Manganese Violet

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Subchronic oral toxicity study of potassium pyrophosphate as a preliminary to long-term carcinogenicity studies in F344 rats

Author:

Shimoji N, Matsushima Y, Imaida K, Hasegawa R, Kurokawa Y and Hayashi Y.

Year:

1988

Bibliographic source:

ESKHA5 Eisei Shikenjo Horoku. Bulletin of the Institute of Hygienic Sciences. 106: 66-72

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

A 13 week sub-chronic toxicity study of potassium pyrophosphate was carried out in male and female F344 rats at the dose levels of 10, 5, 2.5, 1.25, 0.6 and 0% in the diet. Sixty animals of both sexes were divided into the 6 dosage groups. The purpose of the study was to determine the maximumtolerable dose dose of test substance for use in rats in a long-term carcinogenicity study. Observations were limited to mortality, clinical abnormalities, body weights, food intake, haematology, clinical chemistry, gross pathology (incl. organ weight) and histopathology.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrapotassium pyrophosphate

EC Number:

230-785-7

EC Name:

Tetrapotassium pyrophosphate

Cas Number:

7320-34-5

IUPAC Name:

tetrapotassium diphosphate

Details on test material:

- Name of test material (as cited in study report): potassium pyrophosphate.
- Substance type: Water soluble, colourless or white crystal powder.
- Physical state: Solid.
- Analytical purity: ≥ 95%

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Ltd Japan.
- Age at study initiation: At least 6 weeks old.
- Fasting period before study: No. Fasting is recorded to have been carried out after the last dosing until the animals were sacrificed.
- Housing: 5 animals per plastic cage with soft chip on the floor which was changed every 2 weeks.
- Diet (e.g. ad libitum): CRF-1 (powder), Charles River Ltd. The test substance was mixed in the diet and provided ad libitum.
- Water (e.g. ad libitum): Mains water, ad libitum.
- Acclimation period: 1 week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 - 25°C
- Humidity (%): 50 - 60%
- Air changes (per hr): 18 times per hour.
- Photoperiod (hrs dark / hrs light): 12 hours of light and 12 hours of darkness.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Up to 91 days.

Frequency of treatment:

Ad libitum during the exposure period.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: % (nominal in diet)

Doses / concentrations 2

Dose / conc.:

0.6 other: % (nominal in diet)

Doses / concentrations 3

Dose / conc.:

1.25 other: % (nominal in diet)

Doses / concentrations 4

Dose / conc.:

2.5 other: % (nominal in diet)

Doses / concentrations 5

Dose / conc.:

5 other: % (nominal in diet)

Doses / concentrations 6

Dose / conc.:

10 other: % (nominal in diet)

No. of animals per sex per dose:

10 animals per sex per dose.

Control animals:

yes, plain diet

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily.
- Cage side observations checked: Mortality, general abnormalities.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Daily.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 92.
- Anaesthetic used for blood collection: Yes , ether.
- Animals fasted: Yes
- How many animals: All animals.
- Parameters checked: GOT, GPT, Alp, TTT, T-bil, T-cho, TG, beta-lipopro, T-pro, A/G, BUN, creatinine, uric acid, ZTT, Ca, erythrocyte, Ht, MCV, leukocyte and Hb.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Two males and one female from the 10% groups as a result of renal failure.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two males and one female from the 10% groups as a result of renal failure.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Bodyweight gains of both male and female rats treated with 10% and of male rats treated with 5% were significantly decreased as compared with controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was effected in the 10% and 5% groups. No further details are provided.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There was a trend towards higher uric acid levels in both sexes at higher doses. There were significant changes of the hepato-related parameter levels in the dosed groups.

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were significant deviations in organ weights.

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

KIDNEYS: In the 10% groups both sexes exhibited a mild to severe tubulorrhexis accompanied by calcification, interstitial fibrosis and mononuclear cell infiltration in the area of the cortico-medullary border.
The 5% group exhibited a slight to mild tubulorrhexis accompanied by calcification in all animals.
The 2.5% group atrophy and deciduation of the uriniferous tubule epithelial cells was observed in 7/10 males and 8/10 females.
No other effects were reported at the lower doses.

TONGUE: Ulceration and/or granuloma formation was reported in the mucosal tissue at the root of the tongue for 8/8 males and 7/9 females in the 10% group; and for 6/10 males and 4/10 females the 5%.

SALIVARY GLAND: Hypertrophy and basophilisation of acinar cells of the sublingual gland and submandibular gland. Also noted was a compression/atrophy of the striated duct, intercalated duct and excretory duct. These effects were observed in 7/8 males and 4/9 females in the 10% group and in 2/10 males in the 5% group.

OTHER: Hyperplasia of RES cells and lymphoblasts was reported. A lesion in the submandibular lymph node is recorded in 2/10 males and 1/10 female in the 10% group. No other effects are reported for the other organs

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Effect levels

1

Key result

Dose descriptor:

LOEL

Effect level:

0.6 other: %

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

LOEL

Effect level:

308 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

organ weights and organ / body weight ratios

Remarks on result:

other: Extrapolated from level of compound in diet assuming 0.35 kg rat eats 18 g food/day.

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Any other information on results incl. tables

Reduction in the body weight gain and the trend of higher uric acid levels may be due to the decreased food consumption and the effects in the kidneys. The effects in the tongue would be due to the high alkaline property of the test substance in the solution.

Table 1 - Serum chemistry and hematology results (male)

Dose	10%	5%	2.5%	1.25%	0.6%	control
Effective No.	7	10	10	10	10	8
GOT (KU)	92.0±9.6^a	70.0±7.3	66.2±8.1	67.7±4.8	68.5±8.1	67.5±6.1
GPT (KU)	39.5±9.0^a	27.9±1.4^b	26.2±1.9	25.8±2.8	24.3±2.8	25.8±2.0
Alp (KAU)	21.1±2.0^a	13.3±1.2	11.3±1.2^a	11.8±0.9^a	11.8±1.3^b	13.3±1.0
TTT(SHU)	0.99±0.54^a	0.40±0.16	0.27±0.11	0.45±0.16	0.42±0.29	0.34±0.17
T-bil.(mg/dL)	0.55±0.08^b	0.47±0.08	0.51±0.07	0.52±0.08	0.48±0.06	0.46±0.07
T-cho.(mg/dL)	96.3±5.5^a	51.0±5.4	49.9±2.7	54.2±2.7	50.1±3.8	51.7±3.8
TG(mg/dL)	69.1±16.8	77.6±17.6	77.5±14.3	116.0±18.6^a	94.7±30.7	85.3±17.5
β lipo-pro.(mg/dL)	131.4±26.8	117.5±20.8	106.0±27.1	195.1±147.1	132.4±36.8	127.2±19.8
T-pro.(g/dL)	6.25±0.22^b	6.54±0.13	6.57±0.18	6.65±0.16^b	6.5±0.12	6.48±0.10
A/G	1.70±0.19	1.75±0.05	1.81±0.07	1.85±0.07	1.89±0.09	1.93±0.08
BUN(mg/dL)	34.6±2.1^a	22.7±1.8^a	17.9±2.1	17.6±1.4	17.0±1.3	17.5±1.1
Creatine(mg/dL)	0.85±0.21	0.65±0.25	0.84±0.23	0.94±0.15	0.72±0.45	0.91±0.35
Uric acid(mg/dL)	2.00±0.037^a	1.19±0.29	1.01±0.20	1.29±0.56	1.47±0.63	0.98±0.29
ZTT(KU)	2.79±1.02^a	1.62±0.32^a	1.24±0.19^b	1.41±0.36^b	1.07±0.23	1.08±0.12
Ca(mg/dL)	9.2±0.4^a	10.5±0.3^a	10.7±0.2	10.8±0.4	11.1±0.6	10.9±0.3
Erythrocytes(x10^4/mm^2­­)	706.6±43.7^a	893.1±17.3^a	926.1±24.0	953.4±33.0	942.8±15.1	949.0±22.4
Ht.(%)	375.0±16.4^a	443.6±9.1^a	454.6±11.8	468.4±15.6	465.1±9.5	465.5±10.8
MCV(µ2)	53.3±1.1^a	49.7±0.7^b	49.1±0.3	49.2±0.4	49.0±0.5	49.0±0.0
Leukocyte(x10^2/mm^2)	71.9±14.5	59.9±10.5	85.7±38.5	146.6±41.4^a	98.9±14.2^a	59.3±15.5
Hb.(g/dL)	121.1±20.7^a	154.0±3.7^a	153.8±15.1	160.3±6.1	159.9±3.2	161.3±3.6

^ap<0.01;^bp<0.05

 Table 1 continued - Serum chemistry and hematology results (female)

Dose	10%	5%	2.5%	1.25%	0.6%	control
Effective No.	8	9	10	10	9	10
GOT (KU)	90.3±3.6^a	69.0±3.9^a	70.7±5.8^a	68.8±8.7	69.0±5.2^b	63.3±4.1
GPT (KU)	35.2 ±9.0^a	26.5±4.2^b	24.1±2.8	23.4±1.4	23.0±2.6	23.1±2.0
Alp (KAU)	15.0±1.8^a	8.6±1.1	8.6±0.8	10.0±1.7	9.0±1.2	8.5±1.4
TTT(SHU)	0.43±0.17	0.39±0.14	0.45±0.22	0.48±0.19	0.31±0.09^b	0.44±0.16
T-bil.(mg/dL)	0.47±0.16	0.53±0.12	0.62±0.06	0.55±0.11	0.59±0.07	0.57±0.11
T-cho.(mg/dL)	112.0±10.0^a	83.9±9.2	85.0±5.5	86.4±8.5	80.8±5.1^b	87.7±6.2
TG(mg/dL)	65.0±26.2	53.2±9.7	59.0±10.3	57.0±12.2	54.3±4.6	58.3±9.4
β lipo-pro.(mg/dL)	79.0±30.7^b	52.9±13.7^a	93.3±16.4	92.6±21.8	90.4±8.5	102.8±16.9
T-pro.(g/dL)	6.01±0.20^a	6.19±0.18^b	6.41±0.24	6.45±0.09	6.32±0.19	6.37±0.16
A/G	2.00±0.18	1.90±0.26	1.88±0.09	1.84±0.33	2.02±0.10	1.93±0.12
BUN(mg/dL)	35.6±5.3^a	25.8±2.3^a	19.4±1.4^a	18.3±1.4^a	16.8±1.1	16.1±1.2
Creatine(mg/dL)	0.66±0.14	0.70±0.25	0.76±0.34	0.91±0.22	0.86±0.31	0.64±0.33
Uric acid(mg/dL)	1.75±0.32	1.71±0.35	1.66±0.22	1.73±0.29	1.74±0.37	1.78±0.26
ZTT(KU)	0.80±0.38^a	1.67±0.71	2.18±0.39	2.37±1.58	1.56±0.47	1.98±0.82
Ca(mg/dL)	9.5±0.5^a	10.8±1.1	10.4±0.9	9.8±0.4^a	10.0±0.2^a	10.4±0.4
Erythrocytes(x10^4/mm^2­­)	685.8±34.9^a	862.2±25.6	861.1±23.0	856.6±23.4	846.6±21.4	876.9±32.6
Ht.(%)	372.1±15.5^a	445.4±14.1	444.8±13.5	441.1±14.3	448.3±10.1	452.7±16.4
MCV(µ2)	54.1±0.8^a	51.6±0.5	51.8±0.6	51.6±0.7	51.9±0.3	51.7±0.7
Leukocyte(x10^2/mm^2)	66.8±8.3^a	127.9±64.8	207.5±51.1^a	239.0±33.6^a	160.3±55.3	116.5±23.3
Hb.(g/dL)	116.9±24.5^a	159.3±5.3	160.1±5.6	157.8±5.4	158.8±4.7	159.5±4.0

^ap<0.01;^bp<0.05

Table 2 - Absolute and relative organ weights (male)

Relative organ weights are shown in parenthesises (%)

Dose	10%	5%	2.5%	1.25%	0.6%	Control
Effective No.	8	10	10	10	10	10
Brain	1.86±0.04^a(1.1)^a	1.98±0.06 (0.7)^b	2.01±0.06 (0.6)	1.98±0.05 (0.6)^b	2.02±0.04 (0.6)^b	2.02±0.08 (0.6)
Pituitary	0.007±0.003 (0.004)	0.011±0.004 (0.003)	0.008+0.003 (0.002)	0.008±0.003 (0.002)	0.007±0.002 (0.002)	0.011±0.006 (0.003)
Saliv. Glds.	0.79±0.21^b(0.48)^a	0.82±0.28^b(0.28)^b	0.68±0.12 (0.20)	0.60±0.06 (0.18)	0.62±0.05 (0.19)	0.59±0.06 (0.19)
Thymus	0.12±0.05^a(0.07)	0.20±0.04 (0.07)	0.23±0.03 (0.07)	0.22±0.03 (0.07)	0.26±0.04 (0.08)	0.23±0.05 (0.07)
Lung (R)	0.48±0.03^a(0.29)^a	0.71±0.07 (0.24)	0.77±0.04 (0.24)	0.77±0.07 (0.23)	0.77±0.10 (0.23)	0.75±0.11 (0.24)
Lung (L)	0.26±0.03^a(0.15)^a	0.36±0.04 (0.12)	0.39±0.03 (0.11)	0.38±0.03 (0.12)	0.41±0.06 (0.12)	0.37±0.03 (0.12)
Heart	0.58±0.05^a (0.36)^a	0.87±0.14 (0.30)	1.01±0.06 (0.31)	0.96±0.06 (0.29)	1.01±0.08 (0.30)	0.95±0.12 (0.30)
Spleen	0.40±0.05^a(0.25)^a	0.61±0.05 (0.21)^b	0.64±0.04 (0.19)	0.61±0.04 (0.19)	0.65±0.06 (0.19)	0.62±0.05 (0.20)
Liver	4.13±0.37^a(2.5)^a	7.00±0.58 (2.4)^a	7.62±0.60 (2.3)	7.77±0.47^b (2.4)^b	7.67±0.45 (2.3)	7.22±0.54 (2.3)
Adrenal (R)	0.019±0.002^b (0.012)^a	0.21±0.006 (0.007)	0.022±0.001 (0.006)	0.020±0.005 (0.006)	0.021±0.005 (0.006)	0.024±0.004 (0.008)
Adrenal (L)	0.021±0.005 (0.012)^a	0.021±0.003 (0.007)	0.023±0.001 (0.007)	0.023±0.001 (0.007)	0.023±0.003 (0.007)	0.023±0.008 (0.007)
Kidney (R)	0.78±0.05^a (0.48)^a	1.09±0.05^a (0.38)^a	1.03±0.07 (0.31)	0.99±0.09 (0.30)	1.02±0.05 (0.31)	0.99±0.06 (0.31)
Kidney (L)	0.84±0.08^a (0.52)^a	1.10±0.06^a (0.37)^a	1.04±0.08 (0.32)	1.00±0.07 (0.30)	1.02±0.06 (0.31)	0.99±0.05 (0.31)
Testis (R)	1.17±0.07^a (0.71)^a	1.41±0.08 (0.49)^b	1.48±0.06 (0.45)	1.43±0.10 (0.44)	1.48±0.09 (0.44)	1.42±0.07 (0.45)
Testis (L)	1.22±0.06^a (0.75)^a	1.44±0.08 (0.50)^a	1.50±0.05 (0.46)	1.47±0.06 (0.45)	1.52±0.05 (0.46)^b	1.49±0.07 (0.47)

^ap<0.01;^bp<0.05

Table 2 continued - Absolute and relative organ weights (female)

Relative organ weights are shown in parenthesises (%)

Dose	10%	5%	2.5%	1.25%	0.6%	Control
Effective No.	9	10	10	10	10	10
Brain	1.75±0.07^a (1.5)^a	1.84±0.05 (1.0)	1.86±0.06 (1.0)	1.84±0.05 (1.0)	1.89±0.05^b(1.1)	1.86±0.05 (1.0)
Pituitary	0.006±0.002^a (0.005)	0.010±0.003 (0.006)	0.012±0.003 (0.007)	0.011±0.003 (0.006)	0.010±0.003 (0.006)	0.010±0.003 (0.005)
Saliv. Glds.	0.61±0.18^a (0.54)^a	0.44±0.04^b (0.25)^a	0.40±0.05 (0.22)	0.43±0.08 (0.23)	0.41±0.05 (0.23)	0.40±0.03 (0.22)
Thymus	0.13±0.04^a (0.12)	0.17±0.03 (0.09)	0.17±0.06 (0.09)	0.20±0.05 (0.11)	0.20±0.04 (0.11)	0.19±0.03 (0.10)
Lung (R)	0.40±0.04^a (0.35)^a	0.57±0.07 (0.32)^b	0.54±0.04 (0.30)	0.56±0.06 (0.30)	0.54±0.04 (0.30)	0.54±0.06 (0.29)
Lung (L)	0.21±0.03^a(0.19)^a	0.29±0.03 (0.16)	0.27±0.03 (0.15)	0.28±0.03 (0.15)	0.27±0.02 (0.15)	0.29±0.03 (0.16)
Heart	0.48±0.04^a (0.43)^a	0.63±0.03 (0.35)^a	0.65±0.07 (0.35)^b	0.60±0.05 (0.32)	0.60±0.04 (0.33)	0.61±0.04 (0.33)
Spleen	0.34±0.06^a (0.30)^a	0.48±0.06^b (0.27)^a	0.43±0.03 (0.24)	0.43±0.04 (0.23)	0.42±0.05 (0.23)	0.43±0.03 (0.23)
Liver	3.16±0.24^a (2.8)^a	4.07±0.34^b (2.3)^a	3.97±0.16 (2.2)^b	3.94±0.17 (2.1)	3.77±0.25 (2.1)	3.77±0.28 (2.0)
Adrenal (R)	0.018±0.002^b (0.016)^a	0.022±0.005 (0.012)	0.024±0.004 (0.01)	0.023±0.005 (0.012)	0.024±0.004 (0.013)	0.23±0.005 (0.012)
Adrenal (L)	0.020±0.003^a (0.018)^b	0.25±0.003 (0.014)	0.025±0.006 (0.014)	0.024±0.005 (0.013)	0.027±0.004 (0.015)	0.026±0.004 (0.014)
Kidney (R)	0.64±0.05^a (0.57)^a	0.98±0.06^a (0.55)^a	0.72±0.06^a (0.40)^a	0.63±0.05^b (0.34)^b	0.59±0.03 (0.33)^a	0.57±0.04 (0.31)
Kidney (L)	0.67±0.05^a (0.59)^a	0.97±0.04^a (0.56)^a	0.71±0.05^a (0.39)^a	0.62±0.05 (0.33)	0.60±0.03 (0.33)	0.60±0.04 (0.32)
Testis (R)	0.020±0.005^b (0.018)	0.033±0.010 (0.019)	0.041±0.019 (0.023)	0.042±0.020 (0.023)	0.040±0.009 (0.022)	0.042±0.022 (0.023)
Testis (L)	0.012±0.003 (0.011)	0.040±0.020 (0.022)	0.046±0.021 (0.025)	0.039±0.022 (0.021)	0.045±0.016 (0.025)^b	0.033±0.009 (0.18)

^ap<0.01;^bp<0.05

Table 3 - Summary of histopathological findings in F344 rats (both sexes) after 13 weeks treatment.

Findings	Concentration of potassium pyrophosphate in diet
	10%	5%	2.5%	1.25%	0.6%	0%
Kidney Tubular necrosis, with calcification and interstitial fibrosis	+++	++	±	-	-	-
Tongue Ulceration and/or granuloma formation	+	+ ~ ±	-	-	-	-
Salivary glands (serous) hypertrophy	+	± ~ -	-	-	-	-

+++ severe, ++ moderate, + mild, ± slight

Applicant's summary and conclusion

Conclusions:

LOEL = 0.6%

Executive summary:

A 13 week sub-chronic toxicity study of potassium pyrophosphate was carried out in male and female F344 rats at the dose levels of 10, 5, 2.5, 1.25, 0.6 and 0% in the diet. Sixty animals of both sexes were divided into the 6 dosage groups. The purpose of the study was to determine the maximum tolerable dose of test substance for use in rats in a long-term carcinogenicity study. Observations were limited to mortality, clinical abnormalities, body weights, food intake, haematology, clinical chemistry, gross pathology (incl. organ weight) and histopathology. By the end of the test, two male and one female from the 10% dosage group had died of renal failure. Bodyweight gains of both male and female rats treated with 10% and of male rats treated with 5% were significantly decreased as compared with controls. On histological examination, necrosis and calcification in renal tubules, ulceration and/or granuloma formation in tongue mucosa and hypertrophy of salivary glands were observed at high incidence. Based on these findings, a concentration of 5% in diets was considered to be the maximum tolerable dose for use in a long-term carcinogenicity study.