Diquat dibromide

Endpoint summary

Description of key information

- Oral: NOEL = 0.93 mg/kg bw/day for males/females, dog, 1-year, feeding, OECD 452, Hopkins 1990.

Based on the findings from this study the NOEL is considered to be the NOAEL.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Mar 1988 to 17 Mar 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Version / remarks:

1981

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.4100 (Chronic Toxicity)

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

EU Method B.30 (Chronic Toxicity Studies)

Version / remarks:

1988

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: 21 - 24 weeks
- Weight at study initiation: 9.7 - 14.3 kg for males and 7.5 - 11.2 kg for females
- Housing: Individually in indoor pens, each with a floor measuring 345 x 115 cm and consisting of sleeping quarters (with heated floor) and a separate exercise area.
- Diet: male dogs received 400 g and females received 350 g of Laboratory Diet A
- Water: mains drinking water ad libitum
- Acclimation period: 4 - 5 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 - 27
- Air changes (per hr): 12
- Photoperiod: 11 hours light / 13 hours dark

IN-LIFE DATES: From 8 Mar 1988 To: 17 Mar 1989

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
In order to maintain the achieved dose of substance (as mg/kg/day) as close as possible to target levels, the dietary concentration of test substance was adjusted as necessary. Prior to the preparation of the next batch of diet, the group mean body weights were examined and the achieved dose calculated for each group. If considered necessary, the inclusion level of substance in the diet was adjusted to maintain the target dose level. Experimental diet was prepared by dilution of a pre-mix of the test material in ground Laboratory Diet A, mixed mechanically, pelleted and dispensed into bins. Control diet was prepared in the same way as the test diets, except for the addition of test material. The experimental diets were usually prepared in two lots of 50 kg.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken from all batches of pelleted diet for analysis of substance concentration. The chemical stability of substance in the diet was determined for the high and low concentrations at 0 and 48 days after preparation. Samples were also taken from the high and low dose diets for homogeneity determination. All analyses were performed on pelleted diet.

Duration of treatment / exposure:

1 year

Frequency of treatment:

Daily

Dose / conc.:

0.5 other: mg test substance cation species/kg bw/ day (nominal)

Remarks:

Low dose. Group 2

Dose / conc.:

2.5 other: mg test substance cation species/kg bw/ day (nominal)

Remarks:

Mid dose. Group 3

Dose / conc.:

12.5 other: mg test substance cation species/kg bw/ day (nominal)

Remarks:

High dose. Group 4

No. of animals per sex per dose:

4

Control animals:

yes, plain diet

Details on study design:

ANIMAL ASSIGNMENT
The animals were housed as 8 replicates (randomised blocks), 4 per sex, which corresponded to litters. Each replicate consisted of one dog from each group housed in adjacent pens. The randomisation procedure ensured the even distribution of dogs to treatment groups according to litter and body weight within litter.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS
A detailed clinical examination, including cardiac and pulmonary auscultation, was performed by a veterinarian on all dogs during weeks -1, 13, 26, 39 and 52. The dogs were observed at least twice daily, after dosing and at the end of the working day, for gross clinical or behavioural changes. Detailed clinical observations were made once a week. The appearance and consistency of faeces were recorded daily.

BODY WEIGHT
All dogs were weighed weekly (before feeding) throughout the pre-study period, on day 1 of treatment and thereafter at weekly intervals throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food residues (where present) were weighed, recorded and discarded each day, either within 3 - 4 hours of presentation (during the pre-treatment period) or just prior to giving the next meal (during the treatment period).
The group mean achieved intake of test substance, in terms of mg/kg/day, was calculated from individual body weight and food consumption data and the nominal inclusion level (ppm) of substance in the diet, as follows:
mg/kg/day = (ppm substance (nominal) x food consumed per day (q))/ (body weight (g)),
where body weight = 0.5 x (body weight at start of week + body weight at end of week).

OPHTHALMOSCOPIC EXAMINATION
Eyes of all dogs were examined during weeks -1, 8, 16, 24, 32, 40, 48 and 52.

HAEMATOLOGY AND CLINICAL CHEMISTRY
Jugular vein blood samples were obtained from all animals (before feeding) in weeks -1, 4, 13, 26 and 52.

HAEMATOLOGY
The following parameters were examined in all animals at all time points: haemoglobin, mean cell haemoglobin concentration, haematocrit, platelet count, red blood cell count, total white cell count, mean cell volume, differential white cell count, mean cell haemoglobin, blood cell morphology, prothrombin time, kaolin-cephalin time.

CLINICAL CHEMISTRY
The following parameters were examined in all animals at all time points: urea, alkaline phosphatase activity, creatinine, aspartate aminotransferase activity, glucose, alanine aminotransferase activity, albumin, magnesium, total protein, calcium, cholesterol, phosphorus (as phosphate), triglycerides, sodium, bilirubin, potassium, creatine kinase activity, chloride (not measured week -1).
Jugular vein blood samples were taken from all animals before feeding and 2 - 3 hours after feeding in weeks 13, 39 and 52 of treatment. The plasma was analysed by HPLC for test substance concentration using a modification of a published method (Gill et al, 1983).

URINALYSIS
Urine was collected by catheterisation from all dogs during weeks -1, 26 and 52.
Parameters: Volume, glucose, colour, ketones, appearance, protein, specific gravity, bilirubin, pH, blood, urobilinogen, microscopy of sediment.

Sacrifice and pathology:

GROSS PATHOLOGY
On completion of 52 weeks of treatment, all animals were deeply anaesthetised by intravenous administration of sodium pentobarbitone and then killed by exsanguination. A full necropsy examination was performed on each animal and findings recorded.
From all animals the following organs were removed, trimmed free of extraneous tissue and weighed: adrenal glands, kidneys, brain, liver, epididymides, testes, thyroid with parathyroids.
Paired organs were weighed separately.

HISTOPATHOLOGY:
The following tissues were examined in situ, removed and examined and fixed in an appropriate fixative: Gross lesions including masses, mammary gland (females only), adrenal gland, nerve - sciatic, aorta (abdominal), oesophagus, brain, ovary, bone and bone marrow (sternum), pancreas, caecum, pituitary gland, cervix, prostate gland, colon, rectum, duodenum, salivary gland (submandibular), epididymis, skin, eye, spinal cord, femur (including stifle joint - stored only), spleen, gall bladder, stomach, heart, testis, ileum, thymus, jejunum, thyroid/parathyroid gland, kidney, tongue, liver, trachea, lung, urinary bladder, lymph node (mesenteric, prescapular), uterus, voluntary muscle,
All processed tissues were examined by light microscopy.

Statistics:

Body weight gains and absolute body weights were considered by analysis of variance, separately for males. Clinical biochemistry and haematology data were considered at each time of sampling by analysis of covariance on pre-experimental values except for plasma chloride which, in the absence of pre-experimental data, was considered by analysis of variance at each time of sampling. Male and female data were analysed together and the results examined to determine whether any differences between control and treated groups were consistent between sexes. The covariate adjustment was based on the separate sex pre-experimental group means. 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 differential effects on left and right components.
Analyses of variance and covariance, with the exception of analyses for organ weights, allowed for the replicate structure of the study design. All analyses were carried out using the GLM procedure in SAS (1985) and unbiased estimates of treatment group means were provided by the least square means (LSMEANS option in SAS). Each treatment group mean was compared with the control group mean using a two-sided Student's t-test, based on the error mean square in the analysis.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All clinical observations noted during the study were of a type and frequency normally seen in the beagle studies of this duration and there was no indication of an effect of the treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain of males and females given 12.5 mg/kg bw/day was slightly, but significantly lower than that of respective controls during the first 2 weeks of treatment. There were no other notable differences in group mean body weight gains during the remainder of the treatment period. There was no effect on the body weight gain of animals given 0.5 or 2.5 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no adverse effect on food consumption in treated groups and no indication of unpalatability of the test diets.
The achieved intake of substance by treated groups was slightly lower than nominal in males and slightly higher than nominal in females. Overall, achieved dosages were acceptably close to target dosages throughout the treatment period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Bilateral lenticular opacity (cataract) was found in all animals which received 12.5 mg/kg bw/day; unilateral focal lenticular opacity was noted in two females which received 2.5 mg/kg bw/day.
A characteristic pattern of cataract development was observed. In most animals increased prominence of posterior suture lines preceded the appearance of irregular or triangular opacity; in the latter, the points of the triangular area always coincided with the posterior suture lines. Prominent posterior suture lines were first noted in three males and one female in this group at 16 weeks, while actual lenticular opacity was first noted at 16 weeks in males and at 24 weeks in females. Although the changes were ultimately bilateral in all animals, there was often a slight difference between the two eyes in the early stages of development. Increased extent of lenticular opacity was observed at consecutive examinations in all animals given 12.5 mg/kg bw/day. At weeks 48 and 52 of treatment, bilateral opacity involving the whole lens was recorded in all males and one female. Two females in this group showed large bilateral irregular opacities and the one remaining female showed a smaller triangular opacity in both eyes.
Two females given 2.5 mg/kg bw/day also showed unilateral lenticular opacity. One animal showed an irregular faint star-shaped opacity in the left eye which was unchanged from week 8 of treatment until week 52, when a small triangular opacity similar to the lesions seen in high dose animals was noted (the right lens was unaffected). It is considered that only the latter lesion in this animal is related to substance administration.
The early change is thought to be an incidental finding as it was non-progressive, appeared earlier and was different in appearance from the lesion noted in animals given 12.5 mg/kg bw/day. One other female given 2.5 mg/kg bw/day also showed a small focal opacity in the left eye from week 40 of treatment, but the lesion did not progress (the right lens was unaffected). This lesion may also be related to treatment. Lenticular opacity was not observed in any animal given 0.5 mg/kg bw/day or in controls. A variety of other minor ophthalmoscopic changes were recorded at a very low incidence that was unrelated to substance administration.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no differences in haematological parameters which were considered to be related to treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were no differences in blood clinical chemistry parameters which were considered to be related to treatment.
The test substance was not detected in the plasma of animals given 0.5 mg/kg bw/day or in controls. For animals given 12.5 mg/kg bw/day, levels 2 - 3 hours after feeding showed a dose-related increase over those recorded for animals given 2.5 mg/kg bw/day. Only for animals in the high dose group was substance detected in plasma before feeding.

Endocrine findings:

not examined

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:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant differences in organ weights. The mean kidney weight and kidney:body weight ratio of males and females given 12.5 mg/kg bw/day was greater than that of respective controls, attaining statistical significance for each sex (in females, following exclusion of one animal given 12.5 mg/kg bw/day with one kidney absent). Males in all groups given substance showed lower mean adrenal and epididymis weights when compared to control. A slightly more significant and dose-dependent trend was established following adjustment for body weight. However, one control had a noticeably higher adrenal weight compared to the remaining controls, exclusion of which resulted in a less significant trend in treated groups.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Bilateral lenticular opacity was observed at necropsy in all males and 3 of 4 females given 12.5 mg/kg bw/day but not in any animal in the control or lower dose groups. Uniform thickening of the wall of the small intestine (duodenum, jejunum, ileum) and colon was recorded in one animal of each sex given 0.5 and 12.5 mg/kg bw/day and in two males and one female given 2.5 mg/kg bw/day, but not in any control. In one affected male in both the 2.5 and 12.5 mg/kg bw/day, thickening of the bowel wall appeared to extend into the rectum. A small number of other lesions were observed, none of which was related to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Slight to marked bilateral cataractous change of the ocular lens was observed in six animals given 12.5 mg/kg bw/day and minimal or slight unilateral cataractous change was detected in the two other animals in this dose group. Minimal unilateral cataractous change was also detected in one female given 2.5 mg/kg bw/day. Cataractous change was not detected in the lens of any animal in the control or 0.5 mg/kg bw/day groups. Chronic inflammatory changes were detected in the colon and rectum of all animals given 12.5 mg/kg bw/day consistent with chronic irritation of the bowel by test substance in the ingesta. In most cases the rectum was more severely affected than the colon. Similar changes were also seen in the caecum of all males and two females in this group.
The gross thickening of the bowel wall noted at examination post mortem in a few animals in all treated groups was due to hypertrophy of the muscle layer normally seen as a physiological response to increased workload. The incidence and severity were not dose-related and there was no association with the inflammatory lesions observed in the colon and rectum of the 12.5 mg/kg bw/day animals. Although the etiology of the intestinal findings is uncertain, there was no associated clinical evidence of intestinal dysfunction and these changes were considered not to be of toxicological significance.

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOEL

Effect level:

0.5 mg/kg bw/day (actual dose received)

Based on:

other: test substance cation species

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

ophthalmological examination

Remarks on result:

other:

Remarks:

Original value presented in study

Key result

Dose descriptor:

NOEL

Effect level:

0.93 mg/kg bw/day (actual dose received)

Based on:

other: pure test substance

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

ophthalmological examination

Remarks on result:

other:

Remarks:

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

Critical effects observed:

yes

Lowest effective dose / conc.:

2.5 other: mg test substance cation species/kg bw/day

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.:

4.7 mg/kg bw/day (actual dose received)

System:

eye

Organ:

lens

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

DIET PREPARATION AND ANALYSIS

- Concentration analysis: The majority of diets were found to be within 10 % of target values. Only two batches of Group 2 diet showed a greater deviation from target level, but were within 12.3 % of the nominal concentration.

- Homogeneity: Homogeneity was satisfactory, with intersample variation of 5 % or less.

- Stability: Stability was satisfactory.

 

Calculation of key result

The original effect levels were expressed as cation species of the test 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 test substance divided by 184.2 g/mol molecular weight of test substance cation species):

NOEL =1.868 x 0.5 mg/kg bw /day = 0.93 mg/kg bw /day.

Target system / organ toxicity = 1.868 x 2.5 mg/kg bw /day = 4.7 mg/kg bw /day.

 

Table 1: Intergroup comparison of body weight gain from start of study - selected time points (kg)

	Dose level (mg/kg/day)
	Males				Females
week	0	0.5	2.5	12.5	0	0.5	2.5	12.5
1	0.27	0.32	0.30	0.17	0.25	0.15	0.07	0.00*
2	0.70	0.88	0.75	0.32*	0.47	0.38	0.30	0.15**
3	0.92	1.13	1.00	0.67	0.60	0.55	0.50	0.35
12	2.67	3.30	2.75	2.60	1.82	1.65	1.90	1.47
* 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)

 

Table 2: Group mean dose received (mg/kg/day)

weeks	Target dose (mg/kg/day)
	males			females
	0.5	2.5	12.5	0.5	2.5	12.5
1 - 13	0.47	2.39	11.60	0.52	2.53	12.98
1 - 26	0.46	2.38	11.54	0.52	2.52	13.04
1 - 52	0.46	2.42	11.48	0.53	2.53	13.21

 

Table 3: Intergroup comparison of selected ophthalmoscopy findings

	Dose level (mg/kg/day)
	Males				Females
Organ/Finding	0	0.5	2.5	12.5	0	0.5	2.5	12.5
Bilateral lenticular opacity	0	0	0	4	0	0	0	4
Unilateral lenticular opacity	0	0	0	0	0	0	2	0

The presence of each finding is recorded once for each animal regardless of the number of times observed. Pre-experimental observations not included

 

Table 4: Intergroup comparison of selected organ weights (g)

	Dose level (mg/kg/day)
Organ	Males
	0	0.5	2.5	12.5
Kidney :
Absolute wt (g)	68.5	70.0	68.6	84.7*
Adjusted wt (%)	68.4	70.1	68.5	84.8*
Adrenal :
Absolute wt (g)	1.61 (3)	1.60	1.49	1.57
Adjusted wt (%)	1.70 (3)	1.57	1.57	1.46*
Epididymides :
Absolute wt (g)	7.22	6.67	6.05*	6.47
Adjusted wt (%)	7.44	6.56	6.24*	6.17*
	Females
Kidney :
Absolute wt (g)	57.3	55.3	56.6	67.9* (3)
Adjusted wt (%)	57.7	56.1	55.2	67.7* (3)

Adjusted = adjusted for body weight

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

Number of animals, when less than 4, shown in parentheses

 

Table 5: Intergroup comparison of the incidence of selected microscopic findings

	Dietary Concentration (mg/kg/day)
	Males				Females
Organ/Finding	0	0.5	2.5	12.5	0	0.5	2.5	12.5
Eye:
Bilateral cataractous change
Slight	0	0	0	1	0	0	0	1
Moderate	0	0	0	1	0	0	0	1
Marked	0	0	0	1	0	0	0	1
Unilateral cataractous change
Minimal	0	0	0	1	0	0	1	0
Slight	0	0	0	0	0	0	0	1
Colon:
Chronic enteritis-
Slight	0	0	0	1	0	0	0	2
Moderate	0	0	0	3	0	0	0	2
Rectum:
Chronic proctitis:
Slight	0	0	0	0	0	0	0	2
Moderate	0	0	0	1	0	0	0	1
Marked	0	0	0	3	0	0	0	1

Conclusions:

Administration of test substance to dogs for up to one year resulted in toxicity to the lens and large intestine. Bilateral lenticular opacities (cataract) were observed in all dogs which received 12.5 mg/kg bw/day and unilateral opacities in two females which received 2.5 mg/kg bw/day, corresponding to 4.7 mg/kg bw/day of the pure test substance. Chronic inflammatory changes were seen in the colon and rectum of all dogs which received 12.5 mg/kg bw/day. The no observed effect level (NOEL) for the test substance cation species is reported to be 0.5 mg/kg bw/day, corresponding to 0.93 mg/kg bw/day of the pure test substance, when administered to dogs for one year.

Executive summary:

Groups of 4 male and 4 female beagle dogs received oral doses of test substance in the diet at target doses of 0 (control), 0.5, 2.5 or 12.5 mg test substance cation species/kg bw/day in a study complying to OECD guideline 452 and GLP. The animals were observed daily for any signs of toxicity, detailed observations and body weights were recorded at weekly intervals, daily food consumption was recorded and used to determine the achieved dietary intake of test substance/week. Eyes were examined by indirect ophthalmoscopy at frequent intervals during the study and blood and urine samples were collected at intervals for haematology/clinical bioanalyses. Blood samples were also collected before and after feeding every 3 months during the study for the analysis of test substance in plasma. At termination, specified organs were weighed and a full range of tissues was removed and submitted for histopathological examination.

There were no clinical signs of toxicity during the study. A slight impairment in body weight gain was noted during the first 2 weeks of treatment for males and females at 12.5 mg/kg/day. There were no effects on haematology, clinical biochemistry or urine cytology. Increases in kidney weight were noted at 12.5 mg/kg bw/day and reductions in adrenal and epididymal weight; there were no histopathological lesions. Bilateral lenticular opacity was found in all animals which received 12.5 mg/kg bw/day, unilateral focal lenticular opacity was noted in two females which received 2.5 mg/kg bw/day. Inflammatory lesions were noted in the large intestine of all animals which received 12.5 mg/kg bw/day, consistent with chronic irritation of the bowel mucosa by test substance in the ingesta.

Administration of test substance to dogs for up to one year resulted in toxicity to the lens and large intestine. Bilateral lenticular opacities (cataract) were observed in all dogs which received 12.5 mg/kg bw/day and unilateral opacities in two females which received 2.5 mg/kg bw/day, corresponding to 4.7 mg/kg bw/day of the pure test substance. Chronic inflammatory changes were seen in the colon and rectum of all dogs which received 12.5 mg/kg bw/day. The No Observed Effect Level (NOEL) for the test substance cation species was reported at 0.5 mg/kg bw/day, corresponding to 0.93 mg/kg bw/day of the pure test substance, when administered to dogs for one year.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

0.93 mg/kg bw/day

Study duration:

subchronic

Species:

dog

Quality of whole database:

GLP compliant OECD 452 study

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

1 -year dietary study in dogs

Groups of 4 male and 4 female beagle dogs received oral doses of test substance in the diet at target doses of 0 (control), 0.5, 2.5 or 12.5 mg test substance cation/kg bw/day in a study complying to OECD guideline 452 and GLP. The animals were observed daily for any signs of toxicity, detailed observations and body weights were recorded at weekly intervals, daily food consumption was recorded and used to determine the achieved dietary intake of test substance/week. Eyes were examined by indirect ophthalmoscopy at frequent intervals during the study and blood and urine samples were collected at intervals for haematology/clinical bioanalyses. Blood samples were also collected before and after feeding every 3 months during the study for the analysis of test substance in plasma. At termination, specified organs were weighed and a full range of tissues was removed and submitted for histopathological examination.

There were no clinical signs of toxicity during the study. A slight impairment in body weight gain was noted during the first 2 weeks of treatment for males and females at 12.5 mg/kg/day. There were no effects on haematology, clinical biochemistry or urine cytology. Increases in kidney weight and reductions in adrenal and epididymal weight were noted at 12.5 mg/kg bw/day; there were no histopathological lesions. Administration of test substance to dogs for up to one year resulted in toxicity to the lens and large intestine. Bilateral lenticular opacity (cataract) was found in all animals which received 12.5 mg/kg bw/day, unilateral focal lenticular opacity was noted in two females which received 2.5 mg/kg bw/day, corresponding to 4.7 mg/kg bw/day of pure test substance. Inflammatory lesions were noted in the large intestine of all animals which received 12.5 mg/kg bw/day, consistent with chronic irritation of the intestinal mucosa by test substance in the diet. The No Observed Effect Level (NOEL) reported for the test substance cation was 0.5 mg/kg bw/day, corresponding to 0.93 mg/kg bw/day of pure test substance. Based on the findings in this study, the NOEL is carried forward as NOAEL.

 

 

Combined chronic toxicity/carcinogenicity study in rats

In a combined chronic toxicity/carcinogenicity study similar to OECD 453 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 mixing into the diet for 104 weeks at dietary concentrations of 0 (control), 5, 15, 75 and 375 ppm test substance cation. This corresponded to a dietary concentration of 0.35, 1.08, 5.44 and 27.80 mg pure test substance/kg bw/day for males and 0.45, 1.34, 6.80, 36.31 mg pure test substance/kg bw/day for females.  

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. 

Opthalmoscopic examinations revealed a treatment-related incidence of triangular posterior subcapsular opacities of the lens 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-dependent. 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 of toxicological significance at any of the dose levels. There were no compound-related effects on organ weights. Macroscopic examination or rats found dear in the course of the study, 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.

Overall, 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 was determined to be 15 ppm test substance cation, equivalent to 28.02 ppm pure test substance and  corresponding to a dietary intake of 1.083 and 1.345 mg test substance/kg bw/day for males and females, respectively.

 

Investigatory study for cataract formation in rats

Groups of 12 male and 12 female Sprague-Dawley rats were fed diets containing 0, 30, 60 or 300 ppm test substance cation for 90 consecutive days in an investigatory study for cataract formation performed in compliance with GLP. Mean dietary dose levels for males were: 2.4, 4.7 and 23.2 mg test substance cation/kg bw/day; and for females 2.7, 5.0 and 25.3 mg test substance cation/kg bw/day. 

There were no toxicologically significant clinical observations. At 300 ppm, body weight and food consumption were generally low compared to control throughout the study for males and low compared to control during first week of treatment for females. Posterior lens opacities were present in week 13 for 10/12 males and 9/12 females at 300 ppm. A lens opacity plaque was also seen for one female at this dose level. There were no ophthalmoscopic abnormalities in animals in the control or 30 or 60 ppm test substance groups.

The NOEL for ophthalmoscopic abnormalities in the Sprague Dawley rat was reported to be 60 ppm test substance cation (equivalent to 4.7 mg test substance cation/kg bw/day in males and 5.0 mg test substance cation/kg bw/day in females) corresponding to 8.8 and 9.3 mg pure test substance/kg bw/day in males and females, respectively.

Justification for classification or non-classification

The primary target organ in rats and dogs is the eye (cataractous lesions). Lesions in the mouth and gastro-intestinal tract were also seen and were considered to reflect chronic local irritation. The dog was the most sensitive species. The relevant oral NOEL was 0.93 mg/kg bw/day in the 1-year dog study based on unilateral focal lenticular opacity.

Based on the available information, the test substance is classified as STOT RE 1, H372: Causes damage to the eye through prolonged or repeated oral exposure according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.