferation.
The mid dose was 600 ppm. This follows the convention for carcinogenicity studies of a dose at half MTD and served as a fallback should the high dose provoke excessive toxicity. Some peroxisome proliferation was anticipated at this dose.
The low dose was 100 ppm. This was expected to confirm the lack of effect at this level. No peroxisome proliferation was anticipated at this dose.

- Rationale for animal assignment: The animals were randomly allocated to the cages. This procedure ensured that each litter was equally represented in all dose groups (including controls).
Allocation was done in two batches. The animals were distributed amongst the four experimental groups after ensuring that any litters containing unhealthy individuals and litters containing individuals at the extreme of the weight range were excluded from the randomisation procedure. Males and females were allocated separately. Cards were numbered 1-x where x was the number of litters. The cards were shuffled and a card placed on the cage of each litter to give the order of allocation of the litters to the replicates. Allocation from within the litters was also at random. This was done by using Latin square permutations of the numbers 1 to 4. A rat was picked and allocated to the group and cage indicated by the number in the Latin square sequence and ear-punched with the lowest available number for that cage. This procedure was repeated until all cages in all replicates contained one rat. The procedure was then repeated until each cage contained four rats.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of the study, all rats were examined to ensure that they were normal. Cage-side observations which included recording any changes in clinical condition or behaviour were made twice daily. Detailed clinical observations, included the finding of ‘no abnormalities detected’ were recorded weekly, generally at the same time that the bodyweights were recorded. Any rats requiring euthanasia were killed and examined post mortem. Any rats found dead were examined post mortem as soon as possible after death.

DERMAL IRRITATION: No

BODY WEIGHT: Yes
- Time schedule for examinations: The bodyweight of each rat was recorded immediately before feeding of the experimental diets commenced and then on the same day, where practicable, every subsequent week for weeks 2 - 15 of the study and then every 2 weeks until termination. All rats were weighed at termination.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption for each cage of rats was recorded for the first 14 weeks of the study, week 16 and thereafter every fourth week. Food consumption was calculated, at weekly intervals, as a mean value (g food/rat/day) for each cage.

FOOD EFFICIENCY: Yes
- The food utilisation value per cage was calculated as the bodyweight gained by the rats in the cage per 100g of food eaten for weeks 1 - 4, 5 - 8, 9 - 13 and 1 - 13 (this being the major growth phase).

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At weeks 53 and again at week 79 blood was collected from the tail vein of all rats to produce a blood smear. These were not examined.
At scheduled termination of the study, all surviving rats were bled by cardiac puncture and 1.2 mL of blood was taken into a tube containing EDTA as anticoagulant. A differential white cell count was performed. A blood film was prepared for all animals at scheduled termination and examined where the automated analysis results suggest this was necessary.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All surviving rats.
- Parameters checked: Neutrophils, Lymphocytes, Monocytes, Eosinophils and Basophils.

CLINICAL CHEMISTRY: Yes
- Animals fasted: No data
- How many animals: Samples for biochemistry were taken from 12 animals per sex and group except for group 2 females where 11 samples were taken and group 3 females where 13 samples were taken.
- Parameters checked: Liver biochemistry

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Termination: Except for any found dead, the rats, including any killed prematurely, were killed by over exposure to halothane Ph. Eur. vapour, followed by exsanguination, by cardiac puncture.
- Organ weights: From all animals surviving to scheduled termination, the following organs were removed, trimmed free of extraneous tissue and weighed: Adrenal glands, liver, brain, ovaries, epididymides, spleen, heart, testes, kidneys, uterus including cervix. Paired organs were weighed together.
- Samples for electron microscopy were taken from 12 animals per sex and group except for group 2 females where 11 samples were taken and group 3 females where 13 samples were taken.
- Samples were processed by hand by post-fixing in 1 % osmium, dehydration in dimethoxypropane and propylene oxide and infiltration and embedding in araldite resin. Embedded samples were not examined further.

- All animals were examined post mortem. This involved an external observation and a careful internal examination of all organs and structures.
- The following tissues were examined in situ, removed and examined and fixed in an appropriate fixative:
gross lesions including masses, nose, adrenal gland, oesophagus, aorta, ovary, brain (cerebrum, cerebellum and brainstem), oviduct, bone - femur (including knee joint), pancreas, bone marrow - femur, parathyroid gland, caecum, Peyers patches, cervix, pharynx, colon, pituitary gland, duodenum, prostate, epididymis, rectum, eyes (retina optic nerve), salivary gland, ex-orbital lachrymal gland, seminal vesicle, Harderian gland, skin, heart, spinal cord (cervical, thoracic, lumbar), ileum, spleen, jejunum, sternum, kidney, stomach, larynx, testis, liver, thymus, lung, thyroid gland, lymph node - cervical, trachea, lymph node - mesenteric, urinary bladder, mammary gland - (females only), uterus, nerve - sciatic ,voluntary muscle.

HISTOPATHOLOGY: Yes
The following were submitted for histology:
- All tissues from all animals which were killed or died before scheduled termination after 104 weeks.
- All tissues from control and high dose (1 200 ppm) animals surviving to scheduled termination.
- Livers and all macroscopically abnormal tissues including masses from intermediate dose animals (100 and 600 ppm) surviving to scheduled termination.
Tissues for histology were routinely processed, embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin.

Microscopic examination
The following tissues were examined by light microscopy.
- All tissues from all animals which were killed or died before scheduled termination after 104 weeks.
- All tissues from control and high dose (1 200 ppm) animals surviving to scheduled termination.
- Livers and all macroscopically abnormal tissues including masses from intermediate dose animals (100 and 600 ppm) surviving to scheduled termination.

Other examinations:

Peroxisome Proliferation
A total of 96 frozen rat liver samples previously stored at approximately -70 °C (the Test Material) were tested. This set of samples comprised 4 dose groups (control, 100 ppm, 600 ppm and 1 200 ppm) with 12 male and 12 female animals per group. Six livers from each sex at each dose level were assayed.
Livers were weighed and scissor-minced in ice-cold 1.15 % (w/v) KCl. A 10 % (w/v) homogenate was prepared in SET buffer (0.25 M Sucrose, 5 mM EDTA and 2 0mM TrisHCl, pH 7.4). Heavy pellets were prepared following differential centrifugation (at 2 000 rpm and 11 500 rpm) and were re-suspended in SET buffer at a value of 1 g of original liver/mL.
Cyanide-insensitive acyl CoA oxidation activity was determined spectrophotometrically in freshly prepared samples of liver heavy pellet, using palmitoyl CoA as a substrate, using a modification of the method of Bronfmann M et al. (1979).
Results were expressed as nmol NAD+ reduced/min/mg protein.
The protein concentration of liver heavy pellets was determined in aqueous solutions using a modification of the method of Lowry et al. (1951).
Statistical comparisons between treated and control groups have been undertaken for all numerical data sets.

Statistics:

All data were evaluated using the MIXED procedure in SAS (2004).
(Also see "Any other information on materials and methods incl. tables" for further information.)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were more males in the 1 200 ppm group with dermal or subcutaneous masses than in the control or intermediate dose groups. Other findings are of a type and incidence commonly seen in rats and are considered to be unrelated to administration of the test material.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Survival was good with at least 80 % of males and 70 % of females surviving to scheduled termination in the control, 600 and 1 200 ppm groups. Survival was lower in both males and females in the 100 ppm group, 69 % in males and 58 % in females.
There were no treatment related alterations in the factors which were considered contributory to death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of females in the 1 200 ppm group were lower than controls throughout the study. The maximum difference was 18 % below group mean control weight. Body weights of males in the 1 200 ppm group were slightly reduced during the first year of the study. The maximum difference was 4 % below group mean control weight.
Body weights of females in the 600 ppm group were reduced for most of the study; the maximum difference from control group mean bodyweight was 7 %.
There was no evidence of a reduction in body weight in males in the 100 or 600 ppm groups or females in the 100 ppm group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of females in the 1 200 ppm group was reduced throughout the study. The maximum difference was approximately 10 % below control. Food consumption of males in the 1 200 ppm group was statistically significantly below control on some occasions during the first 13 weeks of the study, thereafter it was similar to control.
For females in the 600 ppm group there was a trend towards lower food consumption in the first year of the study but the differences from control are small and achieved statistical significance on only a few occasions. There was no consistent effect on food consumption in males in the 600 ppm group.
There was no effect on food consumption or food utilisation in males or females in the 100 ppm group.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food utilisation was less efficient in the 1 200 ppm group overall (weeks 1 - 13) and for all intervals in females and for weeks 5 - 8 and 9 - 13 in males.
Food utilisation was lower in females in the 600 ppm group for weeks 9 - 13 and overall.
There was no effect on food consumption or food utilisation in males or females in the 100 ppm group

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There was no evidence of a treatment-related effect on the differential white cell count of treated rats.
Males 62 and 83 (both 100 ppm group) had abnormally high white cell counts. Both animals had pathology findings to account for the abnormal white cell values; 62 had a tubular adenocarcinoma affecting the lymph glands and number 83 had lymphoid proliferation in the spleen.

Clinical biochemistry findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weights were increased in females in the 1 200 ppm group. Adjusted weights were 10 % higher than control. However liver weights in males in the 600 and 1 200 ppm groups were lower than control, by 4.6 and 5.3 % respectively.
Kidney weights were higher than control in all treatment groups, although the difference was not clearly dose-related. The mean value in the 1 200 ppm male group was statistically significantly higher than control after exclusion of abnormally high values for animal numbers 62 and 77 (both in the 100 ppm group and with a kidney or an adrenal/kidney mass). Weights were 10.6, 17.1 and 16.7 % higher than control in the 100, 600 and 1 200 ppm male groups respectively based on adjusted values in exclusion tables. Kidney weights were higher than control in females in the 600 and 1 200 ppm groups by 11.9 and 8.8 % respectively based on adjusted values.
Weights of adrenals, brain, epididymides, heart, ovaries, spleen, testes and uterus including cervix were similar in treated and control animals.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The number of animals recorded with a subcutaneous mass was slightly higher than control in males in the 600 and 1 200 ppm groups but the incidence was lower than control in females in the 1 200 ppm group. In animals surviving to termination the incidence was higher than control only in males in the 1 200 ppm group, but lower in females in the 100 and 1 200 ppm groups. Due to these variations in incidence the higher incidences of subcutaneous masses are considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Significant treatment related effects were recorded in the liver of males and females in the 600 and 1 200 ppm groups. The changes were more pronounced in females. Significant changes were also seen in the kidney of females in the 1 200 ppm group.
Changes in the liver consisted of the presence of hyalinization (defined by a ground glass eosinophilic appearance of hepatocytes), predominantly in centri-lobular regions, which is characteristic of peroxisome proliferators. It was observed in 37 out of 52 females in the 1 200 ppm group but only in three males in this dose group. This change was not seen at lower doses.
In addition, an increase in pigment deposition (within macrophages and centri-lobular hepatocytes) was observed in similar numbers of animals in the 1 200 ppm group but also in a small number of females in the 600 ppm group. The incidence of fat recorded within the liver was decreased in both males and females in the 600 and 1 200 ppm groups. Hypertrophy of liver cells was recorded in approximately 25 % of females in the 1 200 ppm group.
There was a decreased incidence of chronic progressive nephropathy in the kidneys of males in the 1 200 ppm group compared to control. This finding is considered not to be adverse. In females in the 1 200 ppm group there was a higher incidence of a number of findings in the kidneys compared to control (intratubular microlithiasis, tubular basophilia/dilatation and vascular ectasia of the renal pelvis). These changes were minimal in extent and were not seen as part of a pattern in the same animals.
A small number of other findings were also altered in incidence in the 1 200 ppm group but are considered unlikely to be treatment-related. These were an increased incidence of pituitary cysts and vascular ectasia of the adrenal in males, haemosiderin in the spleen in females and mononuclear cell infiltration within the Harderian gland of both sexes. There was also a decreased incidence of increased extra medullary haemopoiesis in the spleen, congestion/haemorrhage in the thymus, acinar cell atrophy in the pancreas and secretion and ectasia of the mammary gland in females.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There was a slightly higher incidence of lipoma (benign fatty tumour) of the subcutis in males in the 1200 ppm group. The incidence was 0, 0, 1 and 4 in the control, 100, 600 and 1 200 ppm groups respectively. This was statistically significant when analysed by the Peto trend test for groups 1 to 4, but not for groups 1 to 3.
There were no differences in the intergroup comparison of overall tumour incidences in any group.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Peroxisome Proliferation
Dietary administration of the test material to rats for two years resulted in dose-dependent increases in hepatic peroxisomal β-oxidation as determined by measurement of cyanide-insensitive palmitoyl CoA (PCO) oxidation. Although this response was significantly different from control animals at 600 ppm in female rats and 1 200 ppm for both sexes, the test material increased PCO in both sexes by less than 2-fold, and was thus a minimal increase in peroxisomal enzyme activity.

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Critical effects observed:

no

Mean Dose Received Test Material/kg/day

	Dietary Concentration (ppm)
	100	600	1 200
Males	5.3	32.0	64.6
Females	6.6	39.0	81.7

 

Historical Control Data

Subcutaneous Tissue: Lipoma (BENIGN)

PR1248 Start August 2002, Incidence: Males 2/52

PR1321 Start January 2005, Incidence: Males 0/52

Conclusions:

Under the conditions of the study, the test material is concluded not to be carcinogenic.

Executive summary:

The carcinogenicity of the test material was assessed according to OECD Test Guideline 451 and in compliance with GLP.

Groups of 52 male and 52 female HsdRCCHan:WIST rats were fed diets containing 0 (control), 100, 600 or 1 200 ppm test material. The surviving animals continued to termination after 104 weeks on test.

Clinical observations, bodyweights and food consumption were measured. All animals, including any found dead or killed prematurely were examined post mortem and tissues were taken for subsequent histopathology examination. At scheduled termination, cardiac blood samples were taken for haematology (white cell count) and selected organs were weighed.

At least 80 % of males and 70 % of females in the control, 600 and 1 200 ppm groups survived to two years. Survival was lower in both sexes in the low dose (100 ppm) group. There were more males in the 1 200 ppm group with dermal or subcutaneous masses than in the control or intermediate dose groups.

Body weights of females receiving 1 200 ppm were reduced compared to controls throughout the study; the maximum difference from control was 18 %. Body weights of males in the 1 200 ppm group were slightly reduced during the first year of the study. Body weights of females receiving 600 ppm were reduced for the majority of the study; the maximum difference from control was 7 %. There was no evidence of a reduction in body weight in males in the 100 or 600 ppm groups of females in the 100 ppm group.

Food consumption of females in the 1 200 ppm group was reduced compared to controls throughout the study. Food consumption of males in the 1 200 ppm group was slightly reduced during the first 13 weeks of the study. The food utilisation during the rapid growth phase was lower than control values in males and females in the 1 200 ppm group and females in the 600 ppm group.

There was no evidence of a treatment-related effect on the differential white cell count of treated rats.

Liver weights were increased compared to controls in females in the 1 200 ppm group. Kidney weights were higher than control in all treatment groups, although the difference was not clearly dose-related.

Hyalinization and pigment deposition and decrease in fat were recorded in the livers of both sexes receiving 1 200 ppm. Changes in the livers of the 600 ppm group were decreased fat in both sexes and pigment deposition in a small number of females. In the kidneys of rats receiving 1 200 ppm there was a decrease in the incidence of chronic progressive nephropathy in males, a higher incidence of intratubular microlithiasis, tubular basophilia/dilatation and vascular ectasia of the pelvis in females.

There was no effect of the test material on the number of tumour bearing animals or overall tumour incidence. The only statistically significant difference from control was a slightly higher incidence of lipoma (benign fatty tumour) of the subcutis in males in the 1200 ppm group. The incidence was 0, 0, 1 and 4 in the control, 100, 600 and 1 200 ppm groups respectively. There were no treatment-related factors that contributed to the death of the animals.

There was no effect of the test material on survival.

Body weights and food consumption were reduced in rats receiving 1 200 ppm test material. In addition there were treatment related microscopic changes in the liver and kidney.

Body weights were reduced in females in the 600 ppm group and there were some minor changes in the livers of male and females.

There were no treatment-related effects at a dose of 100 ppm test material.

The test material had no effect on the overall number of animals with tumours.

There was one statistically significant difference from control which was a slightly higher incidence of lipoma (benign fatty tumour) of the subcutis in males in the 1 200 ppm group. There was no histological change in the skin or subcutaneous tissue in other rats, or in other tissues in which fat is stored, to suggest an effect of the compound on these tissues.

Under the conditions of the study, the test material is concluded not to be carcinogenic.

Reference 2

Endpoint:

carcinogenicity, other

Remarks:

QSAR prediction (carcinogenicity multi cell)

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

results derived from a (Q)SAR model, with limited documentation / justification

Justification for type of information:

1. SOFTWARE: Lazar Toxicity Prediction

2. MODEL: Not reported

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
- Test material: O=C(O)C(Oc(c(cc(c1)Cl)C)c1)C
- Impurities:
2-Propenoic acid, 2-chloro- (also known as 2-chloro acrylic acid): O=C(O)C(=C)Cl
(5-methylbenzene-1,3-dicarboxylic acid): Cc1cc(cc(c1)C(=O)O)C(=O)O
(2-methylpropyl 2-(4-chloro-2-methylphenoxy) propanoate): Cc1c(ccc(c1)Cl)OC(C)C(=O)OCC(C)C
(2-methylpropyl 2-(4-chloro-2-methylphenoxy)propanoate): O=C(O)C(Oc(c(cc(c2)Cl)C)c2)COC(=O)C(Oc(c(cc(c1)Cl)C)c1)C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
The easiest and most consistent way of applying QSAR models is to use ready-made software that implements the models via a user interface (European Commission, 2010). The Lazar is an open-source software programme that makes predictions of toxicological endpoints in a training set (http://lazar.in-silico.de). This model has been used to predict the potential mutagenicity and carcinogenicity of the test material and selected impurities.
Lazar searches the training dataset for similar compounds (neighbors) and calculates the prediction from their measured activities. Lazar calculates predictions using:
· A majority vote (weighted by compound similarity) for classification.
· A local QSAR model based on neighbors for regression.
Significant fragments are highlighted in the structure display as follows:
- Features that occur predominately in compounds with activity: "Non-carcinogen".
- Features that occur predominately in compounds with activity: "Carcinogen".
- Regions, where fragments from different classes overlap inert parts.
Note that predictions are based on the measured activities of neighbours. Significant fragments are solely used to determine activity specific similarities of neighbours.
Key to interpretation of results from simulations of Lazar software tool:
- Confidence: Indicates the applicability domain of a model. Predictions with a high confidence can be expected to be more reliable than predictions with low confidence. Confidence values may take any value between 0 and 1. For most models confidence > 0.025 is a (hard) threshold to distinguish between reliable and unreliable predictions.
- Measured activity: Experimental result(s) from the training dataset.

5. APPLICABILITY DOMAIN
Not reported.

6. ADEQUACY OF THE RESULT
For all predictions the model confidence is > 0.025 i.e. above the cut off to distinguish between reliable and unreliable predictions. This means that, for all substances, the predictions are deemed as reliable.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline required

Principles of method if other than guideline:

QSAR prediction

GLP compliance:

no

Remarks:

Not applicable

Results of Lazar Prediction

Chemical	SMILES	DSSTOX Rodent Carcinogenicity Multi Cell_Call
Test material	O=C(O)C(Oc(c(cc(c1)Cl)C)c1)C	Non-carcinogen (Confidence: 0.0876)
2-Propenoic acid, 2-chloro- (also known as 2-chloro acrylic acid)	O=C(O)C(=C)Cl	Non-carcinogen (Confidence: 0.0651)
(5-methylbenzene-1,3-dicarboxylic acid)	Cc1cc(cc(c1)C(=O)O)C(=O)O	Non-carcinogen (Confidence: 0.0865)
(2-methylpropyl 2- (4-chloro-2-methylphenoxy) propanoate)	Cc1c(ccc(c1)Cl)OC(C)C(=O)OCC(C)C	Non-carcinogen (Confidence: 0.0831)
(2-methylpropyl 2- (4-chloro-2-methylphenoxy) propanoate)	O=C(O)C(Oc(c(cc(c2)Cl)C)c2)COC(=O) C(Oc(c(cc(c1)Cl)C)c1)C	Non-carcinogen (Confidence: 0.0878)

 

Conclusions:

All substances examined were predicted to be non-carcinogenic with the restraints of the models.

Executive summary:

Using the QSAR models within the Lazar software tool to predict the mammalian toxicity and mutagenicity for test material and selected impurities, the results show that;

- For all predictions the model confidence is > 0.025 i.e. above the cut off to distinguish between reliable and unreliable predictions. This means that, for all substances, the predictions are deemed as reliable.

- All substances examined (i.e. the test material and 4 impurities) are predicted to be non-carcinogenic with the restraints of the models.

Reference 3

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

22 December 1992 to 26 July 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

1981

Deviations:

yes

Remarks:

, for high dose animals the study was already terminated after 12 months, due to severely affected body weight gain.

Qualifier:

according to guideline

Guideline:

EU Method B.32 (Carcinogenicity Test)

Version / remarks:

EC Commission Directive 87/302/EEC of 18 November,·1987; Part B: Methods for the determination of Toxicity; Carcinogenicity Test; Official Journal of the European Communities No. L 133, pp. 32-36, 1988.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-2 (Carcinogenicity)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

Mice were selected since this species of mammal is internationally acknowledged, and extensive experience with this species is available. Mice are also proposed as the appropriate test animals by EPA and by OECD. B6C3Fl/CrlBR mice were selected since there is extensive experience in the laboratory with this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: The animals were supplied at an age of 42 days. At the start of the administration period (day 0) the mice were 50 - 51 days old.
- Weight at study initiation: The body weight of the males was in the range of 19.3 - 25.8 g (group mean: 22.4 g) and the body weight of the females was in the range of 17.2 - 22.2 g (group mean: 19.5 g).
- Housing: The mice were housed singly in type MI Makrolon cages with mesh were tops (floor area about 200 cm²).
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: On the day of arrival the animals were subjected to an 8-day or 9-day acclimatisation period during which they received ground food and drinking water ad libitum.

DETAILS OF FOOD AND WATER QUALITY:
- Food analyses: The food used in the study was assayed for chemical as well as for microbiological contaminants.
- Drinking water analyses: The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and the Technical Services of BASF Aktiengesellschaft as well as for the presence of microorganisms by a contract laboratory.
- Bedding analyses: The bedding is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 % relative humidity.
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 06.00 a.m. - 06.00 p.m., 12 hours dark from 06.00 p.m. - 06.00 a.m.).
Technical failures led to transient, minor deviations from these ranges. These had, however, no influence on the results of the study.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: The mixtures were prepared in monthly intervals.
- Mixing appropriate amounts with: For each concentration, the test material was weighed out and thoroughly mixed with a small amount of food in a beaker. Subsequently, a premix was prepared in a BOSCH household mixer by adding an appropriate amount of food and mixing for about 3 min. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations, and mixing was carried out for about 10 minutes in a GEBR. LÖDIGE laboratory mixer.

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC with UV-Detection

Details on analytical verification of doses or concentrations:

The stability of the test material in the diet over a period of 33 days at room temperature was proven prior to the study.
The homogeneity of the test material preparation was proven at the start of the study in each 3 samples of the high concentration (2 500 ppm) and the low concentration (25 ppm).
Concentration control analyses were carried out in samples from all dose groups drawn at the start of administration period, thereafter in intervals of about 3-months during the study, and 4 weeks prior to start of necropsy.

HPLC with UV-Detection
Storage conditions until Analyses: Freezer
In the context of a toxicity test of the test material on mice homogeneity tests and concentration control analyses of the test material in feed had to be conducted. The concentration control analyses yielded mean values between 23 mg/kg and 26 mg/kg for the 25 mg/kg samples, between 229 mg/kg and 258 mg/kg for the 250 mg/kg samples and between 2 382 mg/kg and 2 484 mg/kg for the 2 500 mg/kg samples. These are between 90.9 and 104.6 % of the theoretical values for the 25 mg/kg samples (resp. 91.7 - 103.1 % and 95.3 - 99.4 % for the 250 resp. 2 500 mg/kg samples). An inhomogeneous 25 mg/kg sample was found to be 40.4 resp. 29.7 mg/kg. This result was replaced by the concentration of the retain sample CP23321 with a mean value of 25.7 mg/kg. The results of the homogeneity test varied between 22 mg/kg and 25 mg/kg with a relative standard deviation of ± 6.7 % for the 25 mg/kg samples and between 2 524 mg/kg and 2 620 mg/kg with a relative standard deviation of ± 2.1 % for the 2 500 mg/kg samples.
These results confirm the nominal concentrations and the homogeneity of the samples.

Duration of treatment / exposure:

18 months; animals of the high dose group were killed after 12 months due to severe body weight impairment.

Frequency of treatment:

Administered daily in the diet.

Post exposure period:

None

Dose / conc.:

25 ppm (nominal)

Dose / conc.:

250 ppm (nominal)

Dose / conc.:

2 500 ppm (nominal)

No. of animals per sex per dose:

50 per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: In literature the LD50 value in mice was reported as 300 mg/kg by intraperitoneal application.
In a 4-week feeding study mice received the test material in dietary concentrations of 100, 300, 900 and 2 700 ppm. The following findings were obtained and assessed or discussed as possibly being test material-related:
2 700 ppm: Increase in alkaline phosphatase and cholesterol in both sexes; decrease in platelets in the females; increase in absolute and relative liver weights as well as a central hypertrophy of the hepatocytes in both sexes.
100, 300 and 900 ppm: No test material-induced changes.

Due to these findings a supplementary 4-week range-finding feeding study was carried out to determine the target organs and to find the possible MTD for the long-term study. In the chosen concentrations of 7 000; 4 500 and 2 700 ppm the following possibly test material-related findings were obtained:
7 000 ppm: Retarded body weight gain with decreased body weights in both sexes at the end of the study (27 % in females and 32 % in males); reduced general state in both sexes; death of 1/5 females; increase in alkaline phosphatase, alanine; aminotransferase and albumin in both sexes; increase in aspartate aminotransferase in the males; decrease in leukocytes, lymphocytes and platelets in both sexes; decrease in globulins (males) and glucose (females); increased absolute and relative liver weights in both sexes and adrenal weights in males; diffuse hypertrophy of hepatocytes, degenerative liver cell changes; damages and cytoplasmatic eosinophilia of the hepatocytes as sign of the proliferation of the peroxisomes (both sexes); massive proliferation of the peroxisomes in the hepatocytes and additionally hyperplasia of the mitochondric crista as well as changes in their organisatoric structure.
4 500 ppm: Retarded body weight gain with decreased body weights in both sexes at the end of the study (13 % in females and 11 % in males); increase in alkaline phosphatase in both sexes; increase in alanine aminotransferase and albumin in females; decrease in the platelets (females); increased absolute and relative liver weights as well as diffuse hypertrophy of liver cells with cytoplasmatic eosinophilia as sign of proliferation of peroxisomes (in both sexes).
2 700 ppm: Minimal retarded body weight gain with slight decreased body weights in both sexes at the end of the study (5 % in females and 6 % in males); increase in alkaline phosphatase in both sexes; increase in albumin (females); increased absolute and relative liver weights and central hypertrophy of liver cells in both sexes.

In a 3-month feeding study concentrations of 600; 1 200; 2 400 and 4 500 ppm were chosen. The following possibly test material-related findings were obtained (as no histological examinations were carried out, only the results of macroscopy are available):
4 500 ppm: Retarded body weight gain in both sexes with reduced body weights at the end of the study of about 10 % in the males and about 19 % in the females; reduced haemoglobin, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration in both sexes as well as reduced red blood cells and haematocrit in females and mean corpuscular volume in males; increased alkaline phosphatase in both sexes and alanine aminotransferase in females increased creatinine in both sexes and glucose in males as well as total bilirubin, cholesterol and urea in females; decreased total protein and globulins in males and triglycerides in females; liver enlargement and discolouration and in 4/10 animals liver foci in both sexes; absolute and relative increased liver weights in both sexes.
2 400 ppm: Reduced feed consumption (max. 20 %) in the females; decreased haemoglobin, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration in both sexes; increased alkaline phosphatase in both sexes and glucose in the males as well as creatinine and cholesterol in the females; decreased total protein and globulin in the males and triglycerides in the females; liver discolouration in both sexes and in 1/10 male and 6/10 females enlargement as well as a liver foci in 1/10 male; absolute and relative increased liver weights in both sexes.
1 200 ppm: Reduced feed consumption (max. 9 and 18 %) in males and females; decreased haemoglobin and triglycerides in the females; increased cholesterol in the females.
600 ppm: Reduced feed consumption (max. 13 %) in the females; decreased triglycerides in the females.

In an additional 3-month feeding study with the optically active test material concentrations of 2 500; 1 000 and 100 ppm were chosen. The following test material-related findings were obtained:
2 500 ppm: Statistically significantly reduced body weight (about 10 % in males and 9 % in females on day 91); statistically significantly reduced body weight change in both sexes; increase in alkaline phosphatase, cyanideinsensitive palmitoyl-CoA-oxidation in liver homogenate, urea and cholesterol in both sexes; decrease in trigylcerides in both sexes; increase in creatinine and glucose in the males; increase in alanine aminotransferase in the females; decrease in haemoglobin, mean corpuscular haemoglobin and globulins in the males; statistically significant reduction of terminal body weights in both sexes; statistically significant increase of the absolute and relative liver weight in both sexes; macroscopically diagnosed dark-brown discolouration of the liver in both sexes; in the liver: eosinophilic cytoplasm of hepatocytes in both sexes; in the kidneys: eosinophilic cytoplasm of tubular epithelial cells in both sexes; decrease of lipid storage in the liver of both sexes.
1 000 ppm: Statistically significantly reduced body weight (about 8 % in males and 9 % in females); statistically significantly reduced body weight change in both sexes; increase in urea in both sexes; decrease in triglycerides in both sexes; increase in creatinine in the males in the liver: eosinophilic cytoplasm of hepatocytes in one female decrease of lipid storage in the liver of both sexes.
100 ppm: Increase in urea in the females; decrease in triglycerides in the females.

Thus, for the present study the following concentrations were chosen:
2 500 ppm as high concentrations with expected toxic effects
250 ppm as intermediate concentration
25 ppm as low concentration and expected no observed adverse effect level

- Rationale for animal assignment: One day prior to the start of the administration period, the male and female mice were allocated to the test groups according to weight. The list of randomisation instructions was compiled by a computer.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once a day the animals were examined for evident signs of toxicity or mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a week the animals were subjected to a comprehensive clinical examination (including palpation)

DERMAL IRRITATION: No

BODY WEIGHT: Yes
- Time schedule: once a week during the first 13 weeks, and thereafter at 4-weekly intervals

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: once a week during the first 13 weeks, and thereafter at 4-weekly intervals
Intake of test material: The mean daily intake of the test material (group means) was calculated based upon individual values for body weight and food consumption, according to the following equation:

(FCx x C) / BWx = Substance intake for day x

Where:
BWx = Body weight on day x [g]
FCx = Mean daily food consumption on day x [g]
C = Concentration in the diet [mg/kg]

FOOD EFFICIENCY:
Food efficiency (group means) was calculated based upon individual values for body weight and food consumption, according to the following equation:

((BWx – BWy) / FCy to x) x 100 = Food efficiency for day x

Where:
BWx = body weight on day x [g]
BWy = bodyweight on day y (last weighing date before day x) [g]
FCy to x = mean food consumption from day y to day x; calculated as mean daily food consumption on day x, multiplied with the number of days from day y to day x [g]

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was taken by tail puncture during the administration period by decapitation at the end of the study (18 months) or after 12 months from all animals killed in extremis. Blood was taken from non-fasted, unanesthetised animals and by decapitation at the end of the study from fasted, anesthetised animals for differential blood smears. Only the blood smears of the control groups and 250 ppm groups were evaluated. Furthermore, blood smears were prepared and evaluated from all animals - except of the highest dose groups - killed in extremis during the study.
- Parameters checked: The differential blood count was evaluated visually. The data were transferred to the computer. differential blood counts stained according to Wright; microscopic evaluation.

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. At the end of the 18-month administration period all surviving animals were sacrificed after a fasting period (withdrawal of food for about 16 - 20 hours).
The animals of test group 3 were sacrificed using CO2. No further examinations were carried out with these animals.
The animals of test groups 0 - 2 were sacrificed by decapitation under CO2 anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The animals which died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after death and assessed by gross pathology.

- Organ weights: The weight of the anesthetised animals as well as the weights of liver, kidneys, adrenal glands, testes, ovaries, and brain from all animals sacrificed at scheduled dates were determined.

HISTOPATHOLOGY: Yes. The following organs or tissues were fixed in 4 % formaldehyde solution:
- all gross lesions
- brain
- pituitary gland
- thyroid glands with parathyroid glands
- thymus
- trachea
- lungs
- heart
- aorta
- salivary glands (mandibular and sublingual glands)
- liver
- spleen
- kidneys
- adrenal glands
- pancreas
- testes/ovaries
- uterus/vagina/oviducts
- accessory genital organs (epididymides, prostate, seminal vesicle)
- skin
- esophagus
- stomach (forestomach and glandular stomach)
- duodenum, jejunum, ileum
- cecum, colon, rectum
- urinary bladder
- lymph nodes (mesenteric and mandibular)
- female mammary gland
- skeletal muscle
- sciatic nerve
- sternum with marrow
- bone marrow (femur)
- eyes
- femur with knee joint
- spinal cord (cervical, thoracic and lumbar)
- extraorbital lacrimal glands
- gall bladder
After the organs were fixed, the processing, the examination by light microscopy and the evaluation of findings was performed.
All these organs of all animals that died intercurrently or were sacrificed in a moribund state were evaluated histopathologically.
Of one kidney, of one male, the following special stain was performed: Oil Red O (ORO, for lipids).
A correlation between gross lesions and histopathological findings was performed.

Statistics:

Statistics of clinical examinations: Means and standard deviations of each test group were calculated for the variables food consumption, body weight, body weight change, food efficiency and test material intake.
For food consumption, body weight, body weight change and food efficiency: Parametric one-way analysis using the Ftest (ANOVA) (two-sided). If the resulting p-value was equal or less 0.05, a comparison of each group with the control group using the Dunnett'sy est (two-sided) was performed for the hypothesis of equal means.
Means and standard deviations of each test group were calculated for several parameters.
Statistics of pathology: Means and standard deviations were calculated for the variables of terminal body weight and of absolute and relative organ weights (related to terminal body weight) of the animals in each test group. Further statistical evaluations were carried out:
Weight parameters: A comparison of each group with the control group using Dunnett’s test (two-sided) was performed for the hypothesis of equal means.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 2 500 ppm, the finding 'palpable mass in the abdomen' occurred in both sexes more frequently than in the controls.
In the other treatment groups, all clinical findings occurred equally distributed between control and treatment groups.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

In the 2 500 ppm group, 1 male had died by day 280. However, as body weights were severely impaired in males and females, the animals of this group were killed prematurely on request of the sponsor without any further investigations.
The mortality in the remaining test groups up to day 546 (i.e. 18 months) was:
Control group: 2 % males and 2 % females
25 ppm group: 0 % males and 4 % females
250 ppm group: 0 % males and 6 % females
Thus, the test material did not affect the mortality of the animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males and females receiving 2 500 ppm, body weights were severely impaired, being statistically significant from day 7 (males) or 21 (females) onwards. The values in males on day 343 were about 27 % below controls and in females on day 315 were about 37 % below controls; corresponding body weight change values were about 58 % (males) and 71 % (females) below controls. This was clearly test material related.
In the 250 and 25 ppm groups, statistically significant deviations from controls (either increases or decreases) were seen only sporadically and without dose-response relationship. They were therefore assessed as being incidental.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related effects were obtained. All statistically significant deviations (either increased or decreased values) were assessed as being incidental and not biologically relevant, due to the slightness of the deviation and the lack of a dose response relationship.
During the study there was spilling of food by the mice in all groups, irrespective of the dose. The values given can therefore only be used to estimate the food consumption and do not represent exact food intake. (In order to assure equally spaced intervals for calculation, only the values of days 7, 35, 63, 91 and days 119 to 539 (in test group 3 to day 315), were taken into account for calculation of test material intake.) The calculated test material intake values were as follows:
- Test group 1 (25 ppm): 4 mg/kg bw (males), 4 mg/kg bw (females), 4 mg/kg (combined)
- Test group 2 (250 ppm): 40 mg/kg bw (males), 46 mg/kg bw (females), 43 mg/kg (combined)
- Test group 3 (2 500 ppm)*: 592 mg/kg bw (males), 732 mg/kg bw (females), 662 mg/kg (combined)
* Note only up to day 315

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

In the 2 500 ppm group, food efficiency was generally lower than in controls, statistically significant on several days. This was assessed as being test material-related.
No test material-related effects were obtained in the 250 and 25 ppm groups. All statistically significant deviations (either increased or decreased values) were assessed as being incidental and not biologically relevant, due to the slightness of the deviation and the lack of a dose response relationship.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

After 12 and 18 months of test material administration no treatment-related changes were observed in the white blood cells of the male and female mice given 250 ppm of the test compound.
Red blood cells: At the 12 months interval and at the end of the study no treatment-related changes were seen in the erythrocyte morphology of the animals of the 250 ppm group.
Prematurely killed animals: Changes in white and red blood cells were observed in three female animals of the 250 ppm group killed in extremis. However, these changes are considered to be spontaneous, incidental or age-related. Accordingly, these findings are not associated with the test material administered.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute organ weights: The mean weights of adrenal glands in male mice were significantly decreased in dose groups 1 (25 ppm) (p ≤ 0.01, -18.9 %) and 2 (250 ppm) (p ≤ 0.01, -14.95 %). There was no dose-response relationship.
The mean kidney weight of female mice was significantly increased (p ≤ 0.01, +13.54 %) in dose group 2 (250 ppm).
The mean liver weight was significantly decreased (p ≤ 0.05, -7.65 %) in females of group 1 (25 ppm). The other mean absolute weight parameters did not show significant differences when compared with the control group.

Relative Organ Weights: The mean weights of adrenal glands in male mice were significantly decreased in dose groups 1 (25 ppm) (p ≤ 0.01, -21.05 %) and 2 (250 ppm) (p ≤ 0.01, -15.79 %. There was no dose-response relationship.
The mean kidney weight of female mice was significantly increased in dose group 2 (250 ppm) (p ≤ 0.01, +20.74 %).
The other mean relative weight parameters did not show significant differences when compared with the control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross lesions: Of the various gross lesions noted in treated animals, only a few appeared at a different incidence compared to the control group.
In the liver, different coloured foci occurred in one male of the control group, eighteen males of group 1 (25 ppm), and six males of group 2 (250 ppm). In female animals, different coloured foci were observed in five control mice, in twelve mice of group 1 (25 ppm), and in five mice of group 2 (250 ppm). All these animals were survivors.
A red discolouration of the mesenteric lymph node was only observed in five male mice of group 2 (250 ppm). All these animals were survivors.
All other gross lesions including the macroscopically diagnosed "masses" occurred either sporadically or were equally distributed over the control group and the treatment groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Kidneys: Major non-neoplastic lesion in the kidneys was chronic nephropathy. Chronic nephropathy was characterised by the presence of proteinaceous casts within the tubules, areas of tubular atrophy, regeneration and dilation, and/or interstitial fibrosis. The number of females with chronic nephropathy was increased in the 250 ppm group. Thirteen females of the control group, seven females of group 1 (25 ppm), and 27 females of group 2 (250 ppm) showed this finding. In most of these animals, the degree of severity was minimal (grade 1). In males, the number of animals with chronic nephropathy as well as the degree of severity of this finding were comparable between control and treatment groups.
Small foci of amorphous basophilic or black (H&E) deposits, diagnosed as "calcification", were seen either in the lumen of renal tubules, or in the arterial wall, or in the interstitial tissue, and/or along tubular basement membranes. In females, focal calcification was observed in thirteen animals of the control group, nine animals of group 1 (25 ppm), and in 26 animals of group 2 (250 ppm). In most of these animals, the degree of severity was minimal. In males, the number of animals with focal calcification as well as the degree of severity of this finding were comparable between control and treatment groups.
In most males of the control group and of group 1 (25 ppm), vacuoles of variable size, diagnosed as "lipid vacuoles" were seen in proximal tubular epithelial cells (H&E). Some of these vacuoles, especially the smaller vacuoles stained positive for lipid with Oil Red O (ORO). The large vacuoles were lipid-free. Vacuoles were not observed in top dose males, by light microscopy. The absence of lipid vacuoles in top dose males has no toxicological relevance. All other findings in the kidneys (focal/multifocal lymphoid cell infiltration, cysts, focal ossification, infarction, pyelonephritis, storage nephrosis, and tubular hyperplasia) were either single or few observations or occurred at biologically comparable incidences in treated and untreated animals.

Mesenteric lymph node: In five males of group 2 (250 ppm) macroscopically noted red discolourations were caused by focal angiomatous hyperplasias in three males. For the other two males, there were no histopathological correlations.

Liver: Most of the macroscopically diagnosed different coloured foci turned out to be focal fatty infiltration or foci of cellular alteration, histopathologically. Foci of cellular alterations were classified according to their staining properties as basophilic, eosinophilic, and vacuolated. The clear cell type of focal cellular alterations was not observed in this study. Foci of cellular alterations occurred in nine males and seven females of the control group, in eleven males and four females of group 1 (25 ppm), and in four males and two females of group 2 (250 ppm). Most of these foci were basophilic. Foci of cellular alterations, as well as all other findings in the liver did not show biologically relevant differences between treated and untreated animals.

Glandular stomach: In females, focal glandular hyperplasia was observed in seven control animals and two animals of group 2 (250 ppm). Two control males and one male of group 2 (250 ppm) showed this finding. The increased occurrence of focal glandular hyperplasia in control females is considered as incidental.

Thymus: In males, lymphoid hyperplasia was observed in six control animals and two animals of group 2 (250 ppm). Thirteen control females and five females of group 2 (250 ppm) showed this finding. The increased occurrence of lymphoid hyperplasia in control animals is considered as incidental.

Adrenal glands: All pathological findings in the adrenal cortex and medulla of males were either single or few observations or occurred at biologically comparable incidences. There were no histopathological correlates for the decreased absolute and relative weights of the adrenal glands. Furthermore, the weight decrease of the adrenal glands was not dose related. Therefore, the weight decrease of the adrenal glands in males is considered as incidental.

All other organs: All non-neoplastic findings noted were either single or low observations or they were biologically equally distributed over control and treatment groups.

Decedents: There were no treatment-related histopathological findings that caused an earlier death. Of a total of seven decedents, four animals showed different kinds of tumors in different organs and in three animals, there were no microscopic findings, that could explain the cause of death.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Pancreas: Islet cell adenomas were observed in seven control males and one male and one female of group 2 (250 ppm). All these animals were survivors. Most of these animals additionally showed islet cell hyperplasias. Islet cell adenomas were distinguished from islet cell hyperplasia only by the size of the lesion. The cells were morphologically similar to those in normal islets. The cause for the increased occurrence of islet cell adenomas in control males is not known.

Haemolymphoreticular system: Malignant lymphomas were observed in six control females, in two females of group 1 (25 ppm), and in one female of group 2 (250 ppm). All these animals were survivors. The cause for the increased occurrence of malignant lymphomas in control females is not known.

Other organs: Most of the observed tumors were noted in the liver and in the lungs:
Hepatocellular adenomas were seen in three control males, in four males of group 1 (25 ppm), in five males of group 2 (250 ppm), in one female from each of the control group and group 1 (25 ppm), and in three females of group 2 (250 ppm). Hepatocellular carcinomas occurred in seven control males and three control females, in six males and two females of group 1 (25 ppm) and in eight males and five females of group 2 (250 ppm). One hepatocellular carcinoma of a control female metastasised in the lungs. One male of the control group and of group 1 (25 ppm) showed a hemangioma in the liver. All these animals were survivors.
Adenomas were observed in the lungs of each five males of the control group and of group 1 (25 ppm), in three males of group 2 (250 ppm), and two control females. Carcinomas were diagnosed in the lungs of three males of group 1 (25 ppm), five males of group 2 (250 ppm), one control female, two females of group 1 (25 ppm), and of one female of group 2 (250 ppm). All these animals were survivors.
All other neoplastic findings noted were single or low observations.
All neoplastic findings were considered as incidental and not treatment related.

Tumor bearing animals: The number of animals with neoplasms, with one or two and more neoplasms, the number of animals with benign, malignant, and metastasised neoplasms, as well as the total number of primary, benign, malignant, and metastasised neoplasms was either comparable between control and treated animals or the tumors were single or low observations. In females, the number of animals with systemic neoplasms was higher in the control group when compared with the 250 ppm group. The cause for the increased occurrence of systemic neoplasms in control females is not known.

Other effects:

not examined

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

25 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Increased absolute and relative kidney weights and increases in number of chronic nephropathies in next higher dose group (250 ppm).

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

250 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No test material-related findings in this dose group.

Critical effects observed:

not specified

Organ and relative organ weights from carcinogenicity testing in mice

	0 ppm (m/f)	25 ppm (m/f)	250 ppm (m/f)	2500 ppm (m/f)
Dose in mg/kg bw/day	-/-	4/4	40/46	592/732
liver weight, g	1.64/1.56	1.51/1.44#	1.59/1.51	n.i.
relative liver weight, %	4.45/3.62	3.94/3.57	4.16/3.75	n.i.
kidney weight, g	0.79/ 0.52	0.86/ 0.51	0.91/0.59##	n.i.
relative kidney weight, %	2.10/ 1.22	2.32/ 1.27	2.48/1.47##	n.i.
adrenal glands weight, mg	7.10/9.51	5.76##/8.79	6.04##/9.09	n.i.
relative adrenal glands weight, %	0.019/0.022	0.015##/0.022	0.016##/0.022	n.i.

n.i.: not investigated       # and ##: 5% and 1% significance level (Dunnett´s test)

Analyses

- Stability analyses: The stability of the test substance in the diet over a period of 33 days at room temperature was verified prior to the study. As the mixtures were stored no longer than this time period, the stability was guaranteed.

- Homogeneity analyses: The homogeneity of the mixtures was verified at 2 500 ppm and 25 ppm.

- Concentration control analyses: The correctness of the concentrations was demonstrated. The recovery rates were in an acceptable range (90.9 % - 104.6 % of the target concentrations.

- Food analyses: On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants the food was found to be suitable. Fed. Reg. Vol. 44, No. 91 of May 09, 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The number of microorganisms did not exceed 10^5/g food.

- Drinking water analyses: On the basis of the analytical findings the diet was found to be suitable. German drinking water regulation (Trinkwasserverordnung, Bundesgesetzblatt, December 05, 1990) served as a guideline for maximum tolerable contaminants.

Conclusions:

Under the conditions of the study the no observed adverse effect level (NOAEL) for systemic toxicity was 25 ppm (about 4 mg/kg body weight) in females and 250 ppm (43 mg/kg body weight) in males. The test material was not oncogenic under the conditions of this study. The no observed adverse effect level (NOAEL) for carcinogenicity was therefore 250 ppm (43 mg/kg body weight).

Executive summary:

The carcinogenicity of the test material was assessed according to OECD Test Guideline 451, EPA OPP 83 -2, EU Method B.32, and in compliance with GLP.

During the study, the test material was administered to groups of 50 male and 50 female B6C3Fl/CrlBR mice at dietary concentrations of 0, 25, 250, 2 500 ppm. The groups receiving 0, 25 and 250 ppm were treated for about 18 months. As 2 500 ppm proved to be too high (as evident by severe body weight impairment), the remaining animals of these dose groups were killed after about 12 months of treatment without further examinations.

Food consumption and body weights were determined once a week during the first 13 weeks, and thereafter at 4-weekly intervals. At least once a day the animals were examined for evident signs of toxicity or mortality. Once a week they were subjected to an additionally comprehensive clinical examination (including palpation).

Blood smears were prepared after 12 months and 18 months, and from all animals killed in extremis. The blood smears of the control groups and the 250 ppm groups were evaluated.

At the end of the 18 months of treatment all animals were subjected to gross-pathological assessment, followed by histopathological examination. The following test material-related findings were obtained after 18 months.

250 ppm (about 43 mq/kg body weight): Significant increase of the absolute and relative kidney weights in females, and increased number of females with chronic nephropathy.

25 ppm (about 4 mq/kg body weight): No test material-related effects.

Thus at 250 ppm chronic nephropathy was seen in females. The no observed adverse effect level (NOAEL) was 25 ppm (about 4 mg/kg body weight) in females and 250 ppm (43 mg/kg body weight) in males. The test material was not oncogenic under the conditions of this study.

Reference 4

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

07 November 1995 to 28 May 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

1981

Qualifier:

according to guideline

Guideline:

EPA OPP 83-2 (Carcinogenicity)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.32 (Carcinogenicity Test)

Version / remarks:

1988

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

The stability of the test substance in the diet over a period of 33 days at room temperature was confirmed prior to the study.

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

Mice were selected since this species of mammal is internationally acknowledged, and extensive experience with this species is available. Mice are also proposed as the appropriate test animals by EPA and by OECD. B6C3F1/CrlBR mice were selected since there is extensive experience in the laboratory with this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: B6C3F1/CrlBR mice
- Age at study initiation: At the start of the administration period (day 0) the mice were 49 days old.
- Weight at study initiation: The body weight of the males was in the range of 18.1 g - 26.2 g (mean weight: 22.2 g) and the body weight of the females was in the range of 15. 7 g - 20. 7 g (mean weight: 18.0 g).
- Housing: Singly in type MI Makrolon cages with mesh wire tops, floor area about 200 cm^2 (Becker & CO, Castrop-Rauxel, Germany).
- Diet and water: Ad libitum
- Acclimation period: On the day of arrival the animals were subjected to an 8-day acclimatisation period during which they received ground food and drinking water ad libitum.

DETAILS OF FOOD AND WATER QUALITY:
- Food analyses: The food used in the study was assayed for chemical and for microbiological contaminants.
- Drinking water analyses: The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and the Technical Services of BASF Aktiengesellschaft as well as for the presence of microorganisms by a contract laboratory.
- Bedding analyses: The bedding is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): No data (fully air conditioned rooms).
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 06.00 a.m. - 06.00 p.m., 12 hours dark from 06.00 p.m. - 06.00 a.m.).

Technical failures led to transient, minor deviations from these ranges. These had, however, no influence on the results of the study.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: The mixtures were prepared in monthly intervals.
- Mixing appropriate amounts with: For each concentration, the test material was weighed out and thoroughly mixed with a small amount of food in a beaker. Subsequently, a premix was prepared in a BOSCH household mixer by adding an appropriate amount of food and mixing for about 3 min. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations, and mixing was carried out for about 10 minutes in a GEBR. LÖDIGE laboratory mixer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test material in the diet over a period of 33 days at room temperature was confirmed prior to the study. The homogeneity of the test material preparation was proven in the early phase of the study in each of three samples at both concentrations.
Concentration control analyses were carried out in samples from all dose groups drawn at the start of administration period, after 4 weeks as well as after 3, 6, 7, 8, 9, 10, 12, 15 and 18 months after the start of the study. The recovery rates were in an acceptable range (90.5 % -101.0 % of the target concentrations), and the correctness of the concentrations was thus demonstrated.

Duration of treatment / exposure:

18 months

Frequency of treatment:

The test material was administered daily in the diet.

Post exposure period:

None

Dose / conc.:

700 ppm (nominal)

Remarks:

Males

Dose / conc.:

800 ppm (nominal)

Remarks:

Females

No. of animals per sex per dose:

Male animals: 0 ppm and 700 ppm (50 males per dose)
Female animals: 0 ppm and 800 ppm (50 females per dose)

Control animals:

yes, plain diet

Details on study design:

The study was carried out as a supplementary study to a previously conducted carcinogenicity study in order to test the oncogenic potential of the test material at a dose level fulfilling the criteria of a maximum tolerated dose (MTD).

- Dose selection rationale: In a sub-chronic oral toxicity study, the test material was administered to groups of 10 male and 10 female B6C3F1 mice in the diet for 3 months at concentrations of 0 ppm; 100 ppm; 1 000 ppm and 2 500 ppm. The following test-material-related findings were obtained:
2 500 ppm: Statistically significantly reduced body weight (about 10 % below controls in males and 9 % below controls in females on day 91); statistically significantly reduced body weight change in both sexes; increase in serum alkaline phosphatase, cyanide-insensitive palmitoyl-CoA oxidation in liver homogenate, urea and cholesterol in both sexes; decrease in serum triglycerides in both sexes; increase in serum creatinine and glucose in the males; increase in serum alanine aminotransferase in the females; decrease in haemoglobin, mean corpuscular haemoglobin and globulins in the males; statistically significant reduction of the terminal body weight in both sexes; statistically significant increase of the absolute and relative liver weight in both sexes; macroscopically diagnosed dark-brown discolouration of the liver in both sexes; in the liver: eosinophilic cytoplasm of hepatocytes in both sexes; in the kidneys: eosinophilic cytoplasm of tubular epithelial cells in both sexes; decrease of lipid storage in the liver of both sexes.
1 000 ppm: Statistically significantly reduced body weight (about 8 % below controls in males and 9 % below controls in females on day 91) statistically significantly reduced body weight change in both sexes; increase in serum urea in both sexes; decrease in serum triglycerides in both sexes; increase in serum creatinine in the males; in the liver: eosinophilic cytoplasm of hepatocytes in one female; decrease of lipid storage in the liver of both sexes.
100 ppm: Increase in serum urea in the females; decrease in serum triglycerides in the females.
In an 18-month carcinogenicity study the test material was administered to groups of 50 male and 50 female B6C3F1 mice in the diet at concentrations of 0 ppm, 25 ppm, 250 ppm and 2 500 ppm. 2 500 ppm led to a severe impairment of body weight in both sexes (up to 27 % below controls in males and 37 % below controls in females) indicating that the maximum tolerated dose (MTD) was clearly exceeded. The animals of this dose group were therefore sacrificed prematurely due to humane reasons. 250 ppm resulted in a significant increase of the absolute and relative kidney weight in females, and an increased number of females with chronic nephropathy. At 25 ppm, no test material-related findings were obtained.
Based upon the above-mentioned findings, 2 500 ppm proved to be too high as a highest concentration for an oncogenicity study. This was somewhat surprising, as this concentration promised to be an appropriated MTD in the sub-chronic study. Considering therefore that also 1 000 ppm will show overt toxicity after long-term administration, concentrations of 700 ppm (males) and 800 ppm (females) were selected for the present study. The rationale for selecting slightly lower concentration levels for males than for females is based upon general experience with phenoxy-herbicides being slightly more toxic in males than in females.
The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard and is a possible route for human exposure.

- Rationale for animal assignment: One day prior to the start of the administration period, the male and female mice were allocated to the test groups according to weight. The list of randomisation instructions was compiled by a computer (laboratory data processing).

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a week an additional comprehensive clinical examination (including palpation) was carried out.

BODY WEIGHT: Yes
- Time schedule for examinations: During the administration period, body weight was determined on day 0 (start of administration period), at weekly intervals during the first 13 weeks of the study, and thereafter at 4-week intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Food consumption over a period of 7 days was determined weekly during the first 13 weeks and thereafter at 4-week intervals. The values were calculated as mean food consumption in grams per animal and day.
The mean daily intake of the test material (group means) was calculated based upon individual values for body weight and food consumption, according to the following equation:

(FCx x C) / BWx = Substance intake for day x

Where:
BWx = Bodyweight on day x [g]
FCx = Mean daily food consumption on day x [g]
C = Concentration in the diet [mg/kg]

FOOD EFFICIENCY: Food efficiency (group means only) was calculated based upon individual values for body weight and food consumption, according to the following equation:

((BWx - BWy) / FC y to x) x 100 = Food efficiency for day x

Where:
BWx = body weight on day x [g]
BWy = body weight on day y (last weighing date before day x) [g]
FC y to x = mean food consumption from day y to day x; calculated as mean daily food consumptiqn on day x, multiplied with the number of days from day y to day x [g]

HAEMATOLOGY: Yes
Blood was taken by tail puncture from non-fasted, unanesthetised animals during the administration period and by decapitation from fasted, anesthetised animals at the end of the study to prepare differential blood smears. Furthermore, blood smears were prepared and evaluated from animals killed in extremis during the study.
The differential blood count was evaluated visually, stained according to Wright with microscopic evaluation.

Sacrifice and pathology:

At the end of the 18-month administration period all surviving animals were sacrificed after a fasting period (withdrawal of food for about 16 - 20 hours). The animals were sacrificed by decapitation under CO2 anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The animals which died intercurrently or were sacrificed in a moribund state were necropsied as soon as possible after death and assessed by gross pathology.

GROSS PATHOLOGY: Yes
The weight of the anesthetised animals as well as the weights of liver, kidneys, adrenal glands, testes, ovaries, and brain from all animals sacrificed at scheduled dates were determined.

HISTOPATHOLOGY: Yes
The following organs or tissues were fixed in 4 % formaldehyde solution and stained (hematoxylin-eosin): All gross lesions, brain, pituitary gland, thyroid glands with parathyroid glands, thymus, trachea, lungs, heart, aorta, salivary glands (mandibular and sublingual glands), liver, spleen, kidneys, adrenal glands, pancreas, testes/ovaries, uterus/vaginaloviducts, accessory genital organs (epididymides, prostate, seminal vesicle), skin, oesophagus, stomach (forestomach and glandular stomach), duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, lymph nodes (mesenteric and mandibular), female mammary gland, skeletal muscle, sciatic nerve, sternum with marrow, bone marrow (femur), eyes, femur with knee joint, spinal cord (cervical, thoracic and lumbar), extraorbital lacrimal glands, gall bladder.
After the organs were fixed, the processing, the examination by light microscopy and the evaluation of findings was performed. All animals affected were examined for the control group, for the test gropu all animals per group were examined.
A correlation between gross lesions and histopathological findings was performed.

Statistics:

Statistics of clinical examinations: Means and standard deviations of each test group were calculated for food consumption, bodyweight, bodyweight change and food efficiency using comparison of the dose group with the control group using Student's T-Test (two-sided) for the hypothesis of equal means.
Statistics of haematology: Means and standard deviations of each test group were calculated for several haematology parameters.
Statistics of pathology: Means and standard deviations were calculated for the variables of terminal body weight and of absolute and relative organ weights (related to terminal body weight) of the animals in each test group. A comparison of group 1 with the control group using the Wilcoxon test (two-sided) was performed for the hypothesis of equal means.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All clinical findings observed occurred equally distributed between control and treatment groups and were thus incidental in nature.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The administration of the test material did not result in an increase of mortality.
Most animals survived until the scheduled end of the study. Each one control male and female and three females of group 1 were found dead. One male of group 1 was sacrificed in a moribund state.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males (700 ppm) and females (800 ppm), body weight was statistically significantly impaired from day 343 (males) or 203 (females) onwards. The values on day 546 were 4.8 % (males) and 8.4 % (females) below controls. Corresponding body weight change values were 13.0 % (males) and 18.6 % (females) below controls. This was assessed as being related to treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were several minor, but statistically significantly increased or decreased values in males and females. However, as no consistent effect was observed, all these deviations were assessed as being incidental and biologically not relevant.
During the study there was spilling of food by the mice in all groups, irrespective of the dose. The values given in the tables can therefore only be used to estimate the food consumption and do not represent exact food intake.
The mean daily test material intake in mg/kg body weight (calculated from body weight and food consumption) ovar the entire study period was:
700 ppm (males): 112 mg/kg bodyweight/day.
800 ppm (females): 188 mg/kg bodyweight/day.
In order to assure equally spaced intervals for calculation, only the values of days 7, 35, 63, 91 and days 119 to 539 were taken into account for calculation of test material intake.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

There were several minor, but statistically significantly increased or decreased values in males and females. However, as no consistent effect was observed, all these deviations were assessed as being incidental and biologically not relevant.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

White blood cell: After 12 and 18 months of test material administration no treatment-related changes were observed in the white blood cells of the male and female mice given 700 ppm and 800 ppm of the test material, respectively.
Red blood cells: At the 12 month interval and at the end of the study no treatment-related changes were seen in the erythrocyte morphology of the animals receiving the test material.
Prematurely killed animals: There was only one blood smear of an animal killed in extremis. Evaluation of this blood smear revealed no treatment-related changes in the white and red blood cells of the differential blood count.

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute weights: The mean terminal body weight was significantly decreased in males (p ≤ 0.05, -4.90 %) and females (p ≤ 0.01, -10.11 %) of dose group 1.
The mean testis weight was significantly increased (p ≤ 0.05, +4.56 %) in dose group 1.
The mean kidney weight was significantly increased (p ≤0.01, +15.13 %) in females of group 1.
The increased weights of testes in males are considered as incidental. The brain/testes ratio for the test group was less than that for controls, reinforcing the incidental nature of the increase in absolute testes weights.
The other mean absolute weight parameters did not show significant differences when compared with the control group.

Relative organ weights
The mean liver weight was significantly increased in males (p ≤ 0.01, +12.21 %) and females (p ≤ 0.01, +13.75 %) of dose group 1.
The mean kidney weight was significantly increased in males (p ≤ 0.05, +4.69 %) and females (p ≤ 0.01, +26.41 %) of dose group 1.
The mean testis weight was significantly increased (p ~ 0.01, +9.98 %) in dose group 1.
The mean brain weight was significantly increased in males (p ≤ 0.05, +5.35 %) and females (p ≤ 0.01, +9.58 %) of dose group 1.
The increased weights of testes in males and the increased liver and brain weights in males and females do not represent a treatment related effect as they are only a consequence of the reduced terminal body weight of the animals.
The other mean relative weight parameters did not show significant differences when compared with the control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the liver, masses occurred in eight males of the control group and in fifteen males of group 1. In female animals, masses were observed in nine mice of group 1. Of these animals, one control male and each one male and female of group 1 were decedents. There were no masses recorded in female control animals.
All other gross lesions including the macroscopically diagnosed "masses" occurred either singly or were biologically equally distributed over the control group and the treatment group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Kidneys: The major non-neoplastic lesion in the kidneys was chronic nephropathy. Chronic nephropathy was characterised by the presence of areas of tubular atrophy, regeneration and dilation, proteinaceous casts within the tubules, and/or interstitial fibrosis.
The number of males and females with chronic nephropathy was increased in dose group 1.
In males, 38 animals of the control group and all animals of group 1 showed chronic nephropathy.
In females, chronic nephropathy was observed in eight control animals and in 38 animals of group 1. In most animals, the degree of severity was minimal or slight, whereby in treated animals a slight increase was evident.
A minimal or slight focal calcification in the lumen of renal tubules, in the arterial wall, in the interstitial tissue, and/or along tubular basement membranes was observed in 25 males of the control group and in 48 males and 13 females of group 1. The degree of severity was slightly increased in males of group 1.
The occurrence of calcification was often correlated with nephropathy. In most males of the control group, lipid vacuoles were seen in proximal tubular epithelial cells. Lipid vacuoles were not observed in males of group 1 by light microscopy. All other findings in the kidneys (focal lymphoid cell infiltration, cysts, focal ossification, amyloid nephrosis, necrosis, and pyelonephritis were either single observations or occurred at biologically comparable incidences in treated and untreated animals.

Liver: Some of the macroscopically diagnosed masses turned out to be foci of cellular alterations, histopathologically. Basophilic foci of cellular alteration occurred in four control males and in eleven males and four females of group 1. All other findings in the liver were single or low observations or did not show biologically relevant differences between treated and untreated animals.

Pancreas: In male control animals of this strain (B6C3F1), the incidence of islet cell hyperplasia was quite high (18 of 50 males). In dose group 1, eleven males showed this finding.

All other organs: All non neoplastic findings noted were either single observations or they were biologically equally distributed over control and treatment groups.

Decedents: There were no treatment-related histopathological findings.
Five animals were found dead: One female (group 1) showed a massive abscess-forming inflammation of ovaries, two females (groups 0 and 1) showed erosions/ ulcers in the glandular stomach, one control male a focal liver cell necrosis of excessive size, and in one female of group 1, there were macroscopically diagnosed erosions in the glandular stomach.
One male of group 1 was sacrificed in a moribund state. There were no microscopic findings, that could explain the moribund state.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Liver:
Many of the macroscopically diagnosed masses turned out to be hepatocellular adenomas or carcinomas, histopathologically. Hepatocellular adenomas were observed in each four males of the control group and of group 1 and in five females of group 1. One of the affected males of group 1 was sacrificed in a moribund state. Hepatocellular carcinomas occurred in two control males and in each four males and females of group 1. One female of group 1 died prematurely. Cause of death was a massive abscessforming inflammation of the ovaries. All other neoplastic findings noted were observed singly or at a low incidence. They were considered as incidental and not treatment related.

Tumour bearing animals:
The number of males and females with neoplasms, the number of males with benign neoplasms, the number of males and females with malignant and systemic neoplasms, the total number of primary and benign neoplasms in males, as well as the total number of malignant and systemic neoplasms in males and females were either comparable between control and treated animals or the tumors were recorded singly or at a low incidence. The number of females with benign neoplasms as well as the total number of primary and benign neoplasms was slightly increased in treated females due to the occurrence of five hepatocellular adenomas in this group.

Other effects:

not examined

Details on results:

Body weight/body weight change was impaired in males and females in the second half of the study. The values on day 546 were 4.8 % (males) and 8.4 % (females) below controls. Corresponding body weight change values were 13.0 % (males) and 18.6 % (females) below controls. As a late onset of body weight impairment is known for phenoxy-herbicides, this was assessed as being related to treatment. The impairment of body weight change clearly fulfilled the criteria for a maximum tolerated dose (MTD).
No treatment-related changes in the white and red blood cells of the differential blood count were observed.
The only observed treatment-related histological findings were of chronic toxic nature and they occurred in the kidneys: Chronic nephropathy and focal calcification in the kidneys are often seen in aged mice. However, in this study, the number of males and females with chronic nephropathy as well as the degree of severity were increased in the test groups. Focal calcification seems to be connected with chronic nephropathy and may be an additional facette of chronic nephropathy. An increase of focal calcification was observed in treated males and females. The increase of chronic nephropathy in male and female animals is considered treatment-related.
The mean relative kidney weight in males and the mean absolute and relative kidney weights in females were significantly increased in treated animals. Although there were no histopathological correlates (in most animals, the chronic nephropathy was only minimal or slight) for the increased kidney weights, a treatment-related effect may be conceived.
In most males of the control group, lipid vacuoles occurred in proximal tubular epithelial cells. Lipid vacuoles were not observed in treated males by light microscopy. The absence of lipid vacuoles in treated males has no toxicological relevance.
In females, hepatocellular adenomas and carcinomas occurred only in the liver of treated animals: five females showed hepatocellular adenomas and in four females hepatocellular carcinomas were observed. In the control group, there were no hepatocellular tumors. The incidence of hepatocellular adenomas (10 %) lies within the range of historical control data for female B6C3F1 mice (0 % - 10 %). The incidence of hepatocellular carcinomas (8 %) exceeded slightly the range of historical control data for female B6C3F1 mice (0 % - 6 %). Only one of the four affected animals died prematurely, but the cause of death was a massive abscess-forming inflammation of the ovaries and not the liver tumor. In addition, none of the carcinomas in the liver has metastasized, indicating a low level of malignancy. In males, the number of animals with hepatocellular adenomas or carcinomas was comparable between control group and treatment group. Although slightly elevated, the occurrence of hepatocellular tumors in female mice is considered to be incidental. The absence of liver tumors in female control animals in this study is highly unusual for this strain of mice. In an oncogenicity study which was carried out at the same time and in the same laboratory, the incidence of liver tumors in untreated female controls was 10 %.
Basophilic foci of cellular alterations were observed in four control males and in eleven males and four females of substance-treated groups. The incidence of basophilic foci of cellular alterations (8 %) lies within the range of historical control data for female B6C3F1 mice (0 % - 14 %), put in males (22 %), the range of historical control data for male B6C3F1 mice (6 % - 20 %) was slightly exceeded. The slightly increased occurrence of basophilic foci of cellular alterations in treated males is considered as incidental.

In conclusion, the following substance-related findings were obtained:
800 ppm (about 188 mg/kg body weight) females: impairment of body weight (8.4 % below control) and body weight change (18.6 % below control) until the end of the study; significant increase of the mean absolute and relative kidney weights in females; increased number of female mice with chronic nephropathy and slightly increased degree of severity of this finding.
700 ppm (about 112 mg/kg body weight) males: impairment of body weight (4.8 % below control) and body weight change (13.0 % below control) until the end of the study; significant increase of the mean relative kidney weight in males; increased number of male mice with chronic nephropathy and slightly increased degree of severity of this finding.
Thus, toxic signs fulfilling the criteria for an MTD were seen in males and females. The test substance was not oncogenic under the conditions of this study.

Dose descriptor:

NOAEL

Effect level:

700 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No indications for carcinogenicity found at this dose level, which fulfilled the criteria of a maximum tolerated dose (MTD), and which was the highest dose level tested for male mice.

Dose descriptor:

NOAEL

Effect level:

800 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No indications for carcinogenicity found at this dose level, which fulfilled the criteria of a maximum tolerated dose (MTD), and which was the highest dose level tested for female mice.

Critical effects observed:

no

Analyses

- Stability analyses: The stability of the test material in the diet over a period of 33 days at room temperature was verified. As the mixtures were stored no longer than this time period, the stability was guaranteed.

- Homogeneity analyses: The homogeneity of the mixtures was verified.

Concentration control analyses: The correctness of the concentrations was demonstrated. The recovery rates were in an acceptable range (90.5 % - 101.0 % of the target concentrations.

- Food analyses: On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants the food was found to be suitable. Fed. Reg. Vol. 44, No. 91 of May 09, 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The number of microorganisms did not exceed 10^5/g food. Individual results are to be found in the archives of the Department of Toxicology of BASF Aktiengesellschaft.

- Drinking water analyses: On the basis of the analytical findings the water was found to be suitable. German drinking water regulation (Trinkwasserverordnung, Bundesgesetzblatt, Dec. 05, 1990) served as a guideline for maximum tolerable contaminants.

- Bedding analyses: The bedding is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals). On the basis of these results the bedding was considered to contain no substances which would interfere with the conduct of the study.

Male Group Mean Bodyweight Day 0 to Day 56

Group		Day 0 (g)	Day 7 (g)	Day 14 (g)	Day 21 (g)	Day 28 (g)	Day 35 (g)	Day 42 (g)	Day 49 (g)	Day 56 (g)
Group 0 males (0 ppm)	M	21.9	23.9	24.2	25.7	26.1	27.4	27.1	28.2	28.2
	%	100	100	100	100	100	100	100	100	100
	SD	1.5	1.5	1.8	1.5	.19	2.3	2.0	2.0	2.1
	N	50	50	50	50	50	50	50	50	50
Group 1 males (700 ppm)	M	22.4	24.3	24.1	26.2	26.0	27.3	27.6	28.3	28.9
	%	102.1	101.4	99.9	102.0	99.6	99.4	402.2	100.3	102.3
	SD	1.6	1.2	1.9	1.4	2.2	1.7	2.0	1.9	2.0
	N	50	50	50	50	50	50	50	50	50

 

Male Group Mean Bodyweight Day 63 to Day 203

Group		Day 63 (g)	Day 70 (g)	Day 77 (g)	Day 84 (g)	Day 91 (g)	Day 119 (g)	Day 147 (g)	Day 175 (g)	Day 203 (g)
Group 0 males (0 ppm)	M	28.9	29.4	30.2	30.8	31.2	32.9	34.3	34.7	36.5
	%	100	100	100	100	100	100	100	100	100
	SD	2.0	2.2	2.3	2.4	2.5	2.7	3.2	3.2	3.3
	N	50	50	50	50	50	50	50	50	50
Group 1 males (700 ppm)	M	28.9	29.2	30.2	31.0	31.4	33.1	34.4	35.5	35.4
	%	99.8	99.4	100.1	100.4	100.5	100.7	100.4	99.3	96.9
	SD	1.9	2.2	2.2	2.5	2.6	3.0	3.3	3.5	3.3
	N	50	50	50	50	50	50	50	50	50

 

Male Group Mean Bodyweight Day 231 to Day 455

Group		Day 231 (g)	Day 259 (g)	Day 287 (g)	Day 315 (g)	Day 343 (g)	Day 371 (g)	Day 399 (g)	Day 427 (g)	Day 455 (g)
Group 0 males (0 ppm)	M	37.6	39.2	40.2	39.3	40.7	38.8	39.4	40.3	40.2
	%	100	100	100	100	100	100	100	100	100
	SD	3.4	3.3	3.3	3.3	3.2	3.4	3.4	3.6	3.7
	N	50	50	50	50	50	50	50	50	50
Group 1 males (700 ppm)	M	36.9	38.1	39.4	38.1	39.3*	37.9	37.8*	37.6***	36.7***
	%	97.9	97.2	98.0	96.9	96.7	97.7	96.0	91.2	91.2
	SD	3.8	4.0	3.7	3.7	3.6	3.3	3.1	3.4	3.1
	N	50	50	50	50	50	50	50	50	49

 

Male Group Mean Bodyweight Day 483 to Day 546

Group		Day 483 (g)	Day 511 (g)	Day 539 (g)	Day 546 (g)
Group 0 males (0 ppm)	M	40.6	41.0	41.5	40.7
	%	100	100	100	100
	SD	3.8	3.8	3.9	4.2
	N	49	49	49	49
Group 1 males (700 ppm)	M	37.3***	38.2***	39.2**	38.7*
	%	91.9	93.3	94.5	95.2
	SD	3.5	3.7	3.9	4.1
	N	49	49	49	49

Statistics: Student's t-test (two-sided): * P < = 0.05; ** P < = 0.01; *** P < = 0.001

 

Female Group Mean Bodyweight Day 0 to Day 56

Group		Day 0 (g)	Day 7 (g)	Day 14 (g)	Day 21 (g)	Day 28 (g)	Day 35 (g)	Day 42 (g)	Day 49 (g)	Day 56 (g)
Group 0 females (0 ppm)	M	17.9	19.6	19.1	20.9	20.9	22.6	23.2	22.9	23.6
	%	100	100	100	100	100	100	100	100	100
	SD	1.1	1.1	1.8	1.1	2.3	1.4	1.5	1.4	1.5
	N	50	50	49	49	49	49	49	49	49
Group 1 females (800 ppm)	M	18.0	9.7	19.1	21.2	21.9**	22.4	22.9	23.2	23.7
	%	100.7	100.5	99.9	101.6	104.7	99.2	99.0	101.1	100.3
	SD	1.0	1.0	1.8	1.1	1.2	1.2	1.3	1.2	1.3
	N	50	50	49	49	49	49	49	49	49

 

Female Group Mean Bodyweight Day 63 to Day 203

Group		Day 63 (g)	Day 70 (g)	Day 77 (g)	Day 84 (g)	Day 91 (g)	Day 119 (g)	Day 147 (g)	Day 175 (g)	Day 203 (g)
Group 0 females (0 ppm)	M	23.9	24.0	24.6	25.1	25.4	26.4	27.3	27.7	29.0
	%	100	100	100	100	100	100	100	100	100
	SD	1.4	1.7	1.7	1.8	1.9	2.4	2.6	3.0	3.4
	N	49	49	49	49	49	49	49	49	49
Group 1 females (800 ppm)	M	24.0	24.0	24.5	24.8	24.7	25.8	26.4	27.3	27.2**
	%	100.5	100.2	99.4	98.9	97.5	97.8	96.9	98.6	93.8
	SD	1.2	1.3	1.3	1.5	1.7	1.7	1.9	2.0	2.2
	N	49	49	49	49	49	48	48	48	48

 

Female Group Mean Bodyweight Day 231 to Day 455

Group		Day 231 (g)	Day 259 (g)	Day 287 (g)	Day 315 (g)	Day 343 (g)	Day 371 (g)	Day 399 (g)	Day 427 (g)	Day 455 (g)
Group 0 females (0 ppm)	M	29.7	30.8	32.4	32.0	32.3	31.4	32.0	32.9	33.3
	%	100	100	100	100	100	100	100	100	100
	SD	4.0	4.3	4.7	5.0	4.6	4.4	4.3	4.9	5.2
	N	49	49	49	49	49	49	49	49	49
Group 1 females (800 ppm)	M	28.0*	28.6**	30.1**	30.1**	30.2**	29.9*	30.4*	30.4**	30.2***
	%	94.4	92.8	92.9	94.0	93.5	95.0	95.1	92.3	90.8
	SD	2.4	2.8	3.4	3.2	3.4	3.3	3.4	3.7	3.2
	N	48	48	48	48	48	48	48	48	48

 

Female Group Mean Bodyweight Day 483 to Day 546

Group		Day 483 (g)	Day 511 (g)	Day 539 (g)	Day 546 (g)
Group 0 females (0 ppm)	M	34.2	35.0	34.8	34.3
	%	100	100	100	100
	SD	5.3	5.6	5.7	5.8
	N	49	49	49	49
Group 1 females (800 ppm)	M	30.7***	31.5***	31.6**	31.4**
	%	89.7	89.9	90.9	91.6
	SD	3.3	3.9	4.6	4.4
	N	47	47	47	47

Statistics: Student's t-test (two-sided): * P < = 0.05; ** P < = 0.01; *** P < = 0.001

 

 

Males Mean Absolute Organ Weights

Organ		Dose Group 0	Dose Group 1
Terminal body weight (g)	M	37.282	35.457*
	SD	4.066	3.806
	n	49	49
Liver (mg)	M	1394.429	1486.98
	SD	303.244	411.251
	n	49	49
Kidneys (mg)	M	719.408	717.204
	SD	83.082	74.51
	n	49	49
Testes (mg)	M	222.306	232.449*
	SD	26.334	18.479
	n	49	49
Brain (mg)	M	489.571	490.408
	SD	17.519	17.399
	n	49	49
Adrenal glands (mg)	M	5.755	4.98
	SD	1.931	1.09
	n	49	49

Wilcoxon-Test (two-sided) * P <= 0.05 ** P <= 0.01

 

Females Mean Absolute Organ Weights

Organ		Dose Group 0	Dose Group 1
Terminal body weight (g)	M	31.52	28.332**
	SD	5.76	4.01
	n	49	47
Liver (mg)	M	1277.286	1318.681
	SD	126.374	251.716
	n	49	47
Kidneys (mg)	M	485.102	558.511**
	SD	50.3	49.141
	n	49	47
Ovaries (mg)	M	39.347	34.362
	SD	49.935	41.892
	n	49	47
Brain (mg)	M	507.388	506.553
	SD	16.567	21.392
	n	49	47
Adrenal glands (mg)	M	8.939	8.851
	SD	1.725	1.489
	n	49	47

Wilcoxon-Test (two-sided) * P <= 0.05 ** P <= 0.01

 

Males Mean Relative Organ Weights

Organ		Dose Group 0	Dose Group 1
Terminal body weight (g)	M	100	100
	SD
	n	49	49
Liver (mg)	M	3.768	4.228**
	SD	0.86	1.276
	n	49	49
Kidneys (mg)	M	1.941	2.032*
	SD	0.212	0.184
	n	49	49
Testes (mg)	M	0.601	0.661*
	SD	0.079	0.071
	n	49	49
Brain (mg)	M	1.327	1.398*
	SD	0.133	0.149
	n	49	49
Adrenal glands (mg)	M	0.016	0.014
	SD	0.005	0.004
	n	49	49

Wilcoxon-Test (two-sided) * P <= 0.05 ** P <= 0.01

 

Females Mean Relative Organ Weights

Organ		Dose Group 0	Dose Group 1
Terminal body weight (g)	M	100	100
	SD
	n	49	47
Liver (mg)	M	4.13	4.698**
	SD	0.542	0.895
	n	49	47
Kidneys (mg)	M	1.575	1.991**
	SD	0.258	0.184
	n	49	47
Ovaries (mg)	M	0.135	0.118
	SD	0.196	0.131
	n	49	47
Brain (mg)	M	1.659	1.818**
	SD	0.282	0.226
	n	49	47
Adrenal glands (mg)	M	0.029	0.032
	SD	0.008	0.007
	n	49	47

Wilcoxon-Test (two-sided) * P <= 0.05 ** P <= 0.01

Conclusions:

Under the conditions of the study the test material was not oncogenic.

Executive summary:

A carcinogenicity study was conducted according to OECD Test Guideline 451, EPA OPP 83 -2 and EU Method B.32, and in compliance with GLP.

The study was carried out as a supplementary study to a previously conducted carcinogenicity study in order to test the oncogenic potential of the test material at a dose level fulfilling the criteria of a maximum tolerated dose (MTD).

Thus, the test material was administered to groups of 50 male and 50 female B6C3F1/CrlBR mice at dietary concentrations of 0 ppm, 700 ppm (males) and 800 ppm (females) for 18 months. Food consumption and body weights were determined once a week during the first 13 weeks, thereafter at 4-weekly intervals. At least once a day the animals were examined for evident signs of toxicity or mortality. Once a week they were subjected to an additionally comprehensive clinical examination (including palpation). Blood smears were prepared after 12 months and 18 months, and from all animals killed in extremis. At the end of the 18 months of treatment all animals were subjected to gross-pathological assessment, followed by histopathological examination.

The following test material-related findings were obtained:

800 ppm (about 188 mg/kg body weight) females: Impairment of body weight (8.4 % below control) and body weight change (18.6 % below control) until the end of the study; significant increase of the mean absolute and relative kidney weights in females; increased number of female mice with chronic nephropathy and slightly increased degree of severity of this finding.

700 ppm (about 112 mg/kg body weight) males: Impairment of body weight (4.8 % below control) and body weight change (13.0 % below control) until the end of the study; significant increase of the mean relative kidney weight in males; increased number of male mice with chronic nephropathy and slightly increased degree of severity of this finding.

Thus, toxic signs fulfilling the criteria for an MTD were seen in males and females.

Under the conditions of the study the test material was not oncogenic.

Reference 5

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 1984 to 12 June 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Original study report only partly available, but sufficient data are contained in this part, and moreover secondary source (Danish EPA) assesses study as reliable, therefore at least rel. 2 is warranted.

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Version / remarks:

(1981)

Deviations:

yes

Remarks:

, use of two satellite groups of 10 and 15 rats/sex at each dose level for 12 and 24 months, respectively, whereas testing guideline of 1981 stipulates only one high dose satellite group of 20 animals/sex - but no conflict to testing guideline of 2009.

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Remarks:

Chbb = THOM (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 42 days
- Weight at study initiation:
Main groups: Males 167 g (141 g - 192 g); females 136 g (120 g - 156 g)
Satellite group I: Males 164 (14-183); females 135 g (111 g - 149 g)
- Housing: During the test, the rats were housed individually in type DK III stainless steel wire cages, floor area about 900 cm^2 (Becker & CO, Castrop-Rauxel, Germany).
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 14 days acclimatisation period during which they received ground diet and water ad libitum and were accustomed to the environmental conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 % relative humidity
- Air changes (per hr): No data (completely air conditioned rooms).
- Photoperiod (hrs dark / hrs light): The day/night rhythm was 12 hours (12 hours light from 06:00 - 18:00 h, 12 hours dark from 18:00 - 6:00 h).

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks:

Diet pre-mix used

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: To prepare the test material preparations, the test material was weighed out depending on the dose group, and thoroughly mixed with a small amount of the feed in a beaker, using a spatula. The premix was subsequently prepared, initially in a BRAUN mixer (Mx 32) and further in the study also in a BOSCH household mixer. This premix was then adjusted to the required concentration by adding the appropriate amount of feed and was mixed in a laboratory mixer supplied by GEBR. LÖDIGE, for about 10 minutes.

DIET PREPARATION
- Rate of preparation of diet: The feed/substance mix was freshly prepared at intervals of not more than 24 days. The stability of the test material in the feed was verified for a period of 33 days.
- Mixing appropriate amounts with (Type of food): Kliba rats/mice/hamsters maintenance diet.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test material in the maintenance diet was analytically investigated before the study began. To verify homogeneity of the test material preparations, samples were sent to the Analytical Laboratory before the start of the study and than again in the initial phase of the administration period after the mixing procedure had been optimised (additional premix).
Furthermore, samples of each one of the doses were sent to the analytical laboratory at the beginning of the study and thereafter at 3-monthly intervals.
The content of the test material in the feed/test material mixes was determined by means of HPLC.

Duration of treatment / exposure:

24 months

Frequency of treatment:

Continuously in diet

Post exposure period:

None

Dose / conc.:

20 ppm (nominal)

Dose / conc.:

100 ppm (nominal)

Dose / conc.:

400 ppm (nominal)

No. of animals per sex per dose:

Main group: 50 per sex per dose.
Satellite group I: 10 per sex per dose level was dosed for 12 months.
Satellite group II: 15 per sex per dose level was dosed for 24 months.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The selection of the doses was based on the results of a 3-month feeding study with the test material in rats. In this study, dosages of 450, 150 and 50 ppm were used. The following test material-related findings were obtained:
450 ppm: Increase in the creatinine values in the plasma of the female animals; increase in the absolute and relative kidney weights in the male animals.
150 ppm: Increase in the absolute and relative kidney weights in the males; increase in the relative kidney weights in the females.
50 ppm: No changes to be attributable to the test material administration.
On the basis of the above findings, the following dose levels were fixed for the current study on a potential carcinogenic and chronic-toxic effect:
20 ppm as a definite no effect level; 100 ppm was chosen as the intermediate dose and 400 ppm as the highest dose.
Marginal toxic effects on administration of 400 ppm could probably be expected, however, without any adverse effect on the normal lifespan.
- Rationale for animal assignment: 12 days prior to the start of administration (day 0), the male and female rats were allocated to the test groups on the basis of their weights. The list of randomisation instructions was generated by a computer.
- Rationale for selecting satellite groups:
Main group: Determination of the body weight and feed consumption up to 24 months, urinalysis at the end of the study, subsequently necropsy.
Satelite group I: Determination of the body weight and feed consumption up to 12 months, urinalyses. hormone analyses (T3/T4), interim sacrifice after 12 months.
Satelite group II: Clinico-chemical and haematological examinations, necropsy after 24 months.
- Post-exposure recovery period in satellite groups: No.

Positive control:

None

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: The state of health was checked daily; furthermore, the animals were subjected to additional inspection and palpation once a week. A check for mortality was made twice daily (Mondays to Fridays) and once daily (Saturdays, Sundays and public holidays).

BODY WEIGHT: Yes
- Time schedule for examinations: The animals of the main groups and satellite group I were weighed once a week, including week 14 of the administration; subsequently, body weight was determined at 4-weekly intervals. The body weights were determined on the same day of the week each time. There was an additional determination of the amount of feed consumed and of the body weight, which took place at the end of the study outside the 4-weekly cycle (satellite group I and main groups).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: For animals of main group and satellite group I once a week up to 14 weeks and thereafter once a month until the end of the study. The mean amount of daily ingested test material (in mg) per kg body weight was determined at the same intervals as was feed consumption.
To determine the amount of feed consumed, the feed box with contents was weighed and the value obtained was subtracted from the initial value.
The feed consumption of the main groups and satellite group I was weighed once a week, including week 14 of administration, for the course of the preceding week; subsequently it was determined at 4-weekly intervals.
The mean amount of daily ingested test material (in mg) per kg body weight was determined at the same intervals as was feed consumption.
The values given represent a group mean calculated from the amounts of test material ingested by each individual animal, and were determined using the formula:

(FC x D) / BWx

Where:
FC = Mean daily feed consumption (in g) within one week of the study (from day x-7 to day x)
D = Dose in ppm
BWx = Mean bodyweight (in g) on day x of the study

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of the animals in the main groups (control, and highest dose group) were examined for changes to the refracting media using a focusable hand-held slit lamp before the start of the study and after about 6 and 12 months. Further eye examinations were carried out after about 18 months and at the end of the study in each of the first 10 of the surviving animals of both sexes in the control and highest dose group, using a hand-held slit lamp. If changes to the refracting media had either been expected or existed, photographs would have been taken where necessary, using a KOWA camera.
During examinations of the eyes before the start of the study and after about 12 months of the study, the fundus of 10 male and 10 female rats of the control and highest dose

HAEMATOLOGY: Yes
- Time schedule for collection of blood: The blood required was taken from the retroorbital venous plexus of the non-fasted animals in the mornings. The blood samplings and the subsequent analysis of the blood and plasma samples - except for the differential blood counts and the reticulocytes - were carried out in randomised sequence 6 days before the test material was administered (acclimatisation period; blood sampling 0) and about 26, 52, 78, and 104 weeks after the start of the administration (administration period; blood samplings 1, 2, 3, and 4). The lists of randomisation instructions were generated with a random number generator on a computer.
The haematological examinations were carried out before and during the administration period in 15 animals per test group and sex (satellite group II). For blood sampling 4, the animals that died in satellite group II were supplemented to 15 animals per test group and sex by adding main group animals. For the hormone analyses, blood was taken after 52 weeks from 10 animals per group and sex of satellite group I.
- Parameters checked: Haemoglobin, erythrocytes, haematocrit, mean haemoglobin content per erythrocyte, mean cell volume, mean corpuscular haemoglobin concentration, platelets, leukocytes, differential blood count, reticulocytes, prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: The blood required was taken from the retroorbital venous plexus of the non-fasted animals in the mornings. The blood samplings and the subsequent analysis of the blood and plasma samples - except for the differential blood counts and the reticulocytes - were carried out in randomised sequence 6 days before the test material was administered (acclimatisation period; blood sampling 0) and about 26, 52, 78, and 104 weeks after the start of the administration (administration period; blood samplings 1, 2, 3, and 4). The lists of randomisation instructions were generated with a random number generator on a computer.
The clinico-chemical examinations were carried out before and during the administration period in 15 animals per test group and sex (satellite group II). For blood sampling 4, the animals that died in satellite group II were supplemented to 15 animals per test group and sex by adding main group animals. For the hormone analyses, blood was taken after 52 weeks from 10 animals per group and sex of satellite group I.
- Parameters checked: Total bilirubin, creatinine, urea, sodium, potassium, glucose, inorganic phosphate, calcium, chloride, triglycerides, total cholesterol, total protein, albumin, globulins, glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, alkaline phosphatase, triiodothyronine (T 3), thyroxine (T 4).

URINALYSIS: Yes
- Time schedule for collection of urine: The urine of each individual animal was collected overnight from the satellite group I animals about 26 and 52 weeks after the administration began (administration period: Urine collections 1 and 2) and from 10 animals per group of the main group animals about 104 weeks after the administration began (administration period; urine colleection 3).
- Parameters checked: pH, protein, glucose, ketones, bilirubin, blood, nitrite, urobilinogen, sediment.
Except sediment microscopy, all the urine constituents were determined semi-quantitatively with test strips and a reflection photometer. The sediment was evaluated under a microscope.

Sacrifice and pathology:

The rats that survived in satellite groups I were killed at the end of the 12-month administration period and the surviving main and satellite groups II rats at the end of the 24-month administration period after a fasting period (Satellite group I; withdrawal of feed and water, Satellite group II and main group withdrawal of feed).
The animals that survived in satellite group I were subject to an interim kill after about 52 weeks; the rats surviving in the main group and satellite group II were sacrificed after about 104 weeks.

GROSS PATHOLOGY: Yes, all animals
HISTOPATHOLOGY: Yes

Statistics:

Clinical examinations: For the statistical evaluation of the test, means and standard deviation were calculated for the variables feed consumption, body weight and test material intake for the animals in each test group. Statistical significance of the clinical data (body weight) was determined by analysis of variance (ANOVA) followed by a Dunnett's test.

Blood and plasma examinations: Following statistical adjustment (NALIMOV criterion), the means and standard errors were calculated and have been printed out in the tables together with the individual figures.
For judging the significance, the t test (with the exception of the differential blood count) was used to compare the individual dose groups both with the control group and with their corresponding intitial values (blood sampling 0).

Urinalyses
The assessment as to whether specific features are pronounced to various extents in the control and test groups was carried out by the chi^2 test in corresponding two-by-two contingency tables.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The three doses (20, 100 and 400 ppm) administered as addition to the diet did not lead to any disturbances the general state of health of any one of the animals participating in the test. As the duration of the test increased, findings such as decubitus in the tarsal region or alopecia, etc., became more frequent in the animals used. The changes are spontaneous ones, also regularly to be found in untreated older rats.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

The mortality of the male and female animals was not influenced by the administration of the test material.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight of the male and female animals of all doses in the main groups as well as that of the females in satellite group I was comparable to that of the control animals.
There was a significant reduction in body weight between days 154 and 364 in the satellite group I male rats dosed 20 and 100 ppm; a dose-response relationship was not seen, and hence the reduction was assessed as being incidental in nature.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The amount of feed consumed daily by the male and female animals of all dose groups (20, 100, and 400 ppm) in the main groups and satellite group I during the specific duration of the study did not differ substantially when compared to the untreated control rats.
The amounts of test material taken up (mg/kg body weight) each day by the animals in the specific dose groups corresponded to the dose factor chosen. The mean amounts (in mg/kg body weight) of test material taken up in the course of the study are:
20 ppm main group males: 1.1 mg/kg bodyweight.
20 ppm satellite group 1 males: 1.3 mg/kg bodyweight.
20 ppm main group females: 1.4 mg/kg bodyweight.
20 ppm satellite group 1 females: 1.6 mg/kg bodyweight.
100 ppm main group males: 5.5 mg/kg bodyweight.
100 ppm satellite group 1 males: 6.5 mg/kg bodyweight.
100 ppm main group females: 6.9 mg/kg bodyweight.
100 ppm satellite group 1 females: 7.9 mg/kg bodyweight.
400 ppm main group males: 22.2 mg/kg bodyweight.
400 ppm satellite group 1 males: 26.1 mg/kg bodyweight.
400 ppm main group females: 27.9 mg/kg bodyweight.
400 ppm satellite group 1 females: 31.8 mg/kg bodyweight.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The ophthalmological examinations carried out with of a hand-held slit lamp before the start of the test and subsequently every 6 months showed no test material-induced impairment of the refracting media.
At no time was there any difference between the treated and untreated rats in terms of frequency distribution of the corneal findings in the form of remainders of the pupillary membrane or isolated corneal stipplings. Incidentally, these changes are frequently to be found in untreated Wistar rats. The examination with a KOWA camera carried out before the start of the test and after about 12 months showed no changes in the eye fundus.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

From the haematology point of view, the 24-month administration of the test material to male and female rats in doses of 20, 100, and 400 ppm did not cause any changes that could be ascribed to the test material administered.
The significant differences from the control group are not regarded as being related to the test material. Plausibility criteria have been introduced in order to avoid a detailed assessment and discussion of each individual statistically significant difference for each parameter.
The purpose of these criteria is to give all the reasons why there is no relation to administration of the test material, or why such a relation is improbable.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

From the clinical chemistry point of view, the 24-month administration of the test material to male and female rats in doses of 20, 100, and 400 ppm did not cause any changes that could be ascribed to the test material administered.
The significant differences from the control group are not regarded as being related to the test material. Plausibility criteria have been introduced in order to avoid a detailed assessment and discussion of each individual statistically significant difference for each parameter.
The purpose of these criteria is to give all the reasons why there is no relation to administration of the test material, or why such a relation is improbable.

Urinalysis findings:

not specified

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):

400 ppm: Significant increase in the relative kidney weights of the male rats of satellite group I and significant increase in the absolute and relative kidney weights of the males of the main group and satellite group II.
100 ppm: Significant increase in the relative kidney weights of the male rats of satellite group I and significant increase in the absolute kidney weights of the males of the main group and satellite group II.
20 ppm: No changes whatsoever that could be associated with the test material administered.

The 24-month administration of the test material therefore showed a dose-dependent increase in the kidney weights of the male rats of the 100 and 400 ppm groups, while the kidney weights of the female animals remained uninfluenced.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No indications for carcinogenicity found up to the highest dose group (400 ppm).

Dose descriptor:

NOAEL

Effect level:

20 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Effect level:

400 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed at highest dose level tested.

Critical effects observed:

not specified

Treatment related changes in organ weights:

	0 ppm (m/f)	20 ppm (m/f)	100 ppm (m/f)	400 ppm (m/f)
Dose, mg/ kg bw/day*	-/-	1.1/1.4	5.5/6.9	22.2/27.9
kidney weight, g	3.80/2.69	3.83/2.77	4.07^#/1.97	4.31^##/2.82
relative kidney weight, g	0.60/0.76	0.61/0.74	0.62/0.76	0.66^##/0.77

*: calculated for the main group       ^#and^##: 5% and 1% significance level (Dunnett's test)

Analyses

- Analysis of the test material: The active ingredient content of the test material was determined before the start of the study, after about 19 months, and at the end of the study.

The active ingredient content was 92.7 % before the beginning of the study. The stability of the test material was verified after 19 months by the analytical results and at the end of the study HPLC yielded 91.9 % and the gas chromatographic method yielded a content of 94.9 %. In accordance with the study protocol, after one year the test material samples were sent for reanalysis; however, no results were passed on. This is of subordinate significance, since, as it had been expected, the stability of the test material was confirmed by analysis at the end of the test.

The homogeneity of the test material was confirmed analytically before the start of the test.

The technical active ingredient was subjected. to a storage test, which after 2 years of storage at room temperature, at 30, 40, and 50 °C showed that its degree of purity had not declined.

Analysis of the test material preparations: In the analysis of the samples drawn before the start of the study, the homogeneity of the test material preparations could not be unambiguously verified; therefore, new samples were analysed. The results of these investigations confirm the homogeneous distribution of the test material in the vehicle.

The stability of the test material in the maintenance diet over 33 days was demonstrated at the start of study.

- Analysis of feed: The feed was regarded as suitable on the basis of the duration of use and the results of the tests for contaminants. The guideline for maximum tolerable contaminants was the Proposed Guidelines of EPA of May 9, 1979, Fed. Reg. Vol. 44, No. 91, p. 27354.

Only the copper values were found to have dropped; they were however acceptable since they were within the range of error expected with this method of determination.

- Analysis of drinking water: The drinking water used was regarded as suitable on the basis of the results obtained.

Conclusions:

Under the conditions of the study the administration of the test material over a period of 24 months at a dose level of 20 ppm did not lead to any test material-induced changes. Due to the present test results a carcinogenic potential of the test material is to be ruled out.
A dose-dependent increase in the kidney weights of the male rats of the 100 and 400 ppm groups was observed, while the kidney weights of the female animals remained uninfluenced.
 

Executive summary:

The carcinogenicity and chronic repeated dose toxicity of the test material was assessed according to OECD Test Guideline 453 and in compliance with GLP.

Three different doses (20, 100 and 400 ppm) of the test material were administered with the diet to 450 Wistar rats (225 males and 225 females) for 24 months.

Each dose group was split up into a main group (50 animals per sex) and into the satellite groups I and II comprising 10 and 15 animals per sex respectively.

A group of untreated animals (75 males and 75 females) was maintained in parallel for comparison. It was likewise split into a main group and satellite groups I and II. The feed consumption and body weight of the animals in the main group and of satellite group I was determined once a week up to 14 weeks and thereafter once a month until the end of the study. The state of health of all animals was checked daily; furthermore, the animals were subjected to additional inspection and palpation once a week.

At the beginning of the study and then about every 6 months, animals of the main groups (control and highest dose) were examined ophthalmologically.

Blood samples for haematological and clinico-chemical examinations were taken five times altogether from the animals of the satellite group II (at the last blood sampling, dead animals in this satellite group were supplemented by animals of the main groups).

Urinalyses were carried out on the animals of satellite group I two times in the first year, and on 10 animals of each main group about 104 weeks after the beginning of the administration period.

The animals that survived in satellite group I were subject to an interim kill after about 52 weeks; the rats surviving in the main group and satellite group II were sacrificed after about 104 weeks.

All animals used ware assessed by gross pathology. This was followed by a comprehensive histopathological examination.

The following findings were obtained and assessed or discussed to be test material-related:

400 ppm: Significant increase in the relative kidney weights of the male rats of satellite group I and significant increase in the absolute and relative kidney weights of the males of the main group and satellite group II.

100 ppm: Significant increase in the relative kidney weights of the male rats of satellite group I and significant increase in the absolute kidney weights of the males of the main group and satellite group II.

20 ppm: No changes whatsoever that could be associated with the test material administered.

 

Under the conditions of the study the 24-month administration of the test material led to a dose-dependent increase in the kidney weights of the male rats of the 100 and 400 ppm groups, while the kidney weights of the female animals remained uninfluenced. This shows that the male rats responded clearly more sensitively to the test material administered than did the female animals.

The findings obtained confirm the results of the 90-day study on the test material in rats, where increased kidney weights could be seen in the animals of the 450 and 150 ppm groups. In this former study the effects were also more distinct in the male than in the female rats. 

The administration of the test material over a period of 24 months at a dose level of 20 ppm did not lead to any test material-induced changes. Due to the present test results a carcinogenic potential of the test material is to be ruled out.

Reference 6

Endpoint:

carcinogenicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

Please see the read-across justification report in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No indications for carcinogenicity found up to the highest dose group (400 ppm).

Dose descriptor:

NOAEL

Effect level:

20 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Effect level:

400 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed at highest dose level tested.

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5.3 mg/kg bw/day

Study duration:

chronic

Species:

rat

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

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to carcinogenicity.

Additional information

Milburn (2008)

The carcinogenicity of the test material was assessed according to OECD Test Guideline 451 and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Groups of 52 male and 52 female HsdRCCHan:WIST rats were fed diets containing 0 (control), 100, 600 or 1 200 ppm test material. The surviving animals continued to termination after 104 weeks on test.

Clinical observations, bodyweights and food consumption were measured. All animals, including any found dead or killed prematurely were examined post mortem and tissues were taken for subsequent histopathology examination. At scheduled termination, cardiac blood samples were taken for haematology (white cell count) and selected organs were weighed.

At least 80 % of males and 70 % of females in the control, 600 and 1 200 ppm groups survived to two years. Survival was lower in both sexes in the low dose (100 ppm) group. There were more males in the 1 200 ppm group with dermal or subcutaneous masses than in the control or intermediate dose groups.

Body weights of females receiving 1 200 ppm were reduced compared to controls throughout the study; the maximum difference from control was 18 %. Body weights of males in the 1 200 ppm group were slightly reduced during the first year of the study. Body weights of females receiving 600 ppm were reduced for the majority of the study; the maximum difference from control was 7 %. There was no evidence of a reduction in body weight in males in the 100 or 600 ppm groups of females in the 100 ppm group.

Food consumption of females in the 1 200 ppm group was reduced compared to controls throughout the study. Food consumption of males in the 1 200 ppm group was slightly reduced during the first 13 weeks of the study. The food utilisation during the rapid growth phase was lower than control values in males and females in the 1 200 ppm group and females in the 600 ppm group.

There was no evidence of a treatment-related effect on the differential white cell count of treated rats.

Liver weights were increased compared to controls in females in the 1 200 ppm group. Kidney weights were higher than control in all treatment groups, although the difference was not clearly dose-related.

Hyalinization and pigment deposition and decrease in fat were recorded in the livers of both sexes receiving 1 200 ppm. Changes in the livers of the 600 ppm group were decreased fat in both sexes and pigment deposition in a small number of females. In the kidneys of rats receiving 1 200 ppm there was a decrease in the incidence of chronic progressive nephropathy in males, a higher incidence of intratubular microlithiasis, tubular basophilia/dilatation and vascular ectasia of the pelvis in females.

There was no effect of the test material on the number of tumour bearing animals or overall tumour incidence. The only statistically significant difference from control was a slightly higher incidence of lipoma (benign fatty tumour) of the subcutis in males in the 1 200 ppm group. The incidence was 0, 0, 1 and 4 in the control, 100, 600 and 1 200 ppm groups respectively. There were no treatment-related factors that contributed to the death of the animals.

There was no effect of the test material on survival.

Body weights and food consumption were reduced in rats receiving 1 200 ppm test material. In addition there were treatment related microscopic changes in the liver and kidney.

Body weights were reduced in females in the 600 ppm group and there were some minor changes in the livers of male and females.

There were no treatment-related effects at a dose of 100 ppm test material.

The test material had no effect on the overall number of animals with tumours.

There was one statistically significant difference from control which was a slightly higher incidence of lipoma (benign fatty tumour) of the subcutis in males in the 1 200 ppm group. There was no histological change in the skin or subcutaneous tissue in other rats, or in other tissues in which fat is stored, to suggest an effect of the compound on these tissues.

Under the conditions of the study, the test material is concluded not to be carcinogenic.

Supporting Study: Mellert (1996)

The carcinogenicity of the test material was assessed according to OECD Test Guideline 451, EPA OPP 83 -2, EU Method B.32, and in compliance with GLP. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, the test materialwas administered to groups of 50 male and 50 female B6C3Fl/CrlBR mice at dietary concentrations of 0, 25, 250, 2 500 ppm. The groups receiving 0, 25 and 250 ppm were treated for about 18 months. As 2 500 ppm proved to be too high (as evident by severe body weight impairment), the remaining animals of these dose groups were killed after about 12 months of treatment without further examinations.

Food consumption and body weights were determined once a week during the first 13 weeks, and thereafter at 4-weekly intervals. At least once a day the animals were examined for evident signs of toxicity or mortality. Once a week they were subjected to an additionally comprehensive clinical examination (including palpation).

Blood smears were prepared after 12 months and 18 months, and from all animals killed in extremis. The blood smears of the control groups and the 250 ppm groups were evaluated.

At the end of the 18 months of treatment all animals were subjected to gross-pathological assessment, followed by histopathological examination. The following test material-related findings were obtained after 18 months.

250 ppm (about 43 mq/kg body weight): Significant increase of the absolute and relative kidney weights in females, and increased number of females with chronic nephropathy.

25 ppm (about 4 mq/kg body weight):No substance-related effects.

Thus at 250 ppm chronic nephropathy was seen in females. The no observed adverse effect level (NOAEL) was 25 ppm (about 4 mg/kg body weight) in females and 250 ppm (43 mg/kg body weight) in males. The test material was not oncogenic under the conditions of this study.

Supporting Study: Mellert (1999)

A carcinogenicity study was conducted according to OECD Test Guideline 451, EPA OPP 83 -2 and EU Method B.32, and in compliance with GLP. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The study was carried out as a supplementary study to a previously conductedcarcinogenicity study in order to test the oncogenic potential of the test material at a dose level fulfilling the criteria of a maximum tolerated dose (MTD).

Thus, the test material was administered to groups of 50 male and 50 female B6C3F1/CrlBR mice at dietary concentrations of 0 ppm, 700 ppm (males) and 800 ppm (females) for 18 months. Food consumption and body weights were determined once a week during the first 13 weeks, thereafter at 4-weekly intervals. At least once a day the animals were examined for evident signs of toxicity or mortality. Once a week they were subjected to an additionally comprehensive clinical examination (including palpation). Blood smears were prepared after 12 months and 18 months, and from all animals killed in extremis. At the end of the 18 months of treatment all animals were subjected to gross-pathological assessment, followed by histopathological examination.

The following test material-related findings were obtained:

800 ppm (about 188 mg/kg body weight) females: Impairment of body weight (8.4 % below control) and body weight change (18.6 % below control) until the end of the study; significant increase of the mean absolute and relative kidney weights in females; increased number of female mice with chronic nephropathy and slightly increased degree of severity of this finding.

700 ppm (about 112 mg/kg body weight) males: Impairment of body weight (4.8 % below control) and body weight change (13.0 % below control) until the end of the study; significant increase of the mean relative kidney weight in males; increased number of male mice with chronic nephropathy and slightly increased degree of severity of this finding.

Thus, toxic signs fulfilling the criteria for an MTD were seen in males and females.

Under the conditions of the study the test material was not oncogenic.

QSAR: Lye (2011)

Using the QSAR models within the Lazar software tool to predict the mammalian toxicity and mutagenicity for test material and selected impurities, the results show that;

- For all predictions the model confidence is > 0.025 i.e. above the cut off to distinguish between reliable and unreliable predictions. This means that, for all substances, the predictions are deemed as reliable.

- All substances examined (i.e. the test material and 4 impurities) are predicted to be non-carcinogenic with the restraints of the models.

The study was awarded a reliability score of 4 in accordance with the criteria set forth by Klimisch et al. (1997).

Read-Across Study: Kuehborth (1988)

The carcinogenicity and chronic repeated dose toxicity of the read-across material was assessed according to OECD Test Guideline 453 and in compliance with GLP. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Three different doses (20, 100 and 400 ppm) of the test material were administered with the diet to 450 Wistar rats (225 males and 225 females) for 24 months.

Each dose group was split up into a main group (50 animals per sex) and into the satellite groups I and II comprising 10 and 15 animals per sex respectively.

A group of untreated animals (75 males and 75 females) was maintained in parallel for comparison. It was likewise split into a main group and satellite groups I and II. The feed consumption and body weight of the animals in the main group and of satellite group I was determined once a week up to 14 weeks and thereafter once a month until the end of the study. The state of health of all animals was checked daily; furthermore, the animals were subjected to additional inspection and palpation once a week.

At the beginning of the study and then about every 6 months, animals of the main groups (control and highest dose) were examined ophthalmologically.

Blood samples for haematological and clinico-chemical examinations were taken five times altogether from the animals of the satellite group II (at the last blood sampling, dead animals in this satellite group were supplemented by animals of the main groups).

Urinalyses were carried out on the animals of satellite group I two times in the first year, and on 10 animals of each main group about 104 weeks after the beginning of the administration period.

The animals that survived in satellite group I were subject to an interim kill after about 52 weeks; the rats surviving in the main group and satellite group II were sacrificed after about 104 weeks.

All animals used ware assessed by gross pathology. This was followed by a comprehensive histopathological examination.

The following findings were obtained and assessed or discussed to be test material-related:

400 ppm: Significant increase in the relative kidney weights of the male rats of satellite group I and significant increase in the absolute and relative kidney weights of the males of the main group and satellite group II.

100 ppm: Significant increase in the relative kidney weights of the male rats of satellite group I and significant increase in the absolute kidney weights of the males of the main group and satellite group II.

20 ppm: No changes whatsoever that could be associated with the test material administered.

Under the conditions of the study the 24-month administration of the test material led to a dose-dependent increase in the kidney weights of the male rats of the 100 and 400 ppm groups, while the kidney weights of the female animals remained uninfluenced. This shows that the male rats responded clearly more sensitively to the test material administered than did the female animals.

The findings obtained confirm the results of the 90-day study on the test material in rats, where increased kidney weights could be seen in the animals of the 450 and 150 ppm groups. In this former study the effects were also more distinct in the male than in the female rats. 

The administration of the test material over a period of 24 months at a dose level of 20 ppm did not lead to any test material-induced changes. Due to the present test results a carcinogenic potential of the test material is to be ruled out.