Paraquat-dichloride

Endpoint summary

Description of key information

- Oral: reported NOAEL = 0.45 mg/kg bw/day (cation), recalculated NOAEL = 0.62 mg/kg bw/day (pure test substance); male; Beagle dogs; GLP compliant OECD 452; Kalinowski 1983.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 Jan 1981 to 21 Jan 1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 20 to 24 weeks
- Weight at study initiation: group means 12.02 - 12.50 kg
- Fasting period before study: no
- Housing: The animals were housed individually in indoor pens, the floors of which measured 345 cm x 115 cm. Each pen consisted of sleeping quarters (with a heated floor) and a separate exercise area.
- Diet: 400 g laboratory diet, expanded and crushed, which was given to the dogs each morning.
- Water: ad libitum
- Acclimation period: 5 to 6 weeks
- Identification: uniquely identified by tattooed ear numbers

DETAILS OF FOOD AND WATER QUALITY:
Before the study started, the diet the test substance procedure was validated by determining the homogeneity of diets containing nominally 15, 30 and 60 ppm test substance. The stability of the test substance in the laboratory diet was determined using these same diets. Batches of diet were analysed for their test substance content at approximately four-weekly intervals throughout the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20
- Air changes (per hr): 12
- Photoperiod: Natural light was supplemented with artificial light during the working day.

IN LIFE DATES:
6 Jan 1981 to 21 Jan 1982

Route of administration:

oral: feed

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved in 50 mL tap and mixed in 10 kg mixes. The following amounts of technical liquor were used to prepare the selected doses: 0.47 g for 15 ppm, 0.94 g for 30 ppm and 1.56 g for 50 ppm. Samples of diet for test substance analysis were always taken from the first batch of each diet prepared.

DIET PREPARATION
- Rate of preparation of diet: Up to four batches of each diet were prepared successively, at approximately one-week intervals.
- Mixing appropriate amounts with: Laboratory diet.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the study started, the diet preparat ion procedure was validated by determining the homogeneity of diets containing nominally 15, 30 and 60 ppm of test substance. The stability of the test substance in the diet was determined using these same diets. Batches of diet were analysed for their test substance content at approximately four-weekly intervals throughout the study.
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 washing with water, the test substance was eluted from the column with saturated ammonium chloride solution and measured colorimetrically after reduction with sodium dithionite.

Duration of treatment / exposure:

52 weeks

Frequency of treatment:

Daily exposure via food

Dose / conc.:

15 ppm

Remarks:

Group 2. Dietary concentration for test substance cation; equivalent to 0.45 and 0.48 mg/kg bw/day for males and females, respectively.

Dose / conc.:

30 ppm

Remarks:

Group 3. Dietary concentration for test substance cation; equivalent to 0.93 and 1.00 mg/kg bw/day for males and females, respectively.

Dose / conc.:

50 ppm

Remarks:

Group 4. Dietary concentration for test substance cation; equivalent to 1.51 and 1.58 mg/kg bw/day for males and females, respectively.

No. of animals per sex per dose:

6

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose levels for the following study were selected on the basis of a 90-day dietary study in dogs (Sheppard, 1981) in which pulmonary lesions were the principal finding; the no-effect-level was 20 ppm.
- Animal assignment: The randomisation procedure resulted in the even distribution of dogs to treatment groups according to litter and body weight class within litter.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The dogs were observed at least twice daily (in the morning and at the end of the working day) for gross clinical and behavioural abnormalities.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All dogs were given a full clinical examination by a veterinarian pre-experimentally and after 13, 26, 39 weeks and between weeks 48-51. The examination included auscultation of the chest and ophthalmoscopy.

BODY WEIGHT: Yes
- Time schedule for examinations: All dogs were weighed weekly during the acclimatisation period, on the first day of dosing and thereafter at weekly intervals. All weighing was done before giving the meal. The pre-experimental body weights were used only in the selection of animals for the study.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal was determined based on left-overs at the next day of feeding. Food residues were recorded daily prior to giving the next meal. These measurements were made from the week prior to commencement of treatment.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: All dogs were also given a full clinical examination by a veterinarian pre-experimentally and after 13, 26. 39 weeks and between weeks 48-51. The examination included auscultation of the chest and ophthalmoscopy.
- Dose groups that were examined: All groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Jugular vein blood samples were taken from all dogs once pre-experimentally and then in weeks 4, 8, 12, 16, 20, 26, 39 and 52. All samples were obtained prior to giving the meal.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, blood was taken prior to feeding.
- How many animals: All
- Parameters checked: the blood was examined for changes in the cytological and haemostatic profile by determination of haemoglobin, haematocrit, red cell count, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration and total white cell count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Jugular vein blood samples were taken from all dogs, once pre-experimentally and then in weeks 4, 8, 12, 16, 20, 26, 39 and 52.
- Animals fasted: Yes, blood was taken prior to feeding.
- How many animals: All
- Parameters were examined: The following parameters were analysed on plasma: alanine transaminase, aspartate transaminase, creatine kinase and alkaline phosphatase activities; urea, glucose, albumin, total protein and triglycerides; calcium and cholesterol; potassium.

URINALYSIS: Yes
- Time schedule for collection of urine: All dogs were placed in metabolism cages for urine collection pre-experimentally and then in weeks 8, 16, 24, 39 and 50. The collection period was approximately 18 hours.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, water, but not food, was available during the collection period.
- Parameters checked: glucose, ketones, urobilinogen, pH, specific gravity and protein. Further microscopic examinations consisted of: presence or absence of crystals and sperm; erythrocytes, leucocytes, squamous epithelial cells, small epithelial cells and casts.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Organ weights: The following organs were weighed (the left and right components of paired organs being weighed separately): adrenals, brain, gonads, heart, kidneys, liver, lungs (left and right combined with 15 tracheal rings), pituitary, spleen, thymus and thyroids (with parathyroids).

HISTOPATHOLOGY: Yes
Pathology: The following tissues from all dogs were submitted for histopathological examination: adrenals, aorta, bone marrow (rib and costa-chondral junction), brain, caecum, cervix, colon, duodenum, epididymides, eyes, gall bladder, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (bronchial, mesenteric, prescapular), mammary gland (bitches only), oesophagus, ovaries, pancreas, pituitary, prostate, salivary gland (submandibular), sciatic nerves, skin (left flank), spinal cord, spleen, stomach, testes, thymus, thyroids/parathyroids, trachea, urinary bladder, uterus, voluntary muscle (biceps femoris), any abnormal tissue.

Other examinations:

- Test substance concentration in urine, kidney, liver and lung:
Urine samples were obtained from all dogs in week 29 by urinary bladder catheterisation and analysed for the concentration of the test substance. Samples of kidney, liver and lung were taken from all animals at necropsy and analysed for the test substance content.

Statistics:

Body weight gain from the start of the study to each week was considered by analysis of variance, separately for males and females.
Organ weights were considered by analysis of variance and analysis of covariance on final body weight, separately for males and females. The data from paired organs were examined for evidence of differential effects on left and right components. No differences were found and combined data are therefore presented.
Haematology and biochemistry data were considered by analysis of covariance on pre-experimental values at each sampling time. Male and female data were analysed together. The results of the analyses were examined to determine whether the differences between the control and treated groups were consistent between sexes.
All analyses allowed for the replicate design of the study. Group means were adjusted for any missing values. Each paraquat dose group mean was compared to the control group mean using Student's t-test (two-sided) based on the error mean square in the analysis. Where male and female data were analysed together, these comparisons were made for male and female means separately and for the overall means (though these have not been presented in the report). All data were checked for unusual values using simple plots. Where unusual values were detected, the analyses were repeated omitting these values to determine their influence on the conclusions.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Increased respiratory rate or depth (hyperpnoea) was seen in some dogs in all groups, including controls. The incidence of this clinical sign was, however, much greater in the 50 ppm test substance group. There was increased vesicular sound in some dogs in the 30 and 50 ppm groups, the 50 ppm group showing the highest incidence. No dogs in either the control or 15 ppm groups exhibited this latter finding.
Hyperpnoea was most commonly seen during the routine clinical examinations and may in some animals have been partly due to stress. Two males and one female (50 ppm test substance) exhibited hyperpnoea most frequently and it was also often seen in the routine daily observation periods. Hyperpnoea and increased vesicular sound were first observed after thirteen weeks of treatment. No animals were ever in respiratory distress and the symptoms were generally slight.
Some dogs in all groups (five controls; four in the 15 ppm group; four in the 30 ppm group; three in the 50 ppm group) were observed to be either thin or lean for varying periods of time during the study. This included two dogs given 50 ppm test substance in which the respiratory effects became well established.
From week 9 until the end of the study, reddening of the dorsal surface of the tongue was frequently observed in animals of all groups, including controls. Although this was a subjective observation it was evident that there was a dose-related increase in the incidence and frequency of the finding in the 30 and 50 ppm test substance groups. The incidence in the 15 ppm test substance group was less than in the controls. As the study proceeded, there appeared to be an increase in the incidence of gingivitis and tartar on the teeth. Though a consistent relationship between these observations and reddening of the tongue could not be established.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In males, the final mean body weight of all treated groups was slightly lower than that of the control group. There was, however, no evidence of any effect on body weight gain at any time during the study in any of the dose groups.
All female treated groups had slightly greater mean body weight gains than the control group.
Several dogs in all groups, including controls, had fluctuating body weights over the course of the study and some showed either little or no overall weight change and three (one male 15 ppm; two males 30 ppm; one female 50 ppm) showed an overall weight loss.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The majority of animals ate all of the diet given throughout the study. The reduced food consumption of two dogs, both in the 50 ppm test substance group, was considered to be treatment-related. One male frequently left small quantities of food uneaten from the time that hyperpnoea was first observed. A number of females, in all groups including controls, showed reductions in food consumption but only that of one female was considered significant, because of the frequency that moderate to large quantities of diet were left uneaten

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmoscopy revealed no treatment-related changes.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant differences from the control means were seen in several parameters. They were all either small, isolated or not dose-related and none were of haematological significance.
All bone marrow samples examined appeared normal.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Most parameters showed some isolated statistically significant changes which were generally small and not dose-related.
Plasma urea levels of female dogs, particularly in the 15 ppm test substance group, were reduced throughout the study compared to the controls. This change was not dose-related and can probably be attributed to a high control level, rather than a treatment-related effect.
Plasma alkaline phosphatase activities were slightly elevated in the 50 ppm test substance group, particularly in the females. The observation remained consistent throughout the study.
There was also a slight rise at 52 weeks in the female 30 ppm test substance group. There was evidence of increased plasma cholesterol levels, mainly in the 50 ppm test substance group females and to a slightly lesser extent in the females given 15 ppm. The changes were small and not seen with 30 ppm test substance group.
The plasma triglyceride levels of both sexes in the 50 ppm test substance group were slightly raised throughout the study.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The specific gravity of urine in the 50 ppm test substance group was increased slightly, particularly in males in the early stages of the study.
All other urinary parameters appeared normal.

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):

The lung weight of males and females dosed with 50 ppm test substance was increased by 35% and 60% over the control values respectively. There was no evidence of any effect on lung weight in either sex in the 15 and 30 ppm dose groups.
In males, there was a higher mean spleen weight in the 50 ppm test substance group. This was not statistically significantly different from the control group mean and was mainly due to one animal with a high spleen weight. However, with the exclusion of this animal, the mean spleen weight of this group was still slightly raised above the control value. There was no evidence of elevated spleen weight in males of the 15 and 30 ppm test substance groups.
In females, there was also an indication of increased spleen weight in the 50 ppm test substance group. Following exclusion of the high spleen weight, 30 ppm test substance, the mean spleen weight of the females in the 50 ppm test substance dose group was statistically significantly higher than that of the control group.
The reduced kidney weight of the male 15 ppm test substance group was an isolated finding, of no biological significance; there were no other apparent changes in organ weight.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

A wide variety of lesions was observed in dogs from all groups including controls. Dogs from treated groups and controls had lesions of the lungs. The most consistently observed lesion was yellow discolouration and consolidation of areas of the lungs. The extent of this lesion varied considerably from lobe to lobe within the same animal, and from animal to animal. Animals from all groups, including control, showed this lesion but there was a larger number of animals showing the more severe lesions in the 30 and 50 ppm groups. In addition to the areas of yellow consolidation, a number of dogs showed the presence of other lung lesions. Most of these were small focal lesions of a firm pale nodular nature.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A wide variety of histological lesions were observed in dogs in this study. The yellow consolidated lesions of the lungs observed grossly in a number of dogs showed a number of individual histological features, namely:
Peribronchial mononuclear cell infiltration; Peribronchiolar fibrosis; Interalveolar fibrosis; Interalveolar mixed inflammatory cell infiltration – generally relatively sparse; Haemosiderin laden macrophage infiltration intermingled with areas of fibrosis; Alveolar cell hyperplasia and hypertrophy – epithelialisation; Limited inflammatory cell infiltration in airspaces (bronchioles and alveoli). The extent of this was not of such a degree as to warrant the classification of pneumonia.
These changes occurred in close association with each other within any particular area of lung tissue and throughout this report the diagnostic term of chronic pneumonitis has been used to embrace these individual features. Lesions of chronic pneumonitis were graded for overall severity in each lobe of lung for each animal.
Females fed 50 ppm test substance showed a marked increase over controls in the severity of chronic pneumonitis whilst those fed 30 ppm test substance had a slight increase when compared to controls. Females fed 15 ppm test substance did not show any increase over control animals. In males, there was a similar dose response to treatment and, although three dogs fed 15 ppm test substance had slight lesions, two controls also had slight lesions. This suggested that 15 ppm test substance had no effect on the lungs, emphasised by the finding that no control males were completely free of the lesions whereas one 15 ppm test substance male had no lesions.
Several other types of histological lung lesions were observed in both treated and control dogs but none was considered to be treatment related. In both male and female dogs fed 50 ppm and 30 ppm test substance there was a slight increase in the incidence of erythrophagocytosis in the bronchial lymph nodes. This was considered to be related to the similar increase in lung lesions in these groups.
One male dog fed 50 ppm test substance had a reddened tongue at the time of necropsy. This lesion was of a similar appearance to that observed in a number of dogs clinically throughout the course of the study. However, histologically no pathological abnormality was observed and it was therefore concluded that the reddening was passive congestion only. The cause of this lesion is unknown.
Other lesions were present in many other organs both in treated and control animals. However, these occurred in small numbers of animals, were of little pathological importance, or formed part of the normal background pathology in this strain of beagle dog. None of these lesions was considered to be treatment related.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Only two neoplasms were observed in animals in this study. One male from the 30 ppm group had a benign squamous papilloma removed surgically from the right ear pinna at week 20 because of secondary inflammatory changes. At termination one female fed 30 ppm test substance had a parafollicular adenoma of the thyroid gland. Neither of these neoplasms was considered to be related to treatment.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

A dose-related increase in the test substance ion concentration was found in urine obtained in week 29; none was detected in any control urine sample (limit of detection 0.05 µg/mL).
No test substance was found in liver at any dose level and none was found in the kidney of dogs given 15 ppm test substance. The test substance was found in the kidney of dogs in the 30 and 50 ppm groups but the concentrations were not increased in a dose-related manner. There was a dose-related increase in the test substance concentration of the lungs. No test substance was detected in any control animal tissues (limit of detection 0.1 µg/g).

Dose descriptor:

NOAEL

Effect level:

15 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

Original value presented in study. Dietary concentration for test substance cation; equivalent to 0.45 mg test substance cation/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

0.62 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

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

Dose descriptor:

NOAEL

Effect level:

15 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

Original value presented in study. Dietary concentration for test substance cation; equivalent to 0.48 mg test substance cation/kg bw/day.

Dose descriptor:

NOAEL

Effect level:

0.66 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

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

Critical effects observed:

yes

Lowest effective dose / conc.:

30 ppm

System:

respiratory system: lower respiratory tract

Organ:

alveoli

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

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 0.45 mg test substance cation/kg bw/day = 0.62 mg pure test substance/kg bw/day.

(100/72.4) x 0.48 mg test substance cation/kg bw/day = 0.66 mg pure test substance/kg bw/day.

Conclusions:

In this GLP compliant, performed similar to OECD 452, 1-year repeated dose feeding study in dogs, a NOAEL based on the incidence of chronic pneumonitits of 15 ppm (recalculated dietary equivalent value: 0.62 mg pure test substance/kg bw/day (for males)) was found.

Executive summary:

In this GLP compliant study, performed similar to OECD guideline 452, four groups of beagle dogs, each containing six males and six females, received the test substance daily in the diet levels of 0, 15, 30 and 50 ppm for one year. This was equivalent to a dietary exposure of 0, 0.45, 0.93, 1.51 and 0, 0.48, 1.00, 1.58 mg test substance cation/kg bw/day for males and females, respectively. Twice a day animals were checked for gross clinical and behavioural abnormalities. Detailed clinical observations and ophthalmoscopic examinations were performed by a veterinarian 5 times during the study. Body weights were recorded before and during the study at weekly intervals. Compound intake 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, all dogs were subjected to macroscopic and microscopic pathological examinations and a selection of organs was weighed. Test item content in urine (week 29) and several organs was analysed at termination.

A dose related incidence of vesicular sound was noted in the 30 and 50 ppm dose groups. Clinical evidence (hyperpnoea, increased breathing rate and depth) of respiratory dysfunction was seen in animals fed 50 ppm. Histopathological examination showed that these clinical changes correlated with chronic pneumonitis in the 30 and 50 ppm dose groups while there were no lung changes in the 15 ppm dose group. Reduced food intake observed in one male and one female fed 50 ppm test substance was considered to be compound-related. There were no other changes in food consumption or body weight which could be attributed to treatment and no compound-related ocular lesions. Slight increases in plasma alkaline phosphatase, cholesterol and triglycerides were seen in the 50 ppm dose group; these findings are normally associated with an effect on the liver but no histopathological changes were seen and the liver is not a target organ for the test substance. A slight increase in the specific gravity of the urine was also seen in the 50 ppm dose group but all other urinary parameters appeared normal and there were no histopathological changes in the kidney.

There were no other significant treatment-related effects. It was concluded that the dietary administration of 15 ppm test substance cation to dogs for a period of one year is an appropriate NOAEL. The dietary equivalent for the test substance cation are 0.45 mg/kg bw/day. The recalculated NOAEL based on the pure test substance is therefore 0.62 mg/kg bw/day.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.62 mg/kg bw/day

Study duration:

subchronic

Species:

dog

Quality of whole database:

GLP compliant, OECD 452.

System:

respiratory system: lower respiratory tract

Organ:

alveoli

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral

1-year feeding in dogs

In this GLP compliant study, performed to a study protocol similar to that of the OECD guideline 452, four groups of beagle dogs, each containing six males and six females, received the test substance daily in the diet at levels of 0, 15, 30 and 50 ppm (cation) for one year (Kalinowski, 1983). This was equivalent to a dietary exposure of 0, 0.45, 0.93, 1.51 and 0, 0.48, 1.00, 1.58 mg test substance cation/kg bw/day for males and females, respectively.

A dose related incidence of vesicular sound was noted in the 30 and 50 ppm dose groups. Clinical evidence (hyperpnoea, increased breathing rate and depth) of respiratory dysfunction was seen in animals fed 50 ppm. Histopathological examination showed that these clinical changes correlated with chronic pneumonitis in the 30 and 50 ppm dose groups while there were no lung changes in the 15 ppm dose group. Reduced food intake observed in one male and one female fed 50 ppm test substance was considered to be compound-related. There were no other changes in food consumption or body weight which could be attributed to treatment and no compound-related ocular lesions. Slight increases in plasma alkaline phosphatase, cholesterol and triglycerides were seen in the 50 ppm dose group; these findings are normally associated with an effect on the liver but no histopathological changes were seen and the liver was not a target organ for the test substance. A slight increase in the specific gravity of the urine was also seen in the 50 ppm dose group but all other urinary parameters appeared normal and there were no histopathological changes in the kidney.

There were no other significant treatment-related effects. It was concluded that the dietary administration of 15 ppm test substance cation to dogs for a period of one year is an appropriate NOAEL. The dietary equivalent for the test substance cation are 0.45 mg/kg bw/day. The recalculated NOAEL based on the pure test substance is therefore 0.62 mg/kg bw/day.

90-day feeding in dogs

In this GLP compliant study, performed to a study protocol similar to that of the OECD guideline 409, five groups of beagle dogs, each containing three males and three females, received the test substance daily in the diet at levels of 0, 7, 20, 60 and 120 ppm for 13 weeks (Sheppard, 1981). This was equivalent to a dietary exposure of 0, 0.19, 0.56, 1.75, 3.50 and 0, 0.24, 0.71, 1.92, 4.26 mg test substance cation/kg bw/day for males and females, respectively.

Two male and 2 female dogs from the 120 ppm group were killed in extremis between days 16 and 23. All 4 dogs exhibited similar clinical symptoms of marked dyspnoea and body weight loss prior to death. There were no other changes in clinical condition or behaviour that could be attributed to treatment. Both surviving dogs from the 120 ppm group showed an overall body weight loss. There was a slight overall reduction in body weight gain in the female dogs treated at 60 ppm, 20 ppm and 7 ppm when compared to the controls, but these changes were not considered to be treatment-related. There were no effects on food intake at any of the levels employed except for 1 surviving high dose female which showed a loss of appetite from week 8 onwards. There were no distinct changes in any of the haematological, clinical chemistry or urinary parameters examined. Slight haemoconcentration was apparent in 1 high dose incidental kill dog at termination. Retinal blood vessel engorgement was observed in 2 high dose dogs and also in 1 dog from each of the 7 and 20 ppm groups, but this finding could not be directly linked to treatment. Auscultation showed markedly increased respiratory sounds in the 4 high dose dogs that were killed during the study. There was no other conclusive evidence of respiratory distress in any of the other treated dogs. Lung weights were increased in all dogs from the 120 ppm group and in 2 dogs from the 60 ppm group. All other organ weights were in the normal range. Distinct gross and histological treatment-related pulmonary lesions were seen in dogs in the 60 and 120 ppm groups. Very minor renal lesions were also found histologically in a few of these animals. No discernible gross or histological treatment-related pulmonary lesions were seen in dogs in the 7 and 20 ppm groups. The focal pulmonary lesions in these animals were of a type and incidence similar to those in the controls. Determination of homogeneity, stability and achieved concentration of test substance in the animal diet indicated that these were achieved within the limits of reproducibility of the analytical method. Determination of the test substance concentration in dog urine indicated that the substance was excreted in approximate proportion to increasing dietary concentration, and was therefore proof of absorption. No test substance was found in control dog urine.

The NOAEL in this study was considered to be a dietary concentration of 20 ppm (lowest dietary equivalent to 0.56 mg test substance cation/kg bw/day for males). The recalculated NOAEL based on the pure test substance is therefore 0.77 mg pure test substance/kg bw/day.

24-months feeding in rat

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 at levels of 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.

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 bodyweight 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 bodyweight 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, towards 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.

Based on these findings, the NOAEL and the LOAEL for systemic toxicity, for both sexes, are 25 ppm (1.0 mg/kg bw/day) and 75 ppm (3.75 mg/kg bw/day), respectively. The recalculated NOAEL based on the pure test substance is therefore 1.4 mg/kg bw/day.

90-day feeding in rat

In this 90 days feeding study in rats, similar to OECD guideline 408 not performed under GLP, groups of 20 male and 20 female Fischer CDF (F344) rats were fed diets containing 10, 30, 100 or 300 ppm pure test substance for thirteen weeks (Maita, 1980). These levels are equivalent to a dietary dose of 0.68, 1.99, 6.55, 19.6 mg/kg bw/day and 0.72, 2.11, 7.10, 21.1 mg/kg bw/day for males and females, respectively. The control group was fed plain diet.

There were no mortalities or adverse clinical findings during the study. Bodyweight gain was reduced at 300 ppm in both sexes. Food consumption and utilisation were reduced at 300 ppm. Water consumption was reduced in top dose males and in all treated females during the first half of the study. Urine parameters were unaffected. Reductions in erythrocyte parameters consistent with microcytosis were seen in 300 ppm females. A variety of changes in clinical chemistry parameters were seen at 300 ppm, affecting females more than males: decreases in calcium, cholesterol, GPT activity and total protein, with increases in LDH activity, potassium, alkaline phosphatase activity and glucose levels. Autopsy findings were restricted to the 300 ppm dose groups. Organ weights tended to be reduced at this dose, in absolute but not relative terms, though an increase in lung weights in both sexes was seen at 300 ppm. Alveolar epithelial hypertrophy was increased in top dose males. Females at 300 ppm had increases in brown pigment in the spleen and sub-pleural lymphoid hyperplasia, with a reduction in calcification of the renal tubules, the latter showing a dose-response. There was no evidence of adverse effects at 100 ppm or below.

Dietary administration of the substance to rats for 13 weeks produced reduced weight gain, erythrocytic changes and lung lesions at 300 ppm. The NOAEL was found to be 100 ppm for both sexes, equivalent to 6.55 and 7.10 mg/kg bw/day for males and females, respectively.

24-month feeding study in mice

In this lifetime feeding study, performed under GLP to a study protocol similar to that of OECD TG 453, Swiss-derived mice were administered technical test substance incorporated into the diet at concentrations 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 animals per sex per group was designated for interim sacrifice after 52 weeks. At week 36 the top dietary concentration was increased from 100 to 125 ppm because no toxic signs had appeared after 35 weeks of dosing. The applied concentrations corresponded to a dietary dose of 0, 0, 1.87, 5.6 and 15.0/18.7 mg test substance cation/kg body weight/day.

After 52 weeks all surviving mice in the satellite groups were killed and subject to 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 subject to a detailed macroscopic examination, and a comprehensive selection of tissues was taken and examined histopathologically for neoplastic and non-neoplastic changes.

Dietary analyses showed the majority of batches to be within 90-110% 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. Overall, the 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. Tubular effects were prevalent at 125 ppm and pelvic dilatation was 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 finding was probably secondary to degenerative lesions.

A NOAEL of 12.5 ppm test substance cation (1.87 mg test substance cation/kg bw/day = 2.6 mg pure test substance /kg bw/day) can be derived based on dilation of the renal pelvis in males at 37.5 ppm and a variety of kidney effects at 125 ppm. The NOEL for females is 37.5 ppm (5.6 mg test substance cation/kg bw/day = 7.7 mg pure test substance/kg bw/day).

Justification for classification or non-classification

Based on the available information on repeated dose toxicity the test substance is classified as STOT Rep. Exp 1 (H372) for effects on the lungs according EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.