Diquat dibromide

Carcinogenicity

Description of key information

Based on the GLP compliant study similar to OECD TG 453, no excess tumours occurred at any dose level, therefore, there was no indication of a carcinogenic potential.

Oral, NOAEL carcinogenicity >= 375 ppm test substance ion, thus >=700.5 ppm test substance salt (dietary equivalent to 27.80 and 36.31 mg/kg bw/day for males and females, respectively), rats, chronic, Similar to OECD 453, Colley 1985

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Mar 1981 to 12 Apr 1983

Reliability:

1 (reliable without restriction)

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)

Version / remarks:

1981

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity)

Version / remarks:

1998

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.33 (Combined Chronic Toxicity / Carcinogenicity Test)

Version / remarks:

1988

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: 208-211 g males and 163-166 g females
- Housing: 5 per cage (sexes separately) in suspended cages with a wire mesh floor
- Diet: ad libitum, except overnight prior to blood collection and urine collection (other than pre-dose samples)
- Water: ad libitum
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY:
All batches of diet were assayed by the manufacturers for chemical contaminants, microbiological levels and nutritional levels. Routine analysis of water was conducted quarterly.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 10
- Photoperiod (hrs dark / hrs light): 12 /12

IN-LIFE DATES: From: 27 Mar 1981 To: 12 Apr 1983

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was administered by admixture to the diet, and all diet concentrations were based on the test substance cation (ppm w/w). Control animals received untreated diet.

DIET PREPARATION
Each concentration was prepared separately. The diet (powdered) was stirred into a weighed quantity of the test material solution. Further quantities of diet were added to the mixture until 2 kg was reached with thorough stirring between each addition. This 2 kg mix was then added to a pre-weighed quantity of the diet to achieve the required quantity of diet which contained the correct concentration of diquat. This was then mixed in a double-cone blender for a minimum of 7 minutes. Diets were prepared on a weekly basis two weeks prior to feeding and stored in heat-sealed polythene bags at room temperature in the dark. A constant concentration (ppm) was administered in diet throughout the study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to treatment, the diet was analysed to determine homogeneity and stability over a 2-week period at the low (5 ppm) and high (375 ppm) dosage levels. Samples were taken once every 4 weeks during the study, to determine achieved levels at all dietary concentrations.

Duration of treatment / exposure:

- Carcinogenicity study: 104 Weeks
- Satellite group: 52 weeks

Frequency of treatment:

Continuously

Post exposure period:

Not applicable

Dose / conc.:

5 ppm

Remarks:

Group 2. Dietary equivalent to 0.19 and 0.24 mg test substance cation/kg bw/day for males and females, respectively.

Dose / conc.:

15 ppm

Remarks:

Group 3. Dietary equivalent to 0.58 and 0.72 mg test substance cation/kg bw/day for males and females, respectively.

Dose / conc.:

75 ppm

Remarks:

Group 4. Dietary equivalent to 2.91 and 3.64 mg test substance cation/kg bw/day for males and females, respectively.

Dose / conc.:

375 ppm

Remarks:

Group 5. Dietary equivalent to 14.88 and 19.44 mg test substance cation/kg bw/day for males and females, respectively.

No. of animals per sex per dose:

- Carcinogenicity study: 50
- Satellite group: 10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: These levels were chosen by the Sponsor based on the results of a 4-week preliminary study in rats by dietary administration.
- Rationale for selecting satellite groups: Satellite groups, each made up of 10 male and 10 female rats, were destined for interim kill after 1 year of treatment; blood and urine investigations were performed on half the rats in each satellite group before treatment commenced and just prior to termination to relate any changes to histopathology.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
Animals were checked twice daily for moribund/dead animals. Individual animals were given a detailed clinical examination once per working day (not weekends) for the initial 4 weeks of the study and weekly thereafter.

DETAILED CLINICAL OBSERVATIONS:
All rats were palpated every week and the time of appearance, location and dimensions of any palpable masses were recorded.

BODY WEIGHT:
The body weight of each rat was recorded 11 days and one week before the commencement of treatment and at weekly intervals thereafter.

FOOD CONSUMPTION AND TEST SUBSTANCE INTAKE:
Food consumption for each animal was determined. The quantity of food consumed by each cage of rats was recorded once weekly and the group mean weekly intake per rat calculated. Any food wastage was estimated by weight and a corresponding quantity of fresh food added to the diet hopper. Group mean food conversion ratios were calculated for successive four-weekly periods during the first 24 weeks of treatment, as weights of food consumed per unit gains in body weight.

WATER CONSUMPTION:
Daily monitoring by visual examination of the water bottles was maintained throughout the study. Consumption for each cage in the control and high dosage level groups was measured daily for a 5-day period during weeks 6, 12 and 24/25. As no treatment-related changes were recorded, no further measurements were performed.

OPHTHALMOSCOPIC EXAMINATION:
Before dosing commenced and during weeks 13, 26, 52, 78 and 104, the eyes of all animals were examined by indirect ophthalmoscopy. A mydriatic solution was applied to the eyes at least 5 minutes but not more than 30 minutes before ophthalmoscopy was performed.

HAEMATOLOGY:
Prior to the start of treatment and during weeks 26, 52, 78 and 104 samples of blood were withdrawn, under light ether anaesthesia, from the orbital sinus of 10 male and 10 female rats in each main group. Also prior to the start of treatment and in week 52, blood samples were withdrawn from 5 male and 5 female rats from each satellite group. Food was removed overnight from animals to be sampled with the exception of the pre-dose investigations. The following parameters were measured: Haemoglobin; red blood cell count; mean cell volume; mean cell haemoglobin; packed cell volume; activated partial thromboplastin time; mean cell haemoglobin concentration; platelet count; total white cell count; differential white cell count; thrombotest (Measured at week 52 for satellites and week 26 and subsequently for main group animals)

CLINICAL CHEMISTRY:
Prior to the start of treatment and during weeks 26, 52, 78 and 104 samples of blood were withdrawn, under light ether anaesthesia, from the orbital sinus of 10 male and 10 female rats from each main group. (The animals were those indicated under Haematology). Also prior to the start of treatment and in week 52, blood samples were withdrawn from 5 male and 5 female rats from each satellite group. The following parameters were measured:
urea nitrogen; creatinine; glucose; albumin; total protein; cholesterol; total bilirubin; glutamic pyruvic transaminase activity; A/G ratio; alkaline phosphatase activity; calcium; inorganic phosphate phosphorus; sodium; potassium; chloride; glutamic oxaloacetic transaminase activity; total lactic dehydrogenase activity; plasma protein electrophoresis (females only, week 104 samples only)

URINALYSIS:
During weeks 24, 50, 76 and 102 individual overnight urine samples were collected from 10 males and 10 females from each main group. (The animals were those indicated under Haematology). Also in week 50, urine samples were collected from 5 male and 5 female rats from each satellite group. The following parameters were measured: volume; specific gravity; pH; reducing substances; bile pigments; glucose; ketones; protein; urobilinogen; haemoglobin.

Sacrifice and pathology:

INVESTIGATIONS POST MORTEM:
INTERIM DEATHS: Any rat showing signs of severe debility or intoxication, particularly if death appeared imminent was killed by carbon dioxide asphyxiation to prevent loss of tissues through autolytic degeneration or cannibalism. All rats killed in extremis, or found dead in the cage were subjected to detailed macroscopic examination in an attempt to ascertain the cause of death or condition; unless autolysis or cannibalism prevented it, a full spectrum of tissue samples was preserved routinely in buffered 10% formalin.

INTERIM SACRIFICE (SATELLITE GROUP):
On completion of the 52-week laboratory investigations all surviving rats in the satellite groups were killed and subjected to detailed macroscopic examination and organ weight analysis as described below. A full spectrum of tissue samples was preserved and processed as described below.

TERMINAL SACRIFICE:
On completion of the treatment period, all surviving rats were killed (in replicate order) by carbon dioxide asphyxiation and weighed following asphyxiation.

GROSS PATHOLOGY:
All superficial tissues, including the urogenital orifices and tail, each pinna, eye and external auditory meatus, were examined visually and by palpation for distortion, swelling or other evidence of tumour formation; similar attention was given to the mammary glands and the subcutaneous structures. The external nares, buccal cavity and tongue were then examined, and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection, all subcutaneous tissues were examined, including regional lymph nodes, mammary and thyroid/parathyroid glands. The condition of the thoracic viscera was noted, with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal and the urinary bladder was briefly distended with fixative after removal, opened and examined under low-power magnification.
The stomach and caecum were incised and examined, and in addition, portions of duodenum, jejunum, ileum and colon and oesophagus were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination, the lungs were then infused with fixative. The liver was sectioned at intervals of a few millimetres; the kidneys were incised in the transverse plane and examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded. Any lesion suggestive of neoplasia was noted, including details of location, size and multiplicity. Any evidence of adhesion or possible invasion to adjacent structures was noted.

ORGAN WEIGHTS:
The following organs from all animals killed after 52 weeks and at termination were dissected free of fat and weighed: adrenal glands; brain (including brain stem); heart; kidneys; liver; ovaries; testes. Paired organs were weighed together.

TISSUE SUBMISSION:
The following tissues were examined in situ, removed and examined and fixed in an appropriate fixative: All nodules, tissue masses and otherwise macroscopically abnormal tissues, along with samples of adjacent tissue; adrenal gland; aorta; bone (femur); brain (medullary, cerebellar and cortical sections); caecum; mid colon; duodenum; epididymis; eyes; Harderian gland; head (nasal cavity, paranasal sinuses, tongue, oral cavity, nasopharynx, middle ear); heart; ileum; jejunum; kidney; liver (from 3 lobes/ left, median and papillary); lung (all lobes and mainstem bronchi); lymph node - cervical; lymph node - mesenteric; mammary gland; nerve - sciatic; oesophagus; ovary; pancreas; parathyroid gland; pituitary gland; prostate gland; salivary gland; seminal vesicle; skeletal muscle; skin; spinal cord (at least 3 levels, thoracic, lumbar); spleen; sternum (for bone marrow); stomach (glandular and non-glandular); testis; thymus; thyroid gland; trachea; urinary bladder; uterus (corpus and cervix).

MICROSCOPIC EXAMINATION:
Tissues required for microscopic examination were embedded in paraffin wax and sections cut at 5 µm, stained and examined by light microscopy. For satellite group rats dying during the study, macroscopically abnormal tissues were examined microscopically in an attempt to define cause of death. For every rat in the satellite group killed after 52 weeks and for every rat in the main group either dying during the study or killed at termination, all the tissues (with the exception of the head) listed above (including all macroscopically observed lesions) were routinely examined. Three coronal sections through the head were examined initially in 10 males and 10 females from each group killed at termination. If a tumour was found, sections of the head from all main group animals were examined.

Statistics:

See 'Any other information on materials and methods incl. tables'.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant, compound-related effects on the clinical condition of the animals, with the exception of ophthalmoscopic changes. Eye changes were seen in the majority of animals receiving 375 ppm. Initially the eyes appeared more transparent than usual and this was followed by opacity. From week 45 a number of rats with total opacity showed a dark red discolouration of the eyes which became darker with time. This was due to extensive haemorrhage in the anterior chamber of the eye, revealed by ophthalmoscopic examination.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The incidence of mortality was unaffected by treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 375 ppm, body weight gain for females was reduced throughout the study and was reduced for males during the first 13 weeks. In addition, males at 375 ppm lost weight during the final 26 weeks but it is considered that this was probably attributed to senile changes in the rats rather than the effects of the test substance. There was no effect on body weight in rats given 5, 15 or 75 ppm. Results are summarized in Table 1 in 'Any other information on results incl. tables'.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was reduced in rats receiving 375 ppm. Females were affected throughout the treatment period and males were affected during weeks 1-26. There was no effect on food consumption for animals given 5, 15 or 75 ppm. Results are summarized in Table 2 in 'Any other information on results incl. tables'.

Food efficiency:

no effects observed

Description (incidence and severity):

Food utilisation efficiency when compared over the first 13 weeks of the study indicated a slight impairment among rats receiving 375 ppm. Although food conversion ratios tended to be rather variable during weeks 13 to 24, there were no consistent marked differences between values calculated for treated rats when compared with the controls.
The overall ratios calculated for rats from the high dosage group, revealed a marginal impairment of food utilisation efficiency when compared with the controls, but ratios calculated for the remaining treated rats were similar to control ratios. Results are summarized in Table 3 in 'Any other information on results incl. tables'.

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

no effects observed

Description (incidence and severity):

There was no toxicologically significant difference in water consumption between controls and animals given 375 ppm.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Ophthalmoscopic examination revealed a compound-related incidence of triangular posterior subcapsular opacities of the lens in male and female animals receiving 75 or 375 ppm. These opacities were most frequently bilateral and covered the posterior lens face to varying degrees. Among rats receiving 375 ppm, these lesions progressed to total opacity of the lens, affecting all surviving rats receiving 375 ppm when examined at week 104. A low incidence of rats with opacities was seen in the 15 ppm diquat group at 104 weeks only. No compound-related ophthalmological changes were seen in animals given 5 ppm. Results are summarized in Table 4 in 'Any other information on results incl. tables'.
Further details on re-evaluation of these effects are reported under histopathology.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Several minor variations were noted between treated groups and controls but these were considered normal for rats of this age and strain.
There was a reduction in mean cell volume for females receiving 75 ppm at 26 weeks and a reduction in haemoglobin for males receiving 75 ppm at week 26. In the absence of similar effects in the other sex, in either sex at 375 ppm or at later time points these differences are considered unlikely to be compound-induced. No effects were seen in the 15 and 5 ppm groups. Results are summarized in Table 5 in 'Any other information on results incl. tables'.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no differences in blood clinical chemistry parameters which were considered to be related to treatment.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no differences in urine clinical chemistry parameters which were considered to be related to treatment.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no compound-related effects on organ weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examination or rats dying, sacrificed in extremis, or killed at termination, revealed a higher incidence of lenticular opacity and congestion or haemorrhage in the eyes of male and female rats dosed with 375 ppm. There was also a slightly increased incidence of these changes in males receiving 75 ppm. At the interim kill an increased incidence of caecal distension with ingesta or gas was noted in animals of both sexes receiving 375 ppm. There was also a slight increase in the incidence of prominent caecal blood vessels in males receiving 375 or 75 ppm. Similar caecal changes were also observed in two males receiving 375 ppm dying or killed intercurrently in the first year of the study. All other macroscopically observed lesions were considered to be unrelated to treatment. Results are summarized in Table 6 in 'Any other information on results incl. tables'.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

ORIGINAL STUDY
Histopathological examination of the eye revealed cataractous changes in the eyes of rats given 75 and 375 ppm. This was apparent after one year of dosing but the incidence and severity of the lesions increased during the second year of the study. There was a slightly increased incidence of cataractous change at termination only in females given 15 ppm. No changes were seen in males at this level. There were no compound-related lenticular effects at the 5 ppm dose level. Results are summarized in Table 7 in 'Any other information on results incl. tables'.

ADDENDUM 1
In animals receiving 375 ppm test substance ion, there was a clear lenticular effect at week 13, characterised by triangular posterior subcapsular polar opacity and/or total cataract. These changes subsequently were shown to be progressive and bilateral. In animals receiving 75 ppm there was some evidence of an early treatment-related effect at week 13, characterised by triangular posterior subcapsular opacity although such changes were observed in only 1 out of 50 males and 3 out of 50 females. Since the test substance related cataract is known to be time- and dose-dependent, 75 ppm at week 13 is considered to be near the no-effect level, and the real no-effect level is considered to lie nearer 75 ppm than 15 ppm.
The lenticular findings recorded in animals receiving 5 and 15 ppm as discussed above are considered to be unrelated to treatment with the test material.

ADDENDUM 2
The treatment-related ocular effect observed at the end of the study at dosages of 75 and 375 ppm followed a consistent pattern of development, characterised as bilateral triangular posterior subcapsular polar opacities which progressed with time to more extensive opacities and total cataract. Although bilateral cataractous changes do not always occur in both eyes simultaneously, frequently, a lenticular opacity will be observed initially in one eye only with the contralateral eye showing a similar change soon after. This sequential change was noted in several animals on this study.
At 15 ppm there were 7 animals killed at termination and 7 decedents which showed pathological cataractous change to some degree. None of these animals demonstrated the consistent pattern of development described in the previous paragraph as being typical of a test substance-induced lesion.
Furthermore, when all animals were considered together for histopathology, there was no treatment-related increase in overall cataractous change at 15 ppm when compared with controls. It is concluded, therefore, that there was no evidence of a cataractogenic response at 15 ppm.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no histopathological findings to suggest that the test substance had a carcinogenic effect. Benign phaeochromocytoma of the adrenal gland was seen in males receiving 75 or 375 ppm but this was not dose-related, only achieved statistical significance at the 75 ppm and was within the range of background control data. There were two instances in males of statistically significant positive trends with dose: osteosarcoma and thyroid follicular cell adenoma. In both cases these findings reflected elevated incidences at the 375 ppm only which were not statistically significant by Fisher’s exact test and there was no evidence below this dose level of any increasing incidence. These changes are, therefore, considered not to be attributable to administration of the test substance.
For animals dying or killed between 53 and 104 weeks, mammary adenocarcinomas were seen in a small number of animals in all treated groups but in none of the controls. This was considered to be incidental because the incidence was not dose-related, was within the range of background data and at termination a marginally higher incidence of these tumours was seen in control animals.
There was a statistically significant increase in females with multiple neoplasms at 5 ppm and in females with malignant neoplasms at 75 ppm but neither is considered to be treatment-related as the findings involved isolated groups and there was no significant trend with dose.

Key result

Dose descriptor:

NOEL

Remarks:

Systemic toxicity

Effect level:

ca. 28.02 ppm

Based on:

other: pure test substance

Sex:

male/female

Basis for effect level:

ophthalmological examination

Remarks on result:

other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation. Dietary equivalent to 1.083 and 1.345 mg/kg bw/day for males and females, respectively.

Dose descriptor:

NOEL

Remarks:

Systemic toxicity

Effect level:

ca. 15 ppm

Based on:

other: test substance cation species

Sex:

male/female

Basis for effect level:

ophthalmological examination

Remarks on result:

other: Original value presented in study. Dietary equivalent to 0.58 and 0.72 mg/kg bw/day for males and females, respectively.

Key result

Dose descriptor:

NOAEL

Remarks:

Carcinogenicity

Effect level:

>= 700.5 ppm

Based on:

other: pure test substance

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation. Dietary equivalent to 27.80 and 36.31 mg/kg bw/day for males and females, respectively.

Dose descriptor:

NOAEL

Remarks:

Carcinogenicity

Effect level:

>= 375 ppm

Based on:

other: test substance cation species

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Original value presented in study. Dietary equivalent to 14.88 and 19.44 mg/kg bw/day for males and females, respectively.

Critical effects observed:

yes

Lowest effective dose / conc.:

75 other: ppm test substance cation

System:

eye

Organ:

lens

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

140.1 other: ppm. Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation

System:

eye

Organ:

lens

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Verification of test diets

All mean concentrations of the test substance cation In test diet as analysed every four weeks throughout the study were within the specified limits (5 and 15 ppm: 80-120% of nominal, 75 and 375 ppm: 85-110% of nominal). Concentrations of the test substance cation in blended diet prepared to check homogeneity indicated a satisfactory mix of test compound with diet. The stability of the test substance cation in diet, at animal room temperature, was confirmed during storage for 24 days.

Calculation of key result

The original effect levels were expressed as cation species of the registered substance. The key effect levels are re-calculated and corrected to include the counterion species by multiplying with 1.868 (344.0 g/mol molecular weight of registered substance divided by 184.2 g/mol molecular weight of cation species):

1.868 x 15 ppm = 28.02 ppm       dietary equivalent to 1.868 x 0.58 = 1.083 mg/kg bw/day for males

1.868 x 15 ppm = 28.02 ppm      dietary equivalent to 1.868 x 0.72 = 1.345 mg/kg bw/day for females

1.868 x 75 ppm = 140.1 ppm

1.868 x 375 ppm = 700.5 ppm      dietary equivalent to 1.868 x 14.88 = 27.80 mg/kg bw/day for males

1.868 x 375 ppm = 700.5 ppm      dietary equivalent to 1.868 x 19.44 = 36.31 mg/kg bw/day for females

Table 1: Intergroup comparison of body weight gain (g) selected time points

	Dietary Concentration (ppm)
	Males					Females
weeks	0	5	15	75	375	0	5	15	75	375
0-6	227.4	221.8	222.0	217.0	182.5***	99.4	96.8	97.0	96.4	76.1***
6-13	119.7	113.6	113.3	114.9	107.5**	42.7	43.6	44.1	45.3	38.2
13-26	93.4	95.3	90.1	96.0	97.8	37.8	38.9	43.6	40.1	32.4
26-52	116.2	111.9	120.7	109.5	118.5	88.0	91.8	99.8	96.6	63.6**
52-78	77.4	78.6	80.3	66.9	70.8	85.5	76.4	82.4	81.1	58.7**
78-104	21.6	6.3	1.3	-6.9	-10.3	59.4	41.2	74.1	71.6	46.0
** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided) *** Statistically significant difference from control group mean, p<0.001 (Student’s t-test, 2-sided)

Table 2: Intergroup comparison of food consumption (g/rat)

	Dietary Concentration (ppm)
	Males					Females
weeks	0	5	15	75	375	0	5	15	75	375
1-6	1106	1116	1101	1088	982***	805	800	786	780	712***
7-13	1316	1293	1302	1315	1210***	884	890	887	901	824***
14-26	2385	2351	2316	2345	2274*	1603	1615	1627	1623	1533**
27-52	4642	4619	4568	4568	4563	3404	3432	3458	3428	3185***
53-78	4779	4695	4729	4747	4690	3697	3625	3769	3695	3439**
79-104	5015	4814	4911	4843	4780	4062	3960	4149	4162	3746
* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided) ** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided) *** Statistically significant difference from control group mean, p<0.001 (Student’s t-test, 2-sided)

Table 3: Intergroup comparison of food utilisation (g food consumed per unit gains in body weight)

	Dietary Concentration (ppm)
	Males					Females
weeks	0	5	15	75	375	0	5	15	75	375
1-13	7.0	7.2	7.2	7.2	7.6	11.9	12.0	11.8	11.9	13.4
1-26	10.9	11.1	11.1	11.1	11.5	18.3	18.4	17.9	18.2	20.9

Weight of food consumed per unit gains in body weight

Table 4: Intergroup comparison of ophthalmology – selected lens lesions

		Dose Level (ppm)
		Males					Females
Finding	Wk	0	5	15	75	375	0	5	15	75	375
Point posterior subcapsular polar opacities	13	0/50	0/50	0/50	8/50	6/49	0/50	2/50	1/50	5/50	0/50
	26	0/50	0/50	0/50	0/49	0/49	0/50	0/50	0/50	0/50	2/49
	52	0/50	0/49	0/50	1/49	0/48	0/49	0/50	0/47	2/50	0/48
	104	0/22	0/16	0/22	0/21	0/24	0/20	0/22	1/20	0/20	0/27
Posterior subcapsular polar opacities	13	0/50	0/50	1/50	0/50	0/49	0/50	0/50	0/50	0/50	0/50
	26	1/50	2/50	3/50	3/49	0/49	0/50	1/50	0/50	5/50	0/49
	78	0/46	1/44	0/44	0/40	0/40	0/39	0/41	0/42	0/41	0/46
Triangular posterior subcapsular polar opacities	13	0/50	0/50	0/50	1/50	32/49	0/50	0/50	0/50	3/50	40/50
	26	0/50	0/50	0/50	2/49	9/49	0/50	0/50	0/50	5/50	10/49
	52	0/50	0/49	0/50	1/49	3/48	0/49	0/50	0/47	5/50	3/48
	78	0/46	0/44	1/44	3/40	0/40	0/39	0/41	0/42	4/41	1/46
	104	0/22	0/16	1/22	9/21	0/24	0/20	0/22	0/20	1/20	0/27
Total cataract	13	0/50	0/50	0/50	1/50	11/49	0/50	0/50	1/50	0/50	2/50
	26	0/50	0/50	0/50	2/49	38/49	0/50	0/50	0/50	0/50	37/49
	52	0/50	0/49	0/50	2/49	45/48	0/49	0/50	0/47	0/50	43/48
	78	1/46	0/44	0/44	2/40	40/40	0/39	0/41	0/42	2/41	45/46
	104	0/22	0/16	1/22	3/21	24/24	0/20	0/22	1/20	3/20	27/27
Plaque opacities	52	1/50	0/49	3/50	1/49	0/48	0/49	1/50	0/47	0/50	0/48
	78	3/46	2/44	3/44	2/40	0/40	0/39	2/41	0/42	6/41	0/46
	104	5/22	4/16	6/22	3/21	0/24	5/20	3/22	0/20	3/20	0/27

Table 5: Intergroup comparison of haematology – selected parameters (week 26)

	Dose Level (ppm)
	Males					Females
Parameter	0	5	15	75	375	0	5	15	75	375
Mean cell volume	62	62	63	66*	67**	67	65	60***	58***	61***
Haemoglobin	16.3	16.0	15.3**	14.5***	15.1***	15.3	16.0*	15.4	16.1*	15.7
* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided) ** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided) *** Statistically significant difference from control group mean, p<0.001 (Student’s t-test, 2-sided)

Table 6: Intergroup comparison of selected macroscopic findings

		Dose Level (ppm)
		Males					Females
Finding		0	5	15	75	375	0	5	15	75	375
Caecum- distended	M	0 (0)	0 (0)	0 (0)	0 (0)	2 (4)	0 (0)	0 (0)	0 (0)	0 (0)	1(1)
	S	0 (0)	0 (0)	1 (0)	2 (0)	6 (0)	0 (0)	0 (0)	1 (0)	1 (0)	6 (0)
Caecal blood vessels – prominent	M	0 (0)	0 (0)	0 (0)	0 (0)	0 (1)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
	S	2 (0)	1 (0)	3 (0)	6 (0)	4 (1)	1 (0)	0 (0)	2 (0)	1 (0)	2 (0)
Eye – lens opacity	M	0 (1)	0 (3)	2 (0)	3 (3)	24 (25)	0 (0)	0 (0)	1 (0)	2 (0)	27 (21)
	S	0 (0)	0 (0)	0 (0)	0 (0)	9 (0)	0 (0)	0 (0)	0 (0)	0 (0)	9 (0)
Eye – congestion/haemorrhage	M	0 (1)	0 (0)	0 (0)	1 (1)	8 (4)	0 (0)	0 (0)	0 (0)	2 (0)	9 (9)
	S	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (0)	0 (0)	0 (0)	0 (0)
M main study, S satellite group animals Numbers within brackets refer to animals killed in extremis or found dead during the study. Numbers not in brackets refer to rats killed at termination after 104 / 52 weeks. Table 7: Intergroup comparison of cataractous change* detected microscopically (all animals)   Dose Level (ppm)   Males Females Severity 0 5 15 75 375 0 5 15 75 375 Minimal 4 3 4 12 0 2 2 0 18 0 Ungraded / other 1 2 1 2 2 3 0 0 3 2 Slight 1 0 0 7 0 0 0 3 6 0 Moderate 0 0 0 5 1 0 0 0 3 0 Marked 0 0 1 3 6 0 0 1 5 21 Severe 1 2 2 6 48 0 0 2 2 36 Total no. with cataractous change 7 7 8 35 57 5 2 6 37 59 No of animals examined 60 60 60 59 59 60 59 60 60 60 * graded according to most severely affected eye

Conclusions:

Histopathological examination of the eye revealed cataractous changes in the eyes of rats given 75 and 375 ppm. This was apparent after one year of dosing but the incidence and severity of the lesions increased during the second year of the study. There was a slightly increased incidence of cataractous change at termination only in females given 15 ppm. No changes were seen in males at this level. There were no compound-related lenticular effects at the 5 ppm.
There was no evidence of carcinogenic potential. The NOAEL for carcinogenicity is 375 ppm test substance ion, thus 700.5 ppm test substance salt, corresponding to a dietary intake of 27.80 and 36.31 mg/kg bw/day for males and females, respectively. The eye was identified as the target organ and cataractous change was observed at dose levels of 15–375 ppm. After re-evaluation of these effects, the NOAEL for cataract formation was placed on 15 ppm test substance ion, thus 28.02 ppm pure test substance, corresponding to a dietary intake of 1.083 and 1.345 mg/kg bw/day for males and females, respectively.

Executive summary:

In a combined chronic toxicity/carcinogenicity study similar to OECD 453 and in compliance with GLP the test substance was administered to five groups of 60 male and 60 female CD rats of the Sprague Dawley strain, by admixture in the diet for 104 weeks at dietary concentrations of 0 (control), 5, 15, 75 and 375 ppm test substance cation. This correspond to a dietary concentration for males of 0.19, 0.58, 2.91 and 14.88 mg test substance ion/kg bw/day and 0.35, 1.08, 5.44 and 27.80 mg pure test substance/kg bw/day; for females 0.24, 0.72, 3.64 and 19.44 mg test substance ion/kg bw/day or 0.45, 1.34, 6.80, 36.31 mg pure test substance/kg bw/day). Ten male and ten female rats per group were killed after 1 year. Diets were analysed for achieved concentrations, homogeneity and stability. All animals were examined daily for mortality and clinical observations and once a week each animal was given a thorough clinical examination and palpated to enable time of appearance and location of any palpable masses to be noted. All animals were weighed pre-dose and weekly thereafter. Food consumption was recorded weekly and group mean food conversion ratios were calculated for successive 4-week periods during the first 24 weeks of treatment. Water consumption was measured daily for a 5-day period during weeks 6, 12 and 24/25. Ophthalmoscopy was carried out in all main group animals pre-dose and during weeks 13, 26, 52, 78 and 104. Blood for haematological and blood chemistry analyses was collected from 10 males and 10 females per main group pre-dose and during weeks 26, 52, 78 and 104. Blood samples were also taken from 5 rats/sex/satellite group pre-dose and in week 52. During weeks 24, 50, 76 and 102 individual overnight urine samples were collected from the same main group animals used for blood sampling and in week 50 samples were collected from the same 5/sex/satellite animals used for blood sampling. After 52 weeks all surviving rats in the satellite groups were killed. All surviving rats from the main groups were killed after 104 weeks of treatment. Each animal (including unscheduled decedents) was given a detailed macroscopic examination and a selection of organs were weighed. A comprehensive selection of tissues was taken from each animal and examined histopathologically for neoplastic and non-neoplastic changes. 

The incidence of mortality was not affected by treatment and there were no toxicologically significant, compound-related effects on the clinical condition of the animals, with the exception of ophthalmoscopic changes. A reduction in appetite, and marginal impairment of food utilisation efficiency with associated lower weight gain was recorded for rats receiving 375 ppm. There was no treatment-related effect on water consumption. 

There were no changes in haematology, blood chemistry analysis or urinalysis that was considered to be of toxicological significance at any of the dose levels. There were no compound-related effects on organ weights. Macroscopic examination or rats dying, sacrificed in extremis, or killed at termination, revealed a higher incidence of lenticular opacity and congestion or haemorrhage in the eyes of male and female rats dosed with 375 ppm. There was also a slightly increased incidence of these changes in males receiving 75 ppm. At the interim kill an increased incidence of caecal distension with ingesta or gas was noted in animals of both sexes receiving 375 ppm. There was also a slight increase in the incidence of prominent caecal blood vessels in males receiving 375 or 75 ppm. Similar caecal changes were also observed in two males receiving 375 ppm dying or killed intercurrently in the first year of the study. All other macroscopically observed lesions were considered to be unrelated to treatment. There were no histopathological findings to suggest that the test substance had a carcinogenic effect.

Histopathological examination of the eye revealed cataractous changes in the eyes of rats given 75 and 375 ppm. This was apparent after one year of dosing but the incidence and severity of the lesions increased during the second year of the study. There was a slightly increased incidence of cataractous change at termination only in females given 15 ppm. No changes were seen in males at this level. There were no compound-related lenticular effects at the 5 ppm.

 

A treatment-related incidence of triangular posterior subcapsular opacities of the lens was found in male and female animals receiving 375 and 75 ppm. Among rats receiving 375 ppm these lesions progressed to total opacification of the lens, affecting all surviving rats receiving 375 ppm when examined at week 104. A low incidence of rats with opacities was seen in the 15 ppm group at 104 weeks only. Cataractous change was observed in the lenses of rats at the 75 and 375 ppm dose levels. The incidence and severity were dose-related. There was also some evidence that prolonged dietary administration of the test substance at 15 ppm had a minimal cataractogenic effect.

 

In conclusion, there was no evidence of carcinogenic potential. The NOAEL for carcinogenicity is greater than 375 ppm test substance ion, equivalent to 700.5 ppm pure test substance, corresponding to a dietary intake of 27.80 and 36.31 mg/kg bw/day for males and females, respectively.

The eye was identified as the target organ and cataractous change was observed at dose levels of 15–375 ppm. After re-evaluation of these effects, the NOEL for cataract formation determined to be 15 ppm test substance ion, thus 28.02 ppm pure test substance, corresponding to a dietary intake of 1.083 and 1.345 mg/kg bw/day for males and females, respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

27.8 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Study similar to OECD 453 in compliance with GLP

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

Additional information

In a combined chronic toxicity/carcinogenicity study the test substance was administered to five groups of 60 male and 60 female CD rats of the Sprague Dawley strain, by admixture in the diet for 104 weeks at dietary concentrations of 0 (control), 5, 15, 75 and 375 ppm (performed similar to OECD 453 in compliance with GLP). This correspond to a dietary concentration of 0.19, 0.58, 2.91 and 14.88 mg test substance ion/kg bw/day for males (recalculated values for the test substance salt are 0.35, 1.08, 5.44 and 27.80 mg test substance salt/kg bw/day); and 0.24, 0.72, 3.64 and 19.44 mg test substance ion/kg bw/day for females (recalculated values for the test substance salt are 0.45, 1.34, 6.80, 36.31 mg test substance salt/kg bw/day). Ten male and ten female rats per group were killed after 1 year. Diets were analysed for achieved concentrations, homogeneity and stability. All animals were examined daily for mortality and clinical observations and once a week each animal was given a thorough clinical examination and palpated to enable time of appearance and location of any palpable masses to be noted. All animals were weighed pre-dose and weekly thereafter. Food consumption was recorded weekly and group mean food conversion ratios were calculated for successive 4-week periods during the first 24 weeks of treatment. Water consumption was measured daily for a 5-day period during weeks 6, 12 and 24/25. Ophthalmoscopy was carried out in all main group animals pre-dose and during weeks 13, 26, 52, 78 and 104. Blood for haematological and blood chemistry analyses was collected from 10 males and 10 females per main group pre-dose and during weeks 26, 52, 78 and 104. Blood samples were also taken from 5 rats/sex/satellite group pre-dose and in week 52. During weeks 24, 50, 76 and 102 individual overnight urine samples were collected from the same main group animals used for blood sampling and in week 50 samples were collected from the same 5/sex/satellite animals used for blood sampling. After 52 weeks all surviving rats in the satellite groups were killed. All surviving rats from the main groups were killed after 104 weeks of treatment. Each animal (including unscheduled decedents) was given a detailed macroscopic examination and a selection of organs were weighed. A comprehensive selection of tissues was taken from each animal and examined histopathologically for neoplastic and non-neoplastic changes. 

The incidence of mortality was not affected by treatment and there were no toxicologically significant, compound-related effects on the clinical condition of the animals, with the exception of ophthalmoscopic changes. A reduction in appetite, and marginal impairment of food utilisation efficiency with associated lower weight gain was recorded for rats receiving 375 ppm. There was no treatment-related effect on water consumption. 

A treatment-related incidence of triangular posterior subcapsular opacities of the lens was found in male and female animals receiving 375 and 75 ppm. Among rats receiving 375 ppm these lesions progressed to total opacification of the lens, affecting all surviving rats receiving 375 ppm when examined at week 104. A low incidence of rats with opacities was seen in the 15 ppm group at 104 weeks only. Cataractous change was observed in the lenses of rats at the 75 and 375 ppm dose levels. The incidence and severity were dose-related. There was also some evidence that prolonged dietary administration of the test substance at 15 ppm had a minimal cataractogenic effect.

There were no changes in haematology, blood chemistry analysis or urinalysis that was considered to be of toxicological significance at any of the dose levels. There were no compound-related effects on organ weights. Macroscopic examination or rats dying, sacrificed in extremis, or killed at termination, revealed a higher incidence of lenticular opacity and congestion or haemorrhage in the eyes of male and female rats dosed with 375 ppm. There was also a slightly increased incidence of these changes in males receiving 75 ppm. At the interim kill an increased incidence of caecal distension with ingesta or gas was noted in animals of both sexes receiving 375 ppm. There was also a slight increase in the incidence of prominent caecal blood vessels in males receiving 375 or 75 ppm. Similar caecal changes were also observed in two males receiving 375 ppm dying or killed intercurrently in the first year of the study. All other macroscopically observed lesions were considered to be unrelated to treatment. There were no histopathological findings to suggest that the test substance had a carcinogenic effect. Histopathological examination of the eye revealed cataractous changes in the eyes of rats given 75 and 375 ppm. This was apparent after one year of dosing but the incidence and severity of the lesions increased during the second year of the study. There was a slightly increased incidence of cataractous change at termination only in females given 15 ppm. No changes were seen in males at this level. There were no compound-related lenticular effects at the 5 ppm.

In conclusion, there was no evidence of carcinogenic potential. The NOAEL for carcinogenicity is 375 ppm test substance ion, thus 700.5 ppm test substance salt, corresponding to a dietary intake of 27.80 and 36.31 mg/kg bw/day for males and females, respectively. The eye was identified as the target organ and cataractous change was observed at dose levels of 15–375 ppm. After re-evaluation of these effects, the NOAEL for cataract formation was placed on 15 ppm test substance ion, thus 28.02 ppm test substance salt, corresponding to a dietary intake of 1.083 and 1.345 mg/kg bw/day for males and females, respectively.