Pentapotassium bis(peroxymonosulphate) bis(sulphate)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-04-09 - 2002-03-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment. Due to the high mortality the dose of 1000 mg/kg bw/day was reduced to 600 mg/kg bw/day. Therefore, the LOAEL is difficult to derive but assigned to 600 mg/kg bw/d for precautionary reasons.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

guideline 408 “Repeated dose 90-day oral toxicity study in rodents” (May

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Pentapotassium bis(peroxymonosulphate) bis(sulphate)

EC Number:

274-778-7

EC Name:

Pentapotassium bis(peroxymonosulphate) bis(sulphate)

Cas Number:

70693-62-8

Molecular formula:

H3K5O18S4

IUPAC Name:

pentapotassium bis((hydroperoxysulfonyl)oxidanide) hydrogen sulfate sulfate

Test material form:

solid

Test animals

Species:

rat

Strain:

other: Crl:CD(SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, Kent, UK
- Age at study initiation: approx. 6 weeks
- Weight at study initiation: 168 – 215 g (males); 141 – 180 g (females)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Deionised water

Details on oral exposure:

The subchronic toxicity of KMPS triple salt was assessed following oral administration to male and female Crl:CD(SD) IGS BR rats for a period of 13 weeks by gavage in water as the vehicle. Dose levels administered were 0, 25, 200 and 1000 mg/kg bw/day (dose volume: 10 mL/kg bw/day). Following 4 weeks of treatment at 1000 mg/kg bw/day, this dosage level was reduced to 600 mg/kg bw/day due to 4 unscheduled deaths and adverse clinical signs.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability in deionised water was confirmed at nominal concentrations of 1 mg/mL and 100 mg/mL, during ambient temperature.

Duration of treatment / exposure:

91 days

Frequency of treatment:

Once daily, 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

corresponding to 2.5 mg/mL

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

corresponding to 20 mg/mL

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

corresponding to 100 mg/mL; due to 4 unscheduled deaths and adverse clinical signs the high dosage level was reduced to 600 mg/kg bw/day after 4 weeks of treatment

No. of animals per sex per dose:

10 animals/group/sex (40 animals per sex in total)

Control animals:

yes, concurrent vehicle

Details on study design:

Clinical signs of toxicity and mortality/viability were observed daily. Body weights, body weight gains and food consumption was recorded on a weekly basis. A complete haematological and clinical-chemical examination was as well as an ophthalmoscopic examination performed on day 90 and during the Week 12, respectively. Functional and observational battery and motor activity assessments were performed before initiation and during the 12th week of treatment. A shortened battery was performed during Weeks 1 11 and Week 13. On termination of the study, organ weights were determined and a macroscopic as well as microscopic examination of all organs was performed.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

OBSERVATIONS: regularly throughout the study
- Clinical signs. Yes; at least twice daily; a detailed physical examination of each animal was performed weekly
- Mortality. Yes, twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily monitoring by visual appraisal was maintained throughout the dosing period.

OPHTHALMOSCOPIC EXAMINATION: Yes
Performed on all animals following instillation of tropicamide solution before commencement of treatment and during the week 12 of treatment.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 90
- How many animals: all animals
- Parameters checked:
Haematocrit, haemoglobin concentration, red blood cell count (RBC), mean cell haemoglobin concentration (MCHC), MCV, MCH, total and differential leukocyte count (neutrophils, basophils, eosinophils, lymphocytes, monocytes, large unstained cells (LUC), platelet count, prothrombin time, partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 90
- How many animals: all animals
- Parameters checked: sodium, potassium, glucose, urea, creatinine, total protein, albumin, alanine aminotransferase, aspartate aminotransferase, Albumin/Globulin ratio (A/G ratio), alkaline phosphatase, cholesterol

URINALYSIS: No

Sacrifice and pathology:

ORGAN WEIGHTS
yes
organs:
adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus with cervix

GROSS AND HISTOPATHOLOGY
yes
all dose groups
The macroscopic appearance of the tissue of all rats was recorded and samples of tissues were preserved:
adrenals, aorta, brain, caecum, colon, duodenum, epididymides, eyes, femur (with joint), head (including nasal cavity, paranasal sinuses and nasopharynx), heart, ileum, jejunum, kidneys, liver, lung (and bronchi), lymph nodes (mandibular and mesenteric), mammary area, oesophagus, ovaries, pancreas, pituitary gland, prostate gland, rectum, salivary glands, spinal cord, pituitary, prostate, rectum, salvary glands, sciatic nerves, seminal vesicles, skin, spinal cord, spleen, sternum, stomach, testes, thymus, thyroid, parathyroid, trachea, urinary bladder, uterus, (with cervix), other macroscopically abnormal tissue.

Microscopic examination of the prepared slides (from all above indicated tissues) was carried out for all terminal animals. Macroscopic abnormalities were processed and examined for any animal. Stomach was assessed for all dosage groups.

Other examinations:

Functional and observational battery and motor activity assessments were performed during the study at app. the same time of day before initiation of treatment and during the 12th week of treatment (monitoring by using a Coulbourn Infra-Red Activity Monitoring System).
A shortened battery was performed during Weeks 1 11 and Week 13. The battery comprised of 3 sets of observations. The first set was performed when initially handling the animal. The second set of observations was performed in the test arena and the third set comprised handling/specific testing of the animal.
1. Observations in the hand: Ease of removing the animal from the cage, reactivity to handling, occurrence of convulsions, tremors, twitches, salvation/lachrymation, piloerection, vocalisation on handling etc.
2. Observation in the arena: occurrence of convulsions, tremors, twitches, activity counts, rearing counts, assessment of gait/posture etc.
3. Sensory reactivity: approach response, touch response, auditory startle response, tail pinch response, grip strength

Statistics:

The following sequence of statistical tests was used for bodyweight, haematology, blood chemistry, organ weight and functional observational battery data:
If 75 % of the data were the same value, then a frequency analysis was applied. Treatment groups were compared using a Mantel test for a trend in proportions and also pairwise Fisher’s Exact tests for each dose against the control values
If Bartlett’s test for variance homogeneity was not significant at the 1 % level, then parametric analysis was applied. If the F1 test for monotonicity of dose-response was not significant at the 1 % level, William’s test for a monotonic trend was applied. If the F1 test was significant, suggesting that the dose-response was not monotone, Dunnett’s test was performed instead.
If Bartlett’s test was significant at the 1 % level, then logarithmic and square-root transformations were tried. If Bartlett’s test was still significant, then non-parametric tests were applied. If the H1 test for monotonicity of dose-response was not significant at the 1 % level, Shirley's’test for a monotonic trend was applied. If the H1 test was significant, suggesting that the dose-response was not monotone, Dunn’s test was performed instead.
Where appropiate, analysis of covariance was used in place of analysis of variance in the above sequence.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Tables mentioned below are presented under "Remarks on results including tables and figures" and under "Overall remarks"

OBSERVATIONS
Clinical signs
• Post-dose salivation was noted for all animals receiving 1000/600 mg/kg bw/day, with incidence of post-dose salivation also noted among animals receiving 200 mg/kg bw/day.
• During Week 4, piloerection, abnormal gait gasping and hunched posture were noted on occasions for a few animals receiving 1000 mg/kg bw/day. These findings were generally noted from 1 2 hours after dosing but no longer evident the following morning.
• Noisy respiration was noted for 5/10 males and 4/10 females receiving 1000 mg/kg bw/day and one animal receiving 200 mg/kg bw/day during the first 4 weeks of treatment. Following a reduction of the dose level from 1000 to 600 mg/kg bw/day, noisy respiration was noted for 1 male in Weeks 8 and 12 only.
• Incidences of wet coat, paddling of forepaws and piloerection were noted among animals receiving 600 mg/kg bw/day during Week 12and 13.
• Incidental findings of vocalisation and hair loss were noted in all groups.
Mortality
One male (no. 17M; 1000 mg/kg bw/day) was humanely sacrificed in week 3. Two males and one female (no. 14M, 15M ad 64F; 1000 mg/kg bw/day) were also sacrificed due to adverse clinical signs in week 4. Necrotic and inflammatory lesions of the stomach and small intestine were considered to have been contributory to death.

BODY WEIGHT/BODY WEIGHT GAIN
After 4 weeks of treatment, bodyweight gain was lower than that of the concurrent controls for all treated groups of males. The difference from controls was statistically significant for all groups but not dosage related for those animals receiving 25 or 200 mg/kg bw/day. Group mean bodyweight gain by all treated groups of females was however marginally higher than that of controls. Please refer to tables A6.4.1/01-1 and A6.4.1/01-2.

FOOD CONSUMPTION
• Food intake was reduced among males receiving 1000 mg/kg bw/day from Weeks 1 4, continuing to a slightly lesser degree when those animals received 600 mg/kg bw/day from Weeks 5-13.
• Food consumption among males receiving 25 or 200 mg/kg bw/day was slightly less than that by controls but no dose relationship was apparent and the degree of difference from control was minimal.
• Consumption by females at all three dosages was higher than controls to a similar degree and these variations are not thought to be treatment related.
Food conversion efficiency was calculated (please refer to table A6.4.1/01-3):
• The efficiency for animals receiving 25 or 200 mg/kg bw/day was similar to that of controls.
• Food conversion efficiency for animals receiving 600 mg/kg bw/day (previously 1000 mg/kg bw/day) continued to be inferior to that of controls for the period Week 5 13.
The reduction in food utilisation efficiency for animals receiving 1000 mg/kg bw/day and then 600 mg/kg bw/day indicate that the reduced body weight gain was not entirely attributable to reduced food intake and is suggestive of a toxic effect.

WATER CONSUMPTION
No changes in water consumption were observed.

OPHTHALMOSCOPIC EXAMINATION
The incidence and distribution of findings noted in Week 12 fall within background ranges for this age and strain


BLOOD ANALYSIS
Haematology
Week 13 examination:
Please refer to Table A6.4.1/01-4.
There were no adverse treatment-related changes in haematology. The following statistically significant changes in haematology parameters were considered to be non-adverse because of the magnitude or direction of change.
• Eosinophils were minimally increased in males dosed with 1000 mg/kg bw/day. This change was due primarily to one rat with increased WBC as well. Increased eosinophils were possibly related to treatment.
• WBC, basophils, and LUCs were minimally decreased in females dosed with 200 or 1000/600 mg/kg bw/day. These changes were possibly related to treatment. Due to the very small magnitude of response and the lack of dose-response between 200 and 1000/600 mg/kg bw/day these changes were considered to be non-adverse.
• Platelets were increased in females dosed with 1000/600 mg/kg bw/day. This change was considered treatment related. However, minimally increased platelets have no adverse consequences.
• Activated partial thromboplastin time (APTT) was minimally shortened in males dosed with 1000/600 mg/kg bw/day after 13 weeks. Shortened APTT, although likely related to treatment due to the consistency of change, was considered non-adverse because minimal decreases in coagulation times are not associated with toxicity.
• Prothrombin time (PT) was prolonged in males dosed with 200 or 1000/600 mg/kg bw/day. However, all individual values were within the reference interval.

Clinical chemistry
Week 13 examination:
Please refer to Table A6.4.1/01-5
There were no adverse treatment related changes in clinical chemistry parameters.
• ALT was minimally decreased in males dosed with 1000/600 mg/kg bw/day. This was unlikely to be due to treatment due to lack of a dose response.
• Creatinine was minimally decreased in males dosed with 1000/600 mg/kg bw/day. This was not considered to be treatment related due to the magnitude of the change. A minimal creatinine decrease is also not associated with toxicity.
• Glucose was increased in males dosed with 1000/600 mg/kg bw/day. This was not considered to be treatment related. Glucose concentrations of individual rats dosed with 25 or 200 mg/kg bw/day were more variable than those in rats dosed with 1000 mg/kg bw/day. All glucose concentrations of rats dosed with 1000/600 mg/kg bw/day were less than the mean of the reference interval.
• Potassium was decreased in males of all dose groups. However, potassium concentrations in individual rats were equal or greater than the 50th percentile of the reference interval. Therefore, although this change was likely related to treatment, it was not considered adverse.
• Sodium was decreased in females dosed with 1000/600 mg/kg bw/day. Although this change was likely related to treatment, it was not considered adverse due to the magnitude of the change.
• Total protein was minimally decreased in males and females dosed with 1000/600 mg/kg bw/day. This was not considered adverse because the magnitude of change was small. However, this change may be secondary to loss of fluid rich in protein into the gastrointestinal tract. Therefore, although this change was not considered adverse, it was secondary to adverse effects observed histologically.


URINANALYSIS
Not performed


SACRIFICE AND PATHOLOGY
Organ weights
Please refer to table A6.4.01/016.
• There was a significantly higher bodyweight adjusted group mean liver weight for males and females receiving 1000/600 mg/kg bw/day in comparison to controls. This may be treatment related but in the absence of any histopathological change this finding is not considered adverse. No histological changes were observed in the liver to explain the increased organ weight.
• The bodyweight adjusted group mean adrenal weight was also significantly higher for males receiving 1000/600 mg/kg bw/day and the bodyweight adjusted mean brain weights were statistically significantly higher in all treated groups of males. In the absence of dose related trends, consistency between the sexes or related histopathological changes, these differences are not considered to be related to treatment.
• In addition, an increased relative organ weight (see above) might be explainable by reduced absolute body weights.

Gross and histopathology
Macroscopic examination of the 4 decedents:
Please refer to table A6.4.01/01-7.
Stomach: congested in 2/3 males and 1 female
Forestomach: thickened in all decedent animals; oedematous in 1/3 males
Limiting ridge and forestomach: thickened/depression in 1/3 males
Antrum: pale area/white nodules in 1/3 males
Lymph nodes: mandibular, enlarged in 1 female
Lumbar: enlarged in 1/3 males
Caecum: distended in 2/3 males
Duodenum: congested in 1/3 males
Pancreas: dark in 2/3 males
Spleen: pale in 1 female
Abnormal G.I.tract in 1/3 males and distension of G.I.tract in 2/3 males and 1 female.

Macroscopic observations performed on survivors at termination revealed changes to the stomach:
Please refer to table A6.4.1/01-8.
- Forestomach
Thickening of the limiting ridge in 7/7 males and 8/9 females receiving 1000/600mg/kg bw/day and 1/10 females receiving 200 mg/kg bw/day
Generalised thickening in 3/7 males and 1/9 females, with roughening in 4/7 males and depressions in 2/7 males and 2/9 females receiving 1000/600 mg/kg bw/day
All other findings at termination were considered to fall within the background range of macroscopic changes.
Microscopic examination of survivors at termination:
Treatment related effects were observed in the stomach only in the high dose group
In the forestomach inflammation, oedema and haemorrhage in the mucosal and submucosal areas, epithelial necrosis and ulceration (less pronounced in females), epithelial hyperplasia (including the limiting ridge) was noted.
In few cases submucosal inflammation was observed in the glandular stomach in male and female high dose group compared to controls.

Microscopic examination of the Decedents:
Please refer to table A6.4.1/01-9.
In all cases, necrotic and inflammatory lesions of the stomach and, to a lesser extent, the small intestine were considered to be factors contributory to death. The principal findings were epithelial necrosis and subepithelial inflammation and, to a lesser extent, in the glandular region of stomach and villous atrophy and necrosis and also epithelial hyperplasia in the small intestine. Also considered to be associated with the clinical condition of the animals was apotosis, seen in various lymphoid tissues including spleen, mesenteric lymph node, mandibular lymph node and Peyer’s patch in the ileum.


OTHER
FOB and MA examinations:
Treatment with Oxone Monopersulfate Compound for 13 weeks was not associated with any behavioural changes which are considered to be indicative for neuotoxicity.

Effect levels

1

Dose descriptor:

LOAEL

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on the changes in the nonglandular stomach

2

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 1: 90-day gavage study with KMPS triple salt in rats – group mean bodyweight (g)

Week	Control	25 mg/kg bw/day	200 mg/kg bw/day	1000/600 mg/kg bw/day
-	males
0	196.5	188.4	189.7	191.6
1	255.3	242.3	245.5	242.6
2	316.5	296.2	297.1	289.9
3	366.3	334.5	339.2	323.2
4	404.7	367.5	372.0	339.8
5	430.7	389.2	398.0	363.9
6	448.6	406.4	418.5	382.1
7	465.7	414.9	435.3	398.4
8	488.4	434.0	451.9	408.0
9	504.2	450.0	465.6	422.4
10	518.9	461.3	479.7	431.4
11	537.7	473.5	492.5	442.1
12	546.3	483.3	504.6	449.1
13	548.3	487.2	506.8	453.1
-	females
0	157.4	161.2	160.6	163.4
1	183.5	191.4	191.3	194.8
2	207.6	215.9	216.0	217.9
3	224.4	236.2	236.6	239.2
4	237.3	250.2	247.6	253.4
5	250.5	259.0	258.3	265.4
6	257.8	267.7	268.6	273.3
7	264.7	277.6	277.4	278.8
8	269.6	285.6	281.4	283.4
9	278.7	287.5	286.2	292.3
10	282.6	295.1	291.0	293.9
11	288.3	302.7	296.5	295.5
12	287.0	303.9	300.0	298.2
13	288.8	299.0	298.7	298.6

Table 2: 90-day gavage study with KMPS triple salt in rats – group mean bodyweight gain

Week	Control	25 mg/kg bw/day	200 mg/kg bw/day	1000/600 mg/kg bw/day
-	males
0-4	208.2	179.1*	182.3*	147.8**
% of control	-	86	88	71
4-13	143.6	119.7	134.8	113.3*
% of control	-	83	94	79
0-13	351.8	298.8*	317.1*	261.1**
% of control	-	85	90	74
-	females
0-4	79.9	88.9	87.0	89.5
% of control	-	111	109	112
4-13	51.3	48.9	51.0	45.2
% of control	-	95	99	88
0-13	131.4	137.8	138.0	134.7
% of control	-	105	105	102

                          *P< 0.05; **P <0.01 in comparison with controls (Williams’ test)

 

Table 3: Food conversion efficiency – group mean values (%)

Dosage (mg/kg bw/day)	Male				Female
	0	15	200	1000/ 600	0	15	200	1000/ 600
Weeks	-				-
1-4	25.2	23.6	23.6	20.3	15.4	15.7	15.8	15.9
5-13	8.1	7.3	8.0	7.0	4.2	3.8	4.1	3.7
1-13	13.5	12.5	12.9	11.1	7.6	7.5	7.6	7.6

 

Table 4: Haematology

Dosage (mg/kg bw/day)	Males
	Eosinophils x10-9/L		Prothrombin time (sec.)		Activated partial thromboplastin time (sec.)
0	-		-		-
Mean	0.18		12.7		20.0
SD	0.051		0.6		1.49
n	10		10		10
25	-		-		-
Mean	0.21		13.3		20.3
SD	0.077		0.42		1.79
n	10		10		10
200	-		-		-
Mean	0.19		13.5*		19.2
SD	0.058		0.93		1.83
n	10		10		10
1000/600	-		-		-
Mean	0.30*		13.7**		16.9**
SD	0.208		1.04		1.58
n	7		7		7
Dosage (mg/kg bw/day)	Females
	WBC x10-9/L	Basophils x10-9/L		LUC x10-9/L		Plt x10-9/L
0	-	-		-		-
Mean	10.14	0.03		0.26		1014
SD	2.730	0.014		0.054		115.6
n	10	10		10		10
25	-	-		-		-
Mean	8.83	0.02		0.27		955
SD	2.602	0.008		0.097		314.3
n	10	10		10		10
200	-	-		-		-
Mean	6.88*	0.01**		0.19**		1074
SD	1.453	0.005		0.058		132.7
n	10	10		10		10
1000/600	-	-		-		-
Mean	8.59*	0.02**		0.20**		1217++
SD	2.492	0.010		0.071		139.9
n	9	9		9		9

*      p<0.05 in comparison with controls (Williams’ test)                               WBC: White blood cell count
**     p<0.01 in comparison with controls (Williams’ test)                               LUC: Large unstained cells
++     p<0.01 in comparison with controls (Shirley’s test)                                Plt: Platelet count

SD   Standard deviation

 

Table 5: Blood chemistry

Dosage (mg/kg bw/day)	Males
	ALT U/L	Creatinine µmol/L	Glucose mmol/L	K mmol/L	Total Protein g/L
0	-	-	-	-	-
Mean	44	50	5.29	4.4	71
SD	2.5	2.9	0.505	0.21	1.3
n	10	10	10	10	10
25	-	-	-	-	-
Mean	50	50	5.99	4.1*	70
SD	16.3	1.7	0.927	0.24	2.7
n	10	10	10	10	10
200	-	-	-	-	-
Mean	50	50	5.60	4.1*	71
SD	6.1	2.5	1.025	0.28	3.6
n	9	9	9	9	9
1000/600	-	-	-	-	-
Mean	39	47*	6.38**	3.9**	67**
SD	3.6	1.9	0.166	0.08	2.6
n	7	7	7	7	7

*      P<0.05 in comparison with controls (Williams’ test)                              ALT: Alanine aminotransferase
**     P<0.01 in comparison with controls (Williams’ test)                              K: Potassium

 

Table 5 (continued): Blood chemistry 

Dosage (mg/kg bw/day)	Females
	Na mmol/L	Total Protein g/L	Albumin g/L
0	-	-	-
Mean	139	77	40
SD	1.4	4.8	2.2
n	10	10	10
25	-	-	-
Mean	139	77	40
SD	1.3	3.7	1.9
n	10	10	10
200	-	-	-
Mean	138	75	39
SD	0.8	4.5	2.5
n	10	10	10
1000/600	-	-	-
Mean	137*	72*	37*
SD	1.3	3.6	2.3
n	9	9	9

*      p<0.05 in comparison with controls (Williams’ test)                      Na: Sodium

 

Table 6: Organ weights – group mean values (g) for animals killed after 13 weeks of treatment

Dosage (mg/kg bw/day)	Males
	Terminal Body weight	Brain	Adrenals	Liver
0	-	-	-	-
Mean (unadjusted)	542.6	2.15	0.063	20.49
SD	72.4	0.10	0.014	3.87
Mean (adjusted)	-	2.09	0.055	18.13
n	10	10	10	10
25	-	-	-	-
Mean (unadjusted)	483.0	2.15	0.058	19.12
SD	40.4	0.07	0.010	3.70
Mean (adjusted)	-	2.17*	0.061	19.94
n	10	10	10	10
200	-	-	-	-
Mean (unadjusted)	505.3	2.16	0.059	20.02
SD	32.2	0.08	0.012	2.95
Mean (adjusted)	-	2.15*	0.058	19.66
n	10	10	10	10
1000/600	-	-	-	-
Mean (unadjusted)	447.6	2.12	0.068	19.94
SD	40.9	0.10	0.014	2.72
Mean (adjusted)	-	2.19**	0.077**	22.65**
n	7	7	7	7

*       p<0.05 in comparison with controls (Williams’ test)

**      p<0.01 in comparison with controls (Williams’ test)

 

Table 6 (continued): Organ weights – group mean values (g) for animals killed after 13 weeks of treatment 

Dosage (mg/kg bw/day)	Females
	Terminal Body weight	Liver
0	-	-
Mean (unadjusted)	286.2	10.53
SD	24.9	1.18
Mean (adjusted)	-	10.80
n	10	10
25	-	-
Mean (unadjusted)	296.5	10.59
SD	17.0	1.15
Mean (adjusted)	-	10.48
n	10	10
200	-	-
Mean (unadjusted)	295.9	11.34
SD	39.9	1.86
Mean (adjusted)	-	11.25
n	10	10
1000/600	-	-
Mean (unadjusted)	295.5	12.13*
SD	19.3	0.98
Mean (adjusted)	-	12.06**
n	9	9

*       p<0.05 in comparison with controls (Williams’ test)
**      p<0.01 in comparison with controls (Williams’ test)

 

Table 7: Macropathology –group distribution of findings for animals killed or dying during the study

Organ	Males 1000/600 mg/kg bw/day	Females 1000/600 mg/kg bw/day
Caecum          No. examined          distended	3 2	1 0
Pancreas          No. examined          dark	3 2	1 0
Stomach          No. examined          Forestomach thickened          Limiting Ridge, thickened          Forestomach, roughened          Forestomach, depression(s)          congested          Antrum, white nodule(s)          Antrum, pale area(s)          Forestomach oedematous	3 3 1 1 1 2 1 1 1	1 1 0 0 0 1 0 0 0
Miscellaneous          No. examined          Abnormal contents (G.I. tract)          Distension G.I. tract	3 1 2	1 0 1

 

Table 8: Macroscopic observations performed at termination

Dosage (mg/kg bw/day)	Males
	0	25	200	1000/600
Organ	-	-	-	-
Stomach          No. examined          Forestomach, thickened          Limiting ridge, thickened          Forestomach roughened          Forestomach depression(s)          Antrum, white nodule(s)          Cyst(s) Dark area(s)	10 0 0 0 0 2 1 0	10 0 0 0 0 1 0 0	10 0 0 0 0 2 0 0	7 3 7 4 2 1 0 1
Skin          No. examined          Hairloss	10 2	10 1	10 1	7 1
Dosage (mg/kg bw/day)	Females
	0	25	200	1000/600
Organ	-	-	-	-
Stomach          No. examined          Forestomach, thickened          Limiting ridge, thickened          Forestomach depression(s)          Antrum, white nodule(s)          Cyst(s)         Forestomach raised area(s)	10 0 0 0 1 0 0	10 0 0 0 0 0 0	10 0 1 0 4 0 0	9 1 8 2 1 1 1
Skin          No. examined          Hairloss	10 6	10 7	10 3	9 4

 

Table 9: Histopathology –group distribution of findings for animals killed or dying during the study

Organ	Males 1000/600 mg/kg bw/day	Females 1000/600 mg/kg bw/day
Stomach          No. examined          Epithelial hyperplasia-nonglandular region          Submucosal oedema-nonglandular region          Submucosal inflammation-nonglandular region          Epithelial hyperplasia-glandular region          Epithelial necrosis-nonglandular region          Ectopic nonglandular epithelium in glandular          Mucosa, focal          Subepithelial inflammation-glandular region          Epithelial hyperplasia-limiting ridge          Subepithelial inflammation-nonglandular region          Mucosal inflammation-nonglandular region          Mcosal inflammtion-glandular region          Subepithelial oedema-nonglandular region	3 3 3 3 3 3 3 3 1 1 3 1 2 2	1 1 1 1 1 1 1 1 0 0 1 0 0 0
Thymus          No. examined          Apotosis-Cortex          Apotosis Medulla	3 3 2	1 1 0
Necrosis and inflammation of stomach and intestine	3/3	1/1

Applicant's summary and conclusion

Conclusions:

The study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability). Treatment of rats with KMPS triple salt at a combined dosage of 1000/600 mg/kg bw/day for 13 weeks resulted in pathological changes of the stomach. Effects were severe and resulted in the death of 4 animals. Following reduction of the high dose level to 600 mg/kg bw/day there were no more deaths but changes in the nonglandular stomach persisted. There were no toxic effects noted at 200 or 25 mg/kg bw/day and the NOAEL is considered to be 200 mg/kg bw/day. Changes in the stomach of the decedents are considered to be a direct local effect following bolus administration and the oxidative reaction mechanisms of KMPS triple salt at the site of first contact.
No classification and labelling regarding repeated dose toxicity is necessary according to Regulation 1272/2008/EC (CLP).

Executive summary:

Materials and methods

The subchronic toxicity of KMPS triple salt was assessed following oral administration to male and female Crl:CD(SD) IGS BR rats for a period of 13 weeks by gavage in water as the vehicle. Dose levels administered were 0, 25, 200 and 1000 mg/kg bw/day (dose volume: 10 ml/kg bw/day). Following 4 weeks of treatment at 1000 mg/kg bw/day, this dosage level was reduced to 600 mg/kg bw/day due to 4 unscheduled deaths and adverse clinical signs.

Clinical signs of toxicity and mortality/viability were observed daily. Body weights, body weight gains and food consumption was recorded on a weekly basis. A complete haematological and clinical-chemical examination was as well as an ophthalmoscopic examination performed on day 90 and during the Week 12, respectively. Functional and observational battery and motor activity assessments were performed before initiation and during the 12th week of treatment. A shortened battery was performed during Weeks 1 11 and Week 13. On termination of the study, organ weights were determined and a macroscopic as well as microscopic examination of all organs was performed.

 

Results and discussion

Pathological changes resulting from the administration of KMPS triple salt at 1000 mg/kg bw/day was marked, culminating in the death of 4 rats during Week 3 or 4 of treatment. The dose level was reduced to 600 mg/kg bw/day for the surviving animals of this level from Week 5 to termination (Week 13). In all 4 decedent animals, necrotic and inflammatory lesions of the stomach and the small intestine were considered to factors contributory to death. Which is considered to be a direct local effect following bolus administration and the oxidative reaction mechanisms of KMPS at the site of first contact.

Histopathological change was evident in animals receiving 1000/600 mg/kg bw/day for 13 weeks. The target organ was identified as the stomach with inflammatory, degenerative and hyperplastic changes mainly in the nonglandular stomach, the principal findings of which were inflammation, epithelial necrosis and ulceration, and epithelial hyperplasia in the nonglandular stomach. Macropathological changes were also apparent.

The pathological findings seen in the nonglandular area of the stomach are considered to represent damage due to the physical nature of the test substance and as such not considered to be associated with systemic toxicity. The changes in the gut were not of sufficient severity to affect nutrient availability or disturb general homeostasis, when animals were given 600 mg/kg bw/day.

No pathological changes related to treatment were identified in animals receiving 200 mg/kg bw/day. The complete absence of any adverse effect of treatment at this dose level indicates a steep dose-response curve between 200 and 1000 mg/kg bw/day for stomach pathology. The nature of lesions suggests that treatment at 600 mg/kg bw/day was potentiating damage initially caused by treatment at 1000 mg/kg bw/day.