Paraquat-dichloride

Carcinogenicity

Description of key information

- Oral: reported NOAEL >= 7.9 mg/kg bw/day (cation), recalculated NOAEL >= 10.9 mg/kg bw/day (pure test substance); female; Fischer 344 rats; OECD 453; Woolsgrove 1983.

- Oral: reported NOAEL >= 15.0 mg/kg bw/day (cation), recalculated NOAEL >= 20.7 mg/kg bw/day (pure test substance); both sexes; Alpk mice; GLP compliant, OECD 453; Sotheran 1981.

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:

weight of evidence

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

Clinical chemistry, haematology not assessed

GLP compliance:

yes

Species:

mouse

Strain:

other: Alderley Park mice

Remarks:

Swiss-derived mice (SPF)

Details on species / strain selection:

This strain was used, because it was used in a previous study and because background tumour incidences are available from other mouse carcinogenic studies at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 19 days
- Housing: During the study the mice were housed five per cage; the sides of the cages were made of stainless steel sheet, the front, back and floor from stainless steel mesh with 0.9 x 2.5 cm centres and the overall measurements were 18.0 x 32.5 x 12.5 cm. The cages were suspended over collecting trays lined with absorbent paper. Each cage bore a colour coded card identifying the contained animals and their treatment.
- Diet: ad libitum. During the pre-experimental phase and until the week beginning 21 August 1978, a rat diet with a Vitamin E Supplement was used. At this time, a policy decision was made to change from this diet to another diet. The latter diet was fed from then until the end of the study.
- Water: ab libitum
- Acclimation period: The mice were quarantined from the day of the first delivery until 10 days after the last mice arrived. During this period the health status of the mice was carefully observed to detect any adverse reaction to their new environment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23, with occasional values outside this range
- Humidity (%): 35 to 72
- Air changes (per hr): minimum of 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Details on exposure:

- DIET PREPARATION
The appropriate volumes of a 1% w/w aqueous solution of test substance were mixed with the base diet to give the desired concentration of the test substance in each diet. Water was added at 12.5% to 17.5% w/w of total diet to the mix which was then mixed for 15 minutes. The final mix was passed through a pellet mill which produced pellets of 1 cm diameter and variable length. The control diets were prepared in the same manner but without the addition of the test substance. The pelleted diets were dried in an oven at a temperature not exceeding 50 °C for 2.5 hours to 5 hours.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Aqueous extracts of diet were deproteinised with trichloroacetic acid solution and then centrifuged. The supernatants were passed through a cation-exchange column onto which the test substance was absorbed and concentrated. After a wash sequence, the test substance was eluted from the column and measured colorimetrically after reduction with sodium dithionite.

Duration of treatment / exposure:

Interim sacrifice at 52 weeks of treatment.
Terminal sacrifice at 97 to 99 weeks.

Frequency of treatment:

Daily via food

Post exposure period:

None

Dose / conc.:

12.5 ppm

Remarks:

Group 3. Dietary concentration for test substance cation; equivalent to 1.87 mg/kg bw/day for both sexes.

Dose / conc.:

37.5 ppm

Remarks:

Group 4. Dietary concentration for test substance cation; equivalent to 5.6 mg/kg bw/day for both sexes.

Dose / conc.:

100 ppm

Remarks:

Group 5, dose level till week 36. Avererage dietary concentration for test substance cation; equivalent to 15.0 mg/kg bw/day for both sexes.

Dose / conc.:

125 ppm

Remarks:

Group 5, continued at a higher dose level from week 36 onwards. Average dietary concentration for test substance cation; equivalent to 18.7 mg/kg bw/day for both sexes.

No. of animals per sex per dose:

Interim sacrifice: 10 animals/sex/dose (1 control group and all treatment groups).
Terminal sacrifice: 60 animals/sex/dose (2 control groups and all treatment groups).

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were selected from the results of two preliminary feeding studies in mice. 100 ppm was considered to be the likely maximum-tolerated dose to mice over two-years and was expected to produce slight toxic effects. The 12.5 ppm level was selected as the probable no-effect level. The 37.5 ppm treatment was selected as the middle dose level to give a regular progression between the three doses.
- Section schedule rationale (if not random): The study consisted of two parts: Part 1 contained the mice for the main oncogenic assessment and Part 2 contained mice scheduled for interim kill after 52 weeks of treatment to provide information on the concentration of paraquat in tissues and plasma.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Prior to the start of the study. During the study the mice were observed daily for clinical signs, mortality and behavioural abnormalities.
- Any mice which appeared to be ill were observed more frequently and killed if considered necessary.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes, for part 1 mice only.
- Time schedule for examinations: Before experimental diets were first offered and then weekly for the first twelve weeks of the study. Body weights were then recorded once every two weeks until week 36. At this time when the 100 ppm dose level was increased to 125 ppm, individual body weights from all groups were recorded weekly until week 40 to monitor any effects during this period. From week 40 until the end of the study body weights were recorded once every two weeks.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes, for part 1 mice only.
- Time schedule: The food consumption of each cage of mice was recorded weekly for the first twelve weeks of the study and also during weeks 36 to 40. From week 12 to 36 and from week 40 until termination food consumption was recorded weekly once every four weeks. During the periods when food consumption was recorded, daily food wastage was measured.
- Food consumption for each animal determined and mean daily diet consumption calculated as gram food per week
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY: Yes, for part 1 mice only.
- Food utilisation values were calculated from the body weight and food consumption data for each cage of mice as gram weight gain per gram food.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all mice.
Moribund animals and those mice surviving until termination of the study were killed by an overdose of halothane vapour. A full autopsy was carried out on all part 1 mice. An autopsy was also carried out on Part 2 mice but only tissues thought to be abnormal were stored in formal saline.

HISTOPATHOLOGY: Yes, for part 1 mice only.
A full autopsy was carried out on all part 1 mice and the following organs were submitted for histopathological examination: voluntary muscle, salivary glands, cervical lymph nodes, pancreas, spleen, liver with gall bladder, adrenals; kidneys, urinary bladder, heart, lungs, thyroid (with parathyroid in some animals), trachea, oesophagus, thymus, mesenteric lymph node, jejunum, ileum, duodenum, colon, caecum, stomach, pituitary, brain, spinal cord, skin, aorta, eyes, Harderian glands and sciatic nerves. Mammary gland, ovaries, cervix and uterus were submitted from females; testes, epididymes, seminal vesicle, preputial and prostate glands were submitted from males. Any other tissue which appeared to be abnormal at autopsy was also submitted for histopathological examination.
Bone marrow smears were prepared from the left femur of any animal known not to have been dead longer than fifteen minutes. The smears were fixed in methanol, stained by a Romanovsky stain and stored for future reference to assist the pathologist if there were problems classifying any tumour type of the haemopoietic system.

Other examinations:

TEST SUBSTANCE ANALYSIS IN URINE: Yes, in part 1 mice only.
- Time schedule: three-monthly intervals throughout the study.
- How many animals used: Per group two cages from each sex.
- The mice were housed in metabolism cages for 5 hours, water was available ad libitum except during the collection at twelve months when the mice were inadvertently deprived of water. Food was withheld during each collection period to prevent possible contamination of the urine samples.

TEST SUBSTANCE ANALYSIS IN TISSUE AND PLASMA: Yes, in part 2 mice only.
- Time schedule: The mice were killed at week 52.
- Blood was collected by cardiac puncture and individual test substance concentration in the plasma was determined.
- Lungs and kidneys were removed and the test substance concentration was determined.

MICROBIOLOGICAL SCREENING: Yes
After the mice had been selected for the main study (Parts 1 and 2), a further sixty males and sixty females were selected as microbiological sentinels. Four groups of fifteen mice of each sex were fed one of the dietary concentrations of test substance (0, 12.5, 37.5 or 100 (125) ppm) throughout the duration of the main study. These mice were housed in replicates. As far as possible the microbiological sentinel mice were treated in the same way as the animals on the main study.

Statistics:

Weekly food consumption, food wastage and food utilisation during the first 12 weeks of the study and body weight gain throughout the study were considered separately for males and females by analysis of variance. Group means were adjusted for missing values before comparisons were made. Each treated group mean was compared to a combination of the two control group means using Student’s t-test (two sided).
Body weight gain was considered in this way for weeks 1-12 inclusive and then every 4 weeks until week 96. In addition, body weight gain from week 34 was analysed similarly to monitor the effect of the change in the top dose from 100 to 125 ppm. Food utilisation was considered over the periods weeks 1-4; 5-8; 9-12 and 1-12. Food consumption and food wastage were considered for each week they were measured.
Mortality data were analysed separately for males and females using the NCI computer programme. The analysis was performed with the two control groups separately and pooled; the analysis included pairwise comparisons of all groups and an overall test for trend with dose.
The most frequently observed clinical findings and the incidence of neoplastic and non-neoplastic findings were analysed using a one-sided Fisher’s exact test. The data were analysed separately for males and females comparing each treated group to the two control groups pooled. All tumour types at each site were analysed in this manner; where the evidence for increased tumours, the level of tumour incidence and the mortality rates made it appropriate, tumour incidence was also analysed by the logrank test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The most frequent clinical signs were swellings and sores in the genital area of male and (to a lesser extent) female mice, incontinence and loss of hair. These are common findings in this strain of mouse and were seen across all groups, although swelling and sores in the genital area were somewhat more prevalent in the top dose females. The swelling around the genital area in males was subsequently found by microscopic examination generally to be associated with inflammation of the preputial gland or dilation of the ducts.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The mortality rates for the male 37.5 ppm group and the female 125 ppm group differed significantly from the mortality rate of the combined control group. These treated groups showed increased mortality from around week 40 and week 60 respectively. However, with the lack of a corresponding effect on male mortality at the highest dose the male mortality rate increase at 37.5 ppm is unlikely to be related to test substance treatment.
Survival was >45% in all groups at week 80. The mortality rates of the other groups were comparable to or less than that of the controls. The study was terminated between weeks 97 and 99 when 80% mortality was reached in a female control group and was approaching 80% in the study overall.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was no evidence of any detrimental effects on body weight gain in any of the treated groups up to week 34. In fact, males and females receiving 12.5 ppm test substance tended to gain more weight than the controls. Increasing the top dose to 125 ppm at week 36 did not appear to affect the male mice and all male treated groups showed similar weight gains to controls throughout the study. However, there was evidence that the change in the top dose did affect the body weight gain of females in that group, lower body weight gains were seen in top dose females (week 44 onwards). There was also an indication that females receiving 37.5 ppm test substance began to have lower weight gains which became statistically significant from about week 68 onwards.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption data can be considered in two periods. For the first 24 weeks the test substance treated animals ate slightly less food than controls, that consumed by the top dose animals showing the lowest values consistently while the values for the 12.5 and 37.5 ppm groups were not dose-related. After week 24, the female treated groups maintained a similar pattern of response but the male 12.5 and 37.5 ppm groups showed similar values to control.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food utilisation tended to be better for the treated groups than for the controls.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Findings which contributed to the ill health or death of a number of animals were thoracic or abdominal haemorrhages and genitourinary infections. Haemorrhages are common findings in this animal model, particularly in the first year of life, and there was no evidence of a treatment-related effect in this study. Genitourinary infections were relatively frequent in both males and females. In the majority of the male mice these infections appeared to originate in the preputial glands. These glands frequently contained abscesses or marked inflammatory reactions, giving rise to the bulk of subcutaneous lumps or genital swelling found in the males. There was a high incidence of lenticular changes in old animals of all groups. There was no evidence that these degenerative changes increased in test substance-treated animals or gave rise to lenticular opacities (cataracts).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

KIDNEY
The treatment related changes seen in kidney were largely restricted to animals receiving 100/125 ppm test substance and were slightly more marked in males than in females. Renal lesions were initially seen after the dose level had been raised from 100 to 125 ppm in week 36 of the study and consisted of mild hydropic degeneration in some proximal tubules and increased eosinophilia in others. Approximately three months after the dose level had been raised, the pattern of the damage was somewhat different, consisting of mild dilatation and degeneration of tubules in the renal cortex. These tubules had some of the morphological characteristics of distal tubules, although the degenerative changes present made accurate identification difficult. Affected tubules usually showed both dilatation and degenerative change. There was also an increased incidence of tubules showing either dilatation alone or degenerative change without dilatation in animals receiving 100/125 ppm, although similar changes were seen in control kidneys. There was also an increased incidence of degenerating cortical tubules in male mice receiving 37.5 ppm and two animals in this group showed the characteristic treatment-related dilatation and degeneration of tubules seen at a higher incidence in the top dose animals. Other changes in the kidney such as nephropathy, pelvic dilatation, interstitial nephritis and amyloid deposition in glomeruli are common findings in mice of this age and are not considered to be related to treatment although there were occasional statistically significant differences between treated groups and controls.

LUNG
The lungs of many of the animals remained normal up to the termination of the study. Perivascular and alveolar oedema, congestion, mild perivascular inflammation and increased alveolar macrophages were the most common findings in both test and control animals, with no significant differences between the groups. The incidence of hypercellularity of alveolar walls was slightly increased in both males and females receiving 100/125 ppm, but this was a minimal change and was seen in control males. Two male mice receiving 100/125 ppm showed a focal pneumonitis/alveolitis which was similar to the lung lesions normally associated with the administration of high levels of test substance. Similar lesions were seen in three males and one female receiving 37.5 ppm test substance.

OTHER ORGANS
Other non-neoplastic lesions occurring in this study were considered to be typical of the background pathology of the animal model and were not treatment related. Many animals showed a variety of changes in the liver ranging from fatty vacuolation to focal or extensive necrosis, with associated hepatitis. Fatty vacuolation was statistically significantly increased in males receiving 37.5 or 100/125 ppm test substance and isolated other findings in the liver were also significantly increased in the treated animals, but in the absence of any other evidence of a treatment related effect on the liver, it was considered that these changes were part of the normal spectrum of changes in the liver.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was no evidence of an increase in overall total tumours or total number of tumour bearing animals, or in the proportion of malignant tumours found in any of the treatment groups compared with the two control groups. There was similarly no evidence of a treatment related increase in any of these parameters at individual time periods during the study. The range of tumours encountered in the study was typical of that occurring spontaneously in the this animal model.
The tumours occurring most frequently were lymphosarcomas, pulmonary tumours, liver nodules, Harderian gland and pituitary tumours (the latter were found predominantly in females). The incidence of these tumour types was similar to that found in previous studies in this laboratory. The incidence of other tumours was low.

LYMPHOSARCOMAS
These tumours were comprised of lymphocytic, lymphoblastic, reticulum or histiocytic and mixed cell lymphosarcoma. The incidence of any of these tumour types was similar across all the groups and it was, therefore, considered valid to place them in a single category. Lymphocytic or lymphoblastic tumours predominated in the early stages of the study, but there was a tendency for the cell type to become more undifferentiated in older animals. In many animals the thymus was the principal lymphoreticular organ affected, but a proportion of the tumours involved one or more of the lymph nodes without involving the thymus. There were no statistically significant differences between the groups in the incidence of lymphosarcoma, either overall or at individual time periods.

PULMONARY TUMOURS
The majority of these were classified as adenomas but a small proportion showed evidence of malignancy including invasion through the pleura and/or metastasis to mediastinal lymph nodes or other sites in the mediastinum and were classified as carcinoma. The morphology of the tumours was similar to those seen previously in this animal model and are considered to be derived from type II pneumocytes. The overall incidence of pulmonary tumours was similar in test and control groups and no unusual tumour types were encountered in the test groups. The incidence in both males and females receiving 100/125 ppm test substance was somewhat higher than in controls in the time period 79-98 weeks, but was lower than controls in the animals surviving to termination.

LIVER NODULES
Nodular lesions derived from hepatic parenchymal cells were classified as type A or type B. Type A included nodular hyperplasia as well as benign neoplasms. Type B nodules showed evidence of malignant change and some had metastasised, principally to lung; many type B nodules had an angiomatous pattern and were highly haemorrhagic. The incidence of both type A and type B nodules was higher in males than in females. No evidence of a treatment related effect was seen in either sex.

PITUITARY TUMOURS
The majority of these tumours were diagnosed as adenomas; pituitary carcinomas showing invasion of the surrounding brain tissue were found only in four animals. The incidence was considerably higher in females than in males, but was similar across both test and control groups.

HARDERIAN GLAND ADENOMAS
These were slightly increased in female test mice compared with controls and there was also a slight increase in males receiving 12.5 ppm. This increase was statistically significant (one sided Fisher’s exact test) in females receiving 12.5 ppm (p=0.015). There was no evidence of a dose response.

OTHER TUMOURS
Four mammary adenocarcinomas were found in this study, three in females receiving 12.5 ppm and one in a female receiving 100/125 ppm. These tumours occur spontaneously in this strain of mouse and the zero incidence in the control groups on this occasion reflects the variability of tumour incidence in this animal model. Due to the lack of a dose response in this study the incidence in the test groups alone is not regarded therefore as biologically significant. The incidence of renal adenomas was slightly increased in males receiving 100/125 ppm compared with the two control groups. This increase was not statistically significant, using either a one-sided Fisher’s exact test (p=0.071), or a log rank analysis of renal adenomas together with renal carcinomas (p>0.10 for a test of trend with treatment dose). Renal carcinomas were seen only in the two male control groups and in a male receiving 12.5 ppm test substance.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

TEST SUBSTANCE ANALYSIS IN URINE
The results, in terms of test substance excreted (µg/mouse) and test substance concentration (μg/mL) in urine show that dose related absorption of test substance took place in the treated groups during the study. On one occasion, at week 13, a trace of test substance was detected in female control urine. Although exhaustive investigations failed to reveal the source of this contamination it was concluded that no misdosing occurred at that time and the study was not jeopardised in any way.

TEST SUBSTANCE ANALYSIS IN PLASMA AND TISSUES
The samples analysed from animals at the one year interim kill show the presence of small amounts of test substance in plasma and tissues of the treated groups. The plasma concentrations (detection limit 0.006 µg/mL) show a dose related trend particularly in the males. Some difficulties were encountered with the analysis of the tissue samples due to a nonspecific interference with the radio-immunoassay. This interference was thought to have occurred either due to freezing and thawing of samples or to a poor batch of antisera. As a consequence, valid results could not be obtained for some samples from the lower test substance dietary groups or from any controls. The available results for lung and kidney show a relationship with dose.

Dose descriptor:

NOAEL

Effect level:

>= 125 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Original value presented in study. Dietary concentration for test substance cation; equivalent to 15.0 mg test substance cation/kg bw/day for both sexes.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 25.8 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Recalculated value for the dietary concentration for pure test substance, see ‘Any other information on results incl. tables’ for respective calculation

Key result

Critical effects observed:

no

Calculation of key result

The doses of the test substance were expressed in test substance cation, which relates to the cation species in an aqueous solution of the registered substance. The effect levels are already corrected for the amount of water. The key effect levels are calculated by inclusion of the anion species:

(100/72.4) x 18.7 mg test substance cation / kg bw = 25.8 mg pure pure test substance/ kg bw.

Conclusions:

In this lifetime feeding study, performed under GLP and equivalent or similar to OECD 453, no carcinogenic potential of the substance was observed. Therefore the NOAEL for carcinogenicity is higher than 125 ppm (recalculated dietary equivalent value: 20.7 mg pure test substance/kg bw/day (for both sexes)).

Executive summary:

In this lifetime feeding study, performed under GLP similar to OECD 453, Swiss-derived mice, were administered technical test substance incorporated into the diet of to give dose levels of 0, 0 (two control groups), 12.5, 37.5 and 100/125 ppm test substance cation for 97 to 99 weeks (when mortality was approaching 80% in all groups). Each group consisted of 60 animals per sex per dose. In addition, a satellite group of 10 per sex per group was designated for interim sacrifice after 52 weeks. At week 36 the top dose level was increased from 100 to 125 ppm because no toxic signs had appeared after 35 weeks of dosing. These doses corresponded to a dietary intake of 0, 0, 1.87, 5.6 and 15.0/18.7 mg test substance cation/kg body weight/day. The diets were analysed for homogeneity, stability and achieved concentration. All mice had free access to tap water and treated or control diet except when food was removed overnight prior to urine collection. Mice were examined routinely for mortality, clinical abnormalities, masses, body weight and food consumption. Urinary test substance determinations were performed every 13 weeks on 10 mice/sex/group. Tissue (lung and kidney) and plasma test substance levels were determined in samples from the satellite group at termination.

After 52 weeks all surviving mice in the satellite groups were killed and given a macroscopic examination post mortem– only abnormal tissues were preserved. All surviving mice in the main study were killed after 97-99 weeks of treatment. Each animal was given a detailed macroscopic examination and a comprehensive selection of tissues were taken and examined histopathologically for neoplastic and non-neoplastic changes.

Dietary analyses showed the majority of batches to be within 10% of the nominal values, with adequate homogeneity and stability. Clinical signs were consistent between groups though an increase in genital sores and decreased hair loss were noted in the top dose females. Mortality rates were increased in intermediate dose males (week 43 onwards) and top dose females (week 68 onwards) – the former is probably a chance finding due to the lack of a corresponding finding in top dose males. Survival was >45% in all groups at week 80. Body weight gain was increased at 12.5 ppm test substance cation in both sexes at the beginning of the study; lower body weight gains were seen in top dose females after the dose level was increased (week 44 onwards). Food consumption was slightly lower in test substance treated animals than in controls, with food utilisation efficiency increased in males.

Histological examination of animals dying during the study or killed at termination showed the kidney to be the major target organ with tubular effects being prevalent at 125 ppm with pelvic dilatation evident at ≥37 ppm in males. There was no evidence of treatment-related effects on the lungs other than a few instances of alveolar wall thickening/hypercellularity. Ocular lesions were similar in controls and treated groups. There were no significant increases in total neoplastic lesions following test substance administration. Occasional increases in individual tumour incidence were seen (eg. pituitary adenoma at weeks 53-78 and lung adenoma at weeks 78-98), these were of tumours typical of aged mice and not statistically significant nor consistent with time and dose. There was an increase in kidney adenomas in top dose males but not in females, this was probably secondary to degenerative lesions. It is concluded that test substance is not tumourigenic in mice. Therefore the NOAEL for carcinogenicity is higher than 125 ppm (recalculated dietary equivalent value: 20.7 mg pure test substance/kg bw/day (for both sexes)).

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

6 Apr 1978 to 21 Aug 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

no

GLP compliance:

not specified

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5 weeks at arrival.
- Weight at study initiation: 60 to 70 g at arrival.
- Fasting period before study: No.
- Housing: The rats were accommodated in polypropylene cages, (measuring 52 x 35 x 18 cm), with stainless steel mesh floors and lids suspended in batteries of 21 cages above crêpe absorbent paper. The latter was changed three times a week. Five rats of one sex were held in each cage. Cages comprising the different treatment groups were distributed in such a way as to equalise, as far as possible, the effect of any spatially-variable component of the environment.
- Diet: A complete powdered rodent diet, ad libitum prior to and during the treatment period.
- Water: ad libitum via two polythene bottles with chromium plated sipper tubes.
- Acclimation period: 7 days. Rats that failed to gain weight satisfactorily were discarded.

DETAILS OF FOOD AND WATER QUALITY:
The drinking water was sampled and analysed for chlorinated hydrocarbon, polychlorinated biphenyl and organophosphorus contamination on eight, seven and six occasions, respectively, during the treatment period. The food was monitored for chlorinated hydrocarbon, aflatoxin and polychlorinated biphenyl contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
6 Apr 1978 to 21 Aug 1980

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
For the treated groups, the test substance was incorporated into the powdered diet at the required concentrations. A pre-mix was prepared each week, and from this the required dietary concentrations were obtained by direct dilution with further quantities of diet. After Week 44, the pre-mix was wetted before dilution. Homogeneity was achieved by mixing for 20 minutes in a horizontal screw-type mixer.
- Rate of preparation of diet: Weekly.
- Mixing appropriate amounts with: Complete powdered rodent diet.
- Storage temperature of food: After formulation diets were sealed in transparent polythene bags (bearing a colour-coded label identical to that used to identify the cages of the appropriate group) within light-proof polythene bags.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determination of the concentrations of test substance in diet was accomplished by a method supplied by the sponsor. Homogeneity of test substance in the final mix was measured using six samples from each dietary concentration using diet prepared for the first and forty-fourth weeks of treatment. Stability of test substance in the diet stored at room temperature was measured over 14 days using diets prepared for the first week of treatment. The achieved concentrations of test substance in the test diets were determined at fortnightly intervals. All samples were assayed in duplicate, and on each occasion recovery from spiked samples was used as a measurement of assay efficiency. Cumulative recovery values were used as the study progressed. Additional checks of achieved concentration were carried out by the sponsor at weeks 27, 53, 79, 107 and 124.

Duration of treatment / exposure:

Interim sacrifice at 52 weeks of treatment.
Terminal sacrifice of males after 113 weeks of treatment.
Terminal sacrifice of females after 122 weeks of treatment.

Frequency of treatment:

Daily via food

Post exposure period:

None

Dose / conc.:

25 ppm

Remarks:

Group 3. Dietary concentration for test substance cation; equivalent to 1.0 and 1.3 mg/kg bw/day for males and females, respectively.

Dose / conc.:

75 ppm

Remarks:

Group 4. Dietary concentration for test substance cation; equivalent to 3.1 and 3.9 mg/kg bw/day for males and females, respectively.

Dose / conc.:

150 ppm

Remarks:

Group 5. Dietary concentration for test substance cation; equivalent to 6.3 and 7.9 mg/kg bw/day for males and females, respectively.

No. of animals per sex per dose:

Interim sacrifice: 10 animals/sex/dose (1 control group and 3 treatment groups).
Terminal sacrifice: 70 animals/sex/dose (2 control groups and 3 treatment groups).

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dietary concentrations of 25, 75 and 150 ppm of the test substance were selected, based on the results of a preliminary dose-range finding study performed.
- Rationale for animal assignment: The animals were initially segregated into equal weight distribution groups. Any rats in poor health were discarded. Rats were then allocated to the different treatment groups by random selection of five rats of one sex per cage. This procedure ensured that all groups contained populations of rats characterised by similar initial mean and range of body weight.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical signs and mortality: rats were inspected twice daily during the treatment period for evidence of reaction to treatment or ill-health. Any rat displaying severe debility or intoxication was isolated. If death appeared imminent (i.e. where the condition of an animal indicated that it was unlikely to survive overnight) the animal was killed in extremis. From week 118 onwards only one check was carried out on each day of a weekend or public holiday.
- Cage side observations included: Any deviations from normal were recorded in respect of nature and severity, date of onset, duration and progress of the observed condition.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly.
- Detailed clinical observation included: All rats were handled and palpated for the purpose of detecting superficial tumours. The outcome of this examination was recorded for every animal. The date of appearance, location, consistency, size and subsequent history was recorded for all visible and palpable swellings.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded on the day that treatment commenced, at weekly intervals for the first 12 weeks and at fortnightly intervals until week 68, when weekly intervals were again instituted for the remainder of the treatment period to provide more frequent checks on the health status of individual animals.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: The quantity of food eaten by the rats in each cage was calculated weekly from measurements of the amount of food given, that remaining in the food hoppers and an estimate of food spilled.
- The group mean achieved dosages, expressed in terms of mg/kg bw/day, were calculated for the same intervals as those used for recording individual body weights.

FOOD EFFICIENCY: Yes
- The efficiency of food utilisation was assessed from the calculation of the food conversion ratio (i.e. the amount of food consumed per unit of body weight gain). During the early period of rapid growth the food conversion ratio was computed for weekly intervals up to week 12. Overall calculations for weeks 1 - 12, 13-26, 27-40 and 41-52 were also performed.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was subjected to visual observation daily, and quantitative measurements were carried out over three day periods during weeks 1 to 4, 13, 26, 41, 52, 65, 78, 92 and 101.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Before commencement of treatment, both eyes of all rats from each group were examined by means of a binocular indirect ophthalmoscope, approximately 20 minutes after the instillation of tropicamide 0.5%. Several rats found to have retinal lesions at this examination were replaced by spare animals, from the same batch, without ocular defects. At 4, 14, 26, 52 and 79 weeks of treatment, both eyes of the last surviving 20 males and 20 females from each group (ten of each sex from each batch of animals) were examined in a similar manner. The examination at 103 weeks of treatment included all surviving rats as some lenticular changes were seen on previous occasions. As the treatment period was extended beyond 104 weeks, additional ophthalmoscopic examinations of all surviving rats were also performed at approximately eight-week intervals. Each sex was also subjected to a terminal ophthalmic examination before sacrifice.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Before commencement of treatment, blood samples were withdrawn from the retro-orbital sinus of ten male and ten female rats (five spare rats of each sex from each batch) sacrificed for this purpose.
After 14, 26, 40, 53, 66, 79, 92 and 102 weeks of treatment, blood samples were withdrawn from the retro-orbital sinus of ten male and ten female rats from each group.
As the study was extended beyond 104 weeks of treatment, additional blood samples were taken before the sacrifice of either sex. The first five surviving rats of each sex, group and batch were used to provide blood samples, which were examined for the characteristic listed in Table 1 in ‘Any other information on results incl. tables’.
Additional studies were carried out as follows:
After 17 weeks of treatment platelet counts and prothrombin and partial thromboplastin times, for five males from each of Groups 2 (Control) and 4 (75 ppm).
After 29 weeks of treatment, reticulocyte counts for five males and five females from each of Groups 1 (Control) and 5 (150 ppm) and from five females of Group 4 (75 ppm), together with prothrombin and partial thromboplastin times for five females from each of Groups 1 and 4.
After 54 weeks of treatment, prothrombin and partial thromboplastin times for five males from each of Groups 1 and 3 (25 ppm) and for five females from every group.
After 96 weeks of treatment, prothrombin and partial thromboplastin times for five females from each of Groups 2 and 3 (25 ppm).
- Anaesthetic used for blood collection: Yes, under ether anaesthesia, using EDTA as anticoagulant except for prothrombin and partial thromboplastin times, for which citrate was utilised.
- Animals fasted: Not specified.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the same intervals during the treatment period and from the same rats used for haematology, further blood samples were taken, using lithium heparin as anticoagulant.
- Animals fasted: Not specified
- Parameters examined are listed in Table 2 in ‘Any other information on results incl. tables’.

URINALYSIS: Yes
- Time schedule for collection of urine: Before commencement of treatment, urine samples were collected from five spare males and five spare females of each batch.
After completion of 13 (Batch 1 and females of Batch 2), 15 (males of Batch 2), 26, 39, 52, 65, 77 (Batch 1) or 78 (Batch 2) 92 and 101 weeks, samples were collected, if possible, from the ten males and ten females of each group used for haematology (five of each sex from each batch). Additional samples were also collected from five rats from each batch before terminal sacrifice of either sex.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters examined are listed in Table 3 in ‘Any other information on results incl. tables’.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Ten males and ten females from each group after 52 weeks and all rats surviving until the end of the life-span treatment period were killed by carbon dioxide asphyxiation. These rats, together with those sacrificed in extremis and those found dead in their cage, were subjected to the following procedures:
- A detailed necropsy, involving opening of the cranial, thoracic and abdominal cavities was performed. During this, the appearance of normal and abnormal tissues was noted and abnormalities recorded in detail.
The external features of the animal were scrutinised and compared to any relevant comments on the clinical history report. The first incision allowed rapid preparation of a femoral bone marrow smear except in animals found dead in their cages. The eyes, complete with optic nerve, and relevant adnexa, were removed. The cranial cap was lifted and the brain dissected free of meninges. The pituitary was freed from the sella turcica and fixed separately. The ventral abdominal skin was reflected to allow observation of the subcutaneous structures, in particular, mammary glands and superficial lymph nodes. Abdominal and thoracic viscera were examined in situ, and note made of any abnormal position, morphology or interactions. The urinary bladder was slightly inflated with fixative and the urethra ligated. After removal, the bladder mucosa was examined after fixation at the time of embedding.
The entire intestinal tract was re-examined after removal. The stomach was opened along its greater curvature, and rinsed in isotonic saline, prior to fixation. The caecum was similarly treated. After weighing, the major organs were scrutinised and, where appropriate, the cut surfaces were examined. The lungs were slightly inflated with fixative via the trachea, prior to immersion fixation. The vertebral column was removed and fixed intact.
Prior to the disposal of the carcase, a senior prosector checked the retained tissue against the working protocol and considered the necropsy report in detail.
- Organ weight analysis: adrenal glands, brain, heart, ovaries, pituitary gland, spleen, kidneys, testes, liver, lungs, thymus, thyroid glands (after fixation). Paired organs were weighed separately, and their weights combined for analysis. The ratio of organ weight to body weight was calculated for each animal at the interim and terminal sacrifices.

HISTOPATHOLOGY: Yes
Together with all macroscopically observed abnormalities, the following tissues, from all rats dying during the study, or formally sacrificed after 52 weeks or at termination were processed and examined: adrenal glands, aortic arch, blood and bone marrow smears, bone, brain, caecum, colon, duodenum, epididymides, eyes and optic nerves (both were sectioned), harderian gland (left and right), heart, ileum, jejunum, kidneys (left and right), liver (left, right and median lobes), lungs, (left and right lobes, mainstem bronchi), lymph nodes – cervical/mesenteric, mammary gland - posterior/anterior, middle ear, oesophagus, ovaries (left and right), pancreas, pituitary gland, prostate gland, salivary gland (left and right), sciatic nerve (left and right), seminal vesicles, skeletal muscle, skin, spinal cord (cervica, thoracic and lumbar), spleen, stomach, testes (left and right), thymus, thyroid glands, tissue masses and suspected tumours and regional lymph nodes, tongue, trachea, turbinal epithelium, urinary bladder, uterus, uterine cervix.
Blood and bone marrow smears were air-dried and then fixed in methanol, and the eyes were placed in Davidson's fluid. All other tissues were preserved in buffered 4% formaldehyde saline. Blood and marrow smears were stained by a May-Grunwald Giemsa procedure. The remaining tissues were subjected to dehydration and embedding in paraffin wax, sections of the required tissues were cut at 5 µm thickness and stained with haematoxylin and eosin. Both glandular and non-glandular regions of the stomach, and both auricular and ventricular sections of the heart, were thus prepared. The brain was sectioned at three levels (cerebellum, cerebral cortex and medulla). The preserved tissues listed in the section above were examined microscopically from all rats, except those animals which were killed for the specific purpose of taking tissues for determination of their test substance content.

Other examinations:

TEST SUBSTANCE CONCENTRATION IN TISSUES AND URINE
- After 52 weeks of treatment, the five surviving rats with the highest identity numbers of each of Groups 1, 3, 4 and 5 of the first batch were killed by carbon dioxide inhalation. The following tissues were taken and weighed from each rat and individually assayed by the sponsor for test substance content: liver, lungs, kidneys, skin, plasma.
- After 15, 27, 41, 52, 65, 79, 92 and 102 weeks of treatment pooled urine samples were collected from the rats of each sex and group in both batches and analysed for test substance concentrations.

Statistics:

Inter-group variations in mortality and incidence of palpable masses were assessed by 2x2 contingency tests (used as two-tailed tests), deriving a Chi square value. This test was supplemented for analysis of mortality in relation to evaluation of tumour distribution, by Cox's test, incorporating Tarone's test for linear trend. The analysis of incidences of pathological change was performed using 2x2 contingency tables deriving a value for Chi square or by computing the exact probability of the distribution (Fisher's exact test two-tailed).
Each treated group was compared with the combined incidence in the control groups. The analysis of incidences of pulmonary tumours was performed by trend test and Fisher’s exact test (using the National Cancer Institute Statistics Computer programme).
Statistical evaluation, by Student's t-test using a pooled within-group error variance, was performed on the following: Overall food consumption, body weight (at weeks 26, 52, 78, 104, 113 or 117 and 122), haematology, blood chemistry, absolute organ weights, body weight-relative organ weights.
Some organ weight variations were also assessed by analysis of co-variance with body weight. Unless stated otherwise, group mean values were not significantly different from controls (p> 0.05).
Armitages test was used to evaluate trends in the normoblasts recorded during the examination of blood smears taken at termination of the study.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Gross clinical assessments showed that opacity of one or both eyes was observed more frequently among rats receiving 150 ppm and among females receiving 75 ppm than among controls.
The inter-group distribution of rats with one or more palpable masses was not overtly disturbed by treatment. Females receiving 150 ppm bore fewer palpable masses than control females. In other respects the physical condition and behaviour of rats treated with test substance remained similar to those of control animals.
During the second year of the study an icteric condition became evident in a number of animals. These, however, were distributed among the groups without any indication of a treatment-relationship.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The distribution of mortality was unaffected by treatment. There was approximately 50% mortality in all groups at the end of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the first year of treatment moderate depression of body weight gain was recorded for males receiving 150 ppm, leading to significantly lower (p<0.001) mean body weight values after 26 and 52 weeks of treatment. During the second year of treatment the body weight change of these animals remained similar or superior to that of controls, although the mean body weight was significantly lower than the corresponding control value after 78, 104 (p<0.001) and 113 weeks (p<0.05). Minor depression of weight gain was noted in the first six months for males receiving 75 ppm and from week 26 onwards. The group mean body weights of males receiving 25 ppm remained similar to control values.
There was no effect upon the growth of female rats during the first six months of treatment. By the end of week 52, however, the mean body weight of females receiving 150 ppm was slightly but significantly (p<0.05) lower than the overall control mean. The mild depression of body weight gain by females receiving 150 ppm continued during the second year of treatment. The body weight gain of females receiving 75 or 25 ppm was unaffected.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

During the first year of treatment, a slight but persistent depression of food intake was evident among males receiving 150 ppm, while females receiving 150 ppm displayed reduced appetite only during the first six weeks of treatment. The food intake of rats receiving 25 or 75 ppm was not disturbed by treatment.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

There were no marked inter-group differences in food conversion ratios, although from week 13 onwards males and females receiving 150 ppm exhibited slightly inferior efficiency of food utilisation compared with the controls.

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

no effects observed

Description (incidence and severity):

Water consumption was not significantly affected by treatment.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related ocular lesions were not observed before 52 weeks. Afterwards, ophthalmoscopy showed a dosage-related increase in the progression and severity of lenticular cataract in the 150 ppm group and towards the end of the study (from 103 weeks) in the 75 ppm group. Up to and including 103 weeks of treatment no effect was observed in the low dosage group (25 ppm). In the male animals given 25 ppm there were, during the last few weeks of dosing (109-113 weeks), mild ocular changes in some animals that were possibly associated with treatment. However, because of the appearance of this only at the end of the lifespan, and because of the large number of spontaneous cataracts in the F 344 rat, 25 ppm was considered to lie close to the no-effect level for lenticular change.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no inter-group differences of toxicological significance in the haematology.
There were no inter-group differences between treated and control animals that were sufficiently persistent or marked as to be indicative of a response to the treatment. A number of inter-group differences in mean values did attain a level of statistical significance.
Examination of bone marrow smears did not reveal any changes attributable to treatment with the test substance.
After 14 weeks of treatment minor depression of red cell parameters (packed cell volume and erythrocyte count) was recorded for rats receiving 150 ppm. This change was not apparent at subsequent examinations and if related to treatment, was therefore a transient response. Rats receiving 25 or 75 pm were not affected.
After 26, 40 and 53 weeks of treatment slight or moderate depression of total leucocyte count was evident in treated males, predominantly those receiving 150 ppm. This trend was less apparent during the second year of treatment and statistical significance attached only to lower neutrophil counts in the high dosage group after 66 and 92 weeks. Values throughout the study, however, were within normal limits and these changes were of doubtful toxicological significance. Values for treated females were undisturbed.
Slight prolongation of partial thromboplastin time was observed after 66 and 102 weeks of treatment in males receiving 150 ppm. Minor prolongation without attainment of statistical significance was evident in these animals on other occasions, viz. after 40, 53, 92 and 111 weeks. Other treated groups were not affected. Slight depression of platelet count, also in males receiving 150 ppm, was evident after 53 and 66 weeks. Variations from control values were very small, and all values were within the normal range. It was concluded therefore that these changes had no toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A number of the differences between group mean values for control and treated animals attained a level of statistical significance (p<0.05). Most of these differences, however, involved values for the low or intermediate dosage groups and were confined to only one of the sexes, and there was no persistent change in any single parameter. It was concluded, therefore, that these inter-group variations arose by chance and do not reflect any response to the treatment.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis did not reveal any changes relating to treatment.

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):

For the interim sacrifice: most of the group mean body weight-relative organ weights of males receiving 150 ppm were statistically significantly elevated, but this reflected their lower body weight. A notable exception was the liver, which was the only organ of the high dosage males to exhibit significant (p<0.05) depression of absolute weight. These observations suggested that the depression of the liver weight in males receiving 150 ppm was somewhat greater than that to be expected from the decrease in body weight gain, but this inference was not supported by the analysis of co-variance of liver weight and body weight, which indicated that the mean liver weight of males receiving 150 ppm was significantly higher (p<0.01) than the overall control mean, after correction for body weight difference. Similar analysis of liver weights for the corresponding female groups showed that there was no significant difference between the control and high dosage animals. The body weight-relative lung weights of males receiving 75 or 150 ppm were heavier than those of the controls. The group mean values for these treated groups were, however identical to the mean background data value. The differences from control values in this case were therefore not considered to be of toxicological significance.
Other statistically significant organ weight differences in treated males were restricted to the lower and intermediate dosage groups and, in the absence of similar change in the males at the high dosage, were considered to have arisen by chance. The elevation of absolute brain weight among females receiving 150 ppm, which was the only inter-group difference in the female groups to attain a level of statistical significance, was also considered to be a chance event.
Sacrifice at termination: The mean absolute organ weights of males receiving 150 ppm were generally lower than the corresponding control values, largely as a result of the lower body weight of the treated animals. Contrary to expectation, however, the body weight-relative weights of liver and testes in these animals were also significantly depressed. Analysis of co-variance with body weight also revealed significant depression of these organ weights and this conformity may be considered as evidence of a direct effect of treatment upon these organs in addition to any body weight-related change.
Neither of these effects extended to rats receiving 25 or 75 ppm, but analysis of co-variance demonstrated significant depression of liver weight among females receiving 150 ppm although the mean body weight-relative value for these animals was not disturbed.
Apparent increases in the mean relative lung weights in males receiving 75 or 150 ppm and females receiving 150 ppm were not supported by analysis of co-variance, and thus the toxicological significance of this finding is low.
Other inter-group differences which attained statistical significance are ascribable to body weight variation or, as in the case of pituitaries, resulted from marked age-related change in a few individuals.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There was a wide range of lesions present in the animals examined. These were considered not to be significant and did not increase in degree or incidence when the treated animals were compared with the controls. In the animals in the interim sacrifice the small number of changes present were minor in character and were considered incidental since they were typical of those commonly found in this strain of rat.
In the animals at termination, there was a marked treatment-related increase in the incidence of focal subpleural changes (pale subpleural foci/areas). The pulmonary changes were most marked in the highest dosage group male rats and the two highest dosage group female rats. There was a range of commonplace lesions present in the animals examined which were not associated with treatment. Such changes included emaciation; small epididymides; renal cysts and surface irregularity; pulmonary congestion and petechiae; large, congested and prominent cervical lymph nodes; prominent and activated mammary tissue; cystic ovaries; large and haemorrhagic pituitaries; firm and soft subcutaneous masses; large and swollen spleens; gastric ulceration; small, soft, enlarged and also occasionally undescended testes; areas of uterine distension. There was a virtually universal incidence of testicular subcapsular plaques.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Interim sacrifice: There were no lesions that were considered to be related to treatment with the test substance. There was a wide range of banal degenerative and inflammatory changes similar in type and incidence to those commonly found in F 344 rats.
Animals dying or killed in extremis: There were no lesions that were considered to be related to treatment with test substance. There was a range of banal degenerative and inflammatory changes similar in extent and type to those commonly found in F 344 rats.
In animals dying after the interim sacrifice, changes that were considered possibly to be related to treatment with the test substance were as follows: In the brain there was a higher incidence of hydrocephalus in treated female groups than in the controls but dosage-relationship was not evident. In the sciatic nerve there was a higher incidence of degeneration of a small number of fibres, which was not marked or severe (in the individual histopathology reports termed “apparent degeneration of nerve fibres") in males receiving 75 or 150 ppm, the incidence was highest at the high dosage. In treated groups there was a higher incidence of cysts/cystic spaces in the spinal cord.
There was a wide range of mundane degenerative and inflammatory changes present, similar in type and incidence to those considered usual in F 344. The only change considered to be related to treatment was a higher incidence of slight hydrocephalus in females receiving 150 ppm than in the controls.
Changes that possibly associated with treatment with the test substance comprised hydrocephalus, degeneration of the nerve fibres of the sciatic nerve, and an increase in the numbers of cyst/cystic spaces in the spinal cord. However, there were inconsistencies in the incidences of these lesions between animals which died during the treatment period and those killed at termination. Since the distribution of mortality was unaffected by treatment it was considered appropriate to combine the incidences of hydrocephalus for all animals. Analysis in this way demonstrated that there was a statistically significant (p<0.05) incidence of hydrocephalus in females that received 75 or 150 ppm. Males were unaffected at any dosage.
The degree of hydrocephalus (here seen as dilatation of the fourth ventricle) was not marked in any case. Viewed overall, the higher numbers of decedents with degeneration of the sciatic nerve fibres contributed to a statistically significant increase in incidence (p<0.001) in males at the intermediate or high dosage. Females were unaffected at any dosage. However, the disproportionate incidences in decedents and in terminal animals, and the lower incidence in male controls than in female controls indicate that there was probably an element of chance to the group distribution of this mild lesion and its toxicological significance is therefore low.
The group distribution of animals with cysts/cystic spaces in the spinal cord was remarkable for the extremely low incidence amongst animals killed at termination. In addition, although six control females (from a total of 119 animals) had this lesion, it was absent from all control males, and the incidences in the female control and treated groups were similar to the incidences in the male treated groups. It therefore appears most likely that the absence of this lesion in the male control groups was fortuitous, and that the apparently significant incidence in all male treated groups was a consequence of this. It is considered therefore that the incidences of cysts or cystic spaces in the spinal cord were unrelated to the treatment.
Proliferative lung lesions in this study were not easily compartmentalised into non-neoplastic or neoplastic, or into adenoma or carcinoma. Strong evidence suggested that the test substance induced a proliferative lesion of the alveolar epithelium in the mid- and high dose group. There was also some evidence of a possible treatment-related inflammatory reaction in the lungs since, when all incidences of macrophages present in the alveolar tissue were combined for animals which died from week 53 or were killed at termination, the numbers of animals affected in the intermediate or high dosage groups were higher than those in the controls.
The test substance accelerated, in a dosage-related manner, the onset and progression of a cataractous change that is a normal feature of the ageing F 344 rat.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Interim sacrifice: There were a few neoplasms present, none of which was associated with the treatment.
Animals dying or killed in extremis: There were a few neoplasms found in animals which died before the interim sacrifice, none of which was associated with the treatment. In animals dying after the interim sacrifice, there was a low incidence of squamous cell carcinoma in the head region. In some cases the sites of origin could be discriminated (hard palate, auditory canal, skin and subcutis). In other cases such discrimination was not possible (because of extensive involvement of numerous tissues and structures). This uncommon tumour was present in the head region of three control males, two males receiving 25 ppm, three females receiving 75 ppm and six males and two females receiving 150 ppm.
There was a range of other neoplasms present which were similar in type and incidence to those considered usual in F 344 rats. These included mammary gland benign fibroepithelial tumours, pituitary adenomas and carcinomas, thyroid parafollicular cell adenomas, benign adrenal pheochromocytomas, skin and subcutis fibromas and lipomas and monocytic leukaemia. In some treated groups these tumours were present at higher incidences than in the controls. The higher number of pituitary adenomas in females receiving 75 ppm was considered unlikely to constitute an effect of the treatment since the increase was not sustained in the high dosage group.
A slightly higher incidence of fibromas in the intermediate dosage group male was a consequence of an unusually low incidence in the control groups (background data in males is 46/235). The higher incidence of lipomas in males receiving 150 ppm was not supported by the findings at termination.
There were no differences in the distribution of benign and malignant neoplasms or in the number of different neoplasms per animal.
Animals sacrificed at termination: There was a wide range of neoplasms present, similar in type and incidence to those considered usual in F 344 rats. These included mammary gland benign fibroepithelial tumours, pancreatic islet cell adenomas, pituitary adenomas, thyroid parafollicular cell adenomas and carcinomas, testicular interstitial cell tumours, skin and subcutis fibromas and lipomas and monocytic leukaemia. The group distribution of these tumours was unrelated to the treatment, and there were no differences in the group distribution of benign or malignant tumour types.
A wide range of tumour types was observed in treated and control animals, and there was no evidence that treatment with test substance resulted in a marked change in the group distribution of any of these tumours. In some cases it was possible to determine the site of origin, but in others the involvement with numerous tissues was so extensive that discrimination was not possible. However, if the tumours arising at different sites in the head, or those too generalized to categorize, are considered independently, there is no significant difference between the incidence in treated and control animals. Furthermore even if all cases of squamous cell carcinoma are combined (excluding those in the lungs) there is no statistically significant difference between any treated group and the controls, nor is there any evidence for dosage-relationship. It is likely, therefore, that the distribution of these tumours was a chance event.
In the lungs, the most common neoplastic change seen was leukaemia, characterised by large numbers of leucocytes within the major vessels and distending the alveolar capillaries. The term 'incipient leukaemia” was used when the appearance of the lung was suggestive of a leukemic process but there was insufficient evidence on the basis of the lung sections alone to allow a definitive diagnosis. The leukaemia appeared to be predominantly monocytic in type and is well recognised as a common spontaneous disease in F344 rats. It occurred in all groups and was more common in males than females. The incidence of leukaemia did not show a relationship to treatment.
Small numbers of adenomas and carcinomas, which were considered to be primary in the lung, were seen. Carcinomas showed a variety of forms. Most frequent were adenocarcinomas showing proliferation of abnormal epithelium, with glandular or acinar formation, bizarre nuclei with frequent mitoses, obliteration of the normal architecture and invasion of surrounding structures. There was a variable degree of stromal fibrosis and some tumours showed areas of necrosis and dystrophic calcification. Some squamous cell carcinomas were seen also, while other carcinomas appeared to be of mixed glandular and epidermoid (adeno-squamous) type. The incidence of both adenomas and carcinomas did not show an association with the administration of the test substance.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

After 52 weeks of treatment the concentration of test substance in urine samples was found to be dosage-related. No test substance was detected in control urine samples. Test substance was detected in the plasma and kidneys of rats of all treated groups sacrificed after 52 weeks of treatment. In both sexes receiving 150 ppm and males receiving 75 ppm test substance was present in the skin. In rats receiving 75 or 150 ppm, test substance was present in the lungs, but only in females receiving 150 ppm was test substance found in the liver.

Dose descriptor:

NOAEL

Effect level:

>= 150 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Original value presented in study. Dietary concentration for test substance cation; equivalent to 6.3 and 7.9 mg test substance cation/kg bw/day for males and females, respectively.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 10.9 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Recalculated value for the dietary concentration for pure test substance, see ‘Any other information on results incl. tables’ for respective calculation

Key result

Critical effects observed:

no

Verification of homogeneity of experimental diets

Serial analysis of samples of the experimental diets mixed for the first week of the study showed that there was no appreciable change in the concentrations of test substance during a subsequent period of 14 days when the diets were stable at normal animal room temperatures.

Achieved concentrations of test substance in the experimental diets were in tolerable agreement with the intended concentrations. Additional checks were carried out by the sponsor on diets mixed for weeks 27, 53, 79, 107 and 124 and the results of these were also in reasonable agreement with the intended values.

Serial analysis of samples of the test material, carried out by the sponsor at intervals during the study, showed that the test substance cation content remained uniform and hence the batch of material provided for the study did not deteriorate as the study progressed.

Calculation of key result

The doses of the test substance were expressed in test substance cation, which relates to the cation species in an aqueous solution of the registered substance. The effect levels are already corrected for the amount of water. The key effect levels are calculated by inclusion of the anion species:

(100/72.4) x 7.9 mg test substance cation / kg bw = 10.9 mg pure pure test substance/ kg bw.

Conclusions:

In this lifetime feeding study, not performed under GLP or according to a OECD guideline, no carcinogenic potential of the substance was observed. Therefore the NOAEL for carcinogenicity is higher than 150 ppm (recalculated dietary equivalent value: 10.9 mg pure test substance/kg bw/day (for females)).

Executive summary:

In this lifetime feeding study, not performed under GLP, similar to OECD 453, Fischer 344 rats were fed diets containing the test substance for 113-117 weeks (males) and 122-124 weeks (females). At 52 weeks, 10 animals per group were sacrificed for interim assessment. Five groups, two control and three treatment groups, consisted of 80 animals of each sex. The treatment groups were fed a diet containing 25, 75 or 150 ppm test substance. This was equivalent to a dietary exposure of 1.0, 3.1 and 6.3 mg/kg bw/day for males and 1.3, 3.9 and 7.9 mg/kg bw/day for females. Twice a day animals were checked for gross clinical abnormalities. Detailed clinical observations and ophthalmoscopic examinations were performed throughout the study. Body weights, food consumption and efficiency were recorded during the study at weekly intervals. Compound intake was calculated as time-weighted averages from the consumption and body weight gain data. A variety of haematological and biochemical investigations was made at intervals throughout the study. Furthermore, urine was checked biochemically and microscopically. At termination, the rats were subjected to macroscopic and microscopic pathological examinations and a selection of organs was weighed. Test item content in urine and several organs was analysed at termination.

The distribution of mortality was unaffected by treatment. There was approximately 50% mortality in all groups at the end of the study. At 150 ppm, statistically-significant reductions in body-weight gain (10-34% during weeks 11-68 for males and 11-34% during weeks 27-78 for females), food consumption (3-8%), and efficiency of food utilisation in both sexes were observed (12-21% in males and 11-13% in females during weeks 13-52). There was a statistically significant depression of body weight gain in the first year of the study in males receiving 75 ppm. Water consumption was not significantly affected at any dietary level tested.

Microscopic examination of lung tissues indicated that treatment at 150 ppm, in both sexes, and possibly at 75 ppm in males, was associated with proliferative lesions of the alveolar epithelium. These lesions were not easily classified into non-neoplastic or neoplastic, nor into adenoma or carcinoma. This study provided strong evidence for the induction by the test substance of a proliferative lesion of the alveolar epithelium and some controversial evidence for the induction of lung adenomas in female Fischer 344 rats. There was no treatment-related increase in the incidence of lung adenocarcinoma at any dose level in either sex. At 25 ppm, significant increases in the incidence of proliferative lung lesions, compared to the controls, were not observed. Increased relative weight of the lungs (lung/body weight ratio) in the males (16%) and the females (14%) was seen in animals sacrificed at the termination of the study. Slight dilation of the fourth ventricle of the brain was evident in females receiving 150 (36%) or 75 ppm, but not in males at these dosages or in either sex at 25 ppm. A statistically significant increase in the incidence of apparent degeneration of occasional/several sciatic nerve fibers was noted in decedent males receiving 75 or 150 ppm. Both hydrocephalus and nervous tissue changes were considered by the authors of the study possibly to be associated with the treatment. Pathology summaries indicate that atrophy of the testes was recorded in the high-dietary group (5/33) but not in controls at termination, and moderate lymphoid hyperplasia was observed in the respiratory epithelium of males receiving 75 and 150 ppm and dying between 52 weeks and termination.

The above findings show that the test substance enhanced the development of ocular lesions in all of the treated groups. The predominant lesions detected ophthalmoscopically were lenticular opacities and cataracts. These lesions were either not observed or were observed infrequently before week 103. At week 103, dose related statistically significant increases in the incidence of ocular lesions were observed only in the mid-dose and high-dose male and female groups. Based on these findings, no carcinogenic potential of the substance was observed. Therefore the NOAEL for carcinogenicity is higher than 150 ppm (recalculated dietary equivalent value: 10.9 mg pure test substance/kg bw/day (for females)).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

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

Additional information

Rat - 24 months

In this lifetime feeding study, not performed under GLP, similar to OECD 453, Fischer 344 rats were fed diets containing the test substance for 113-117 weeks (males) and 122-124 weeks (females) (Woolsgrove, 1983). At 52 weeks, 10 animals per group were sacrificed for interim assessment. Five groups, two control and three treatment groups, consisted of 80 animals of each sex. The treatment groups were fed a 25, 75 or 150 ppm test substance in their food. This was equivalent to a dietary exposure of 1.0, 3.1 and 6.3 mg/kg bw/day for males and 1.3, 3.9 and 7.9 mg/kg bw/day for females. At termination, the rats were subjected to macroscopic and microscopic pathological examinations and a selection of organs was weighed. Test item content in urine and several organs was analysed at termination.

The distribution of mortality was unaffected by treatment. There was approximately 50% mortality in all groups at the end of the study. At 150 ppm, statistically-significant reductions in body-weight gain (10-34% during weeks 11-68 for males and 11-34% during weeks 27-78 for females), food consumption (3-8%), and efficiency of food utilisation in both sexes were observed (12-21% in males and 11-13% in females during weeks 13-52). There was a statistically significant depression of body weight gain in the first year of the study in males receiving 75 ppm. Water consumption was not significantly affected at any dietary level tested. The test substance accelerated, in a dosage-dependent manner, the onset and progression of cataract changes, ranging from minor opacity to total cataract in both males and females. Treatment related ocular lesions were first seen at 52 weeks. Thereafter, ophthalmoscopy revealed a statistically significant dosage-related increase in the incidence, progression, and severity of lenticular cataract in the 150 ppm group and, toward the end of the study (103 weeks), in the 75 ppm group. However 25 ppm was the NOAEL for lenticular cataracts in both males and females at termination (week 112/113 for males and week 118/119 for females). A statistically significant higher incidence of secondary eye lesions was found at termination in females receiving 75 or 150 ppm when compared to controls.

Haematological investigation indicated no significant effects attributable to the test substance. Urinalysis did not reveal any treatment related changes. Reductions in liver and testicular weights were noted at termination in the 150 ppm dietary group. Macroscopic examination at necropsy revealed a treatment-related increase in the incidence of focal subpleural changes in animals killed at termination in all dietary groups. This effect was most marked in females receiving 75 ppm and in both sexes receiving 150 ppm. Microscopic examination of lung tissues indicated that treatment at 150 ppm, in both sexes, and possibly at 75 ppm in males, was associated with proliferative lesions of the alveolar epithelium. These lesions were not easily classified into non-neoplastic or neoplastic, nor into adenoma or carcinoma. This study provided strong evidence for the induction by the test substance of a proliferative lesion of the alveolar epithelium and some controversial evidence for the induction of lung adenomas in female Fischer 344 rats. There was no treatment-related increase in the incidence of lung adenocarcinoma at any dose level in either sex. At 25 ppm, significant increases in the incidence of proliferative lung lesions, compared to the controls, were not observed. Increased relative weight of the lungs (lung/body weight ratio) in the males (16%) and the females (14%) was seen in animals sacrificed at the termination of the study. Slight dilation of the fourth ventricle of the brain was evident in females receiving 150 (36%) or 75 ppm, but not in males at these dosages or in either sex at 25 ppm. A statistically significant increase in the incidence of apparent degeneration of occasional/several sciatic nerve fibers was noted in decedent males receiving 75 or 150 ppm. Both hydrocephalus and nervous tissue changes were considered by the authors of the study possibly to be associated with the treatment. Pathology summaries indicate that atrophy of the testes was recorded in the high-dietary group (5/33) but not in controls at termination, and moderate lymphoid hyperplasia was observed in the respiratory epithelium of males receiving 75 and 150 ppm and dying between 52 weeks and termination.

The above findings show that the test substance enhanced the development of ocular lesions in all of the treated groups. The predominant lesions detected ophthalmoscopically were lenticular opacities and cataracts. These lesions were either not observed or were observed infrequently before week 103. At week 103, dose related statistically significant increases in the incidence of ocular lesions were observed only in the mid-dose and high-dose male and female groups. Based on these findings,no carcinogenic potential of the substance was observed. Therefore the NOAEL for carcinogenicity is higher than 150 ppm (recalculated dietary equivalent value: 10.9 mg pure test substance/kg bw/day (for females)).

Mice - 24 months

In this lifetime feeding study, performed under GLP similar to OECD 453, Swiss-derived mice, were administered technical test substance incorporated into the diet of to give dose levels of 0, 0 (two control groups), 12.5, 37.5 and 100/125 ppm test substance cation for 97 to 99 weeks (when mortality was approaching 80% in all groups) (Sotheran, 1981). Each group consisted of 60 animals per sex per dose. In addition, a satellite group of 10 per sex per group was designated for interim sacrifice after 52 weeks. At week 36 the top dose level was increased from 100 to 125 ppm because no toxic signs had appeared after 35 weeks of dosing. These doses corresponded to a dietary intake of 0, 0, 1.87, 5.6 and 15.0/18.7 mg test substance cation/kg body weight/day. The diets were analysed for homogeneity, stability and achieved concentration. All mice had free access to tap water and treated or control diet except when food was removed overnight prior to urine collection. Mice were examined routinely for mortality, clinical abnormalities, masses, body weight and food consumption. Urinary test substance determinations were performed every 13 weeks on 10 mice/sex/group. Tissue (lung and kidney) and plasma test substance levels were determined in samples from the satellite group at termination.

After 52 weeks all surviving mice in the satellite groups were killed and given a macroscopic examinationpost mortem– only abnormal tissues were preserved. All surviving mice in the main study were killed after 97-99 weeks of treatment. Each animal was given a detailed macroscopic examination and a comprehensive selection of tissues were taken and examined histopathologically for neoplastic and non-neoplastic changes.

Dietary analyses showed the majority of batches to be within 10% of the nominal values, with adequate homogeneity and stability. Clinical signs were consistent between groups though an increase in genital sores and decreased hair loss were noted in the top dose females. Mortality rates were increased in intermediate dose males (week 43 onwards) and top dose females (week 68 onwards) – the former is probably a chance finding due to the lack of a corresponding finding in top dose males. Survival was >45% in all groups at week 80. Body weight gain was increased at 12.5 ppm test substance cation in both sexes at the beginning of the study; lower body weight gains were seen in top dose females after the dose level was increased (week 44 onwards). Food consumption was slightly lower in test substance treated animals than in controls, with food utilisation efficiency increased in males.

Test substance levels in urine showed dose related values, though not always in direct proportion to administered levels – a positive result in a control sample from week 13 was investigated but the cause could not be found though mis-dosing was not considered to have occurred. Plasma test substance levels at 52 weeks were dose-related in males but in females there was only a minimal difference between the groups. Kidney and lung analyses were subject to interference but available data showed levels of test substance to be proportional to dietary levels. These results indicate absorption of test substance was not saturated at the levels used in this study.

Histological examination of animals dying during the study or killed at termination showed the kidney to be the major target organ with tubular effects being prevalent at 125 ppm with pelvic dilatation evident at ≥37 ppm in males. There was no evidence of treatment-related effects on the lungs other than a few instances of alveolar wall thickening/hypercellularity. Ocular lesions were similar in controls and treated groups. There were no significant increases in total neoplastic lesions following test substance administration. Occasional increases in individual tumour incidence were seen (eg. pituitary adenoma at weeks 53-78 and lung adenoma at weeks 78-98), these were of tumours typical of aged mice and not statistically significant nor consistent with time and dose. There was an increase in kidney adenomas in top dose males but not in females, this was probably secondary to degenerative lesions. It is concluded that test substance is not tumourigenic in mice. Therefore the NOAEL for carcinogenicity is higher than 125 ppm (recalculated dietary equivalent value: 20.7 mg pure test substance/kg bw/day (for both sexes)).