Tetrapotassium pyrophosphate

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

Cross-reference

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study is performed in accordance with an appropriate guideline (OECD 408) and with few deficiencies. No indication of GLP compliance is reported and only group mean data are provided.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

(1) The source and target substances are both inorganic salts of a monovalent cation from Group 1A of the periodic table, sodium or potassium, and pyrophosphoric acid. Thus, they all share the Na+ or K+ cation and the P2O74- anion as common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+ or K+ cations and the P2O74-anion.
(3) The pyrophosphate ion is the simplest form of a condensed phosphate group. A condensed phosphate anion has one or several P-O-P bonds. As the group contains only two phosphate groups, both of the phosphorus ions are classified as “terminal phosphorus”. The pyrophosphate can undergo ionisation with loss of H+ from each of the two –OH groups on each P and therefore can occur in the -1, -2 -3 or -4 state. The degree of ionisation is dependent upon the associated cations and the ambient pH (if in solution). Therefore the above substances have a pyrophosphate anion that is likely to behave in a similar way. In addition, the sodium and potassium cations are key elements in various cellular processes their import and export over cell membranes is regulated via pore systems and usually tightly regulated. As such, the presence of varying quantities of such cations is not expected to have an impact on the toxicity of the substances detailed above therefore as the both ionic components of the substance are common the results of toxicity studies can be reliably read-across within the group. .

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Animals were only dosed for 5 days / week, Individual animal data not reported

GLP compliance:

not specified

Remarks:

Study published in the literature, no details on GLP compliance.

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Orient Bio Co., Ltd. (Gyeonggi-do, Korea)
- Age at study initiation: 5 weeks old
- Weight at study initiation: between 150 and 200 g
- Housing: The animals were housed in groups of two to three in stainless steel wire mesh cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum - commercial rodent chow (2.0 Mrad gamma-ray sterilized EP pellet, Cargill Agri Purina Korea Ltd, Korea).
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3°C
- Humidity (%): 55 ± 5%
- Air changes (per hr): 10 to 18
- Photoperiod (hrs dark / hrs light): 10/14

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once a day, 5 days a week

Remarks:

Doses / Concentrations:
250, 500, 1000 mg/kg bw
Basis:
nominal in water

No. of animals per sex per dose:

10 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: 14-day range-finder study; No toxicity or mortality was observed in any of the five male and female rats treated with tetrasodium pyrophosphate for 14 days.

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day

BODY WEIGHT: Yes
- Time schedule for examinations: once a week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes (time scale for measurements not recorded: Food consumption by each group was measured at the start of treatment and during the 90-day administration period.

FOOD EFFICIENCY: Not applicable

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not applicable

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: Yes; isoflurane
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: No data
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: During dosing week 13 (last week)
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- The following parameters were examined: glucose, bilirubin, ketone body, specific gravity, blood, pH, protein, urobilinogen, nitrite and leukocyte levels, using the Multistix 10SG (Bayer, U.S.A.) and urine analyser (Clinitek 500, U.S.A.).

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Statistical Analysis
The differences in parameters (BWs, organ weights, and the results of the blood biochemistry and haematology) were assessed by a standard two-way analysis of variance (ANOVA). If these showed statistical significance, Duncan’s or Dunnett’s multiple range test were used to compare groups (SPSS version 12.0 [SPSS Inc., Chicago, IL, USA]). P-values < 0.05 were considered statistically significant.

Clinical signs:

no effects observed

Description (incidence and severity):

No mortality. Slight clinical signs noted in female rat treated with 500 and 1,000 mg/kg bw/ day (see 'details on results')

Mortality:

no mortality observed

Description (incidence):

No mortality. Slight clinical signs noted in female rat treated with 500 and 1,000 mg/kg bw/ day (see 'details on results')

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male rats treated with 1,000 mg/kg bw/day had a lower terminal bodyweight than the control animals. No other effects noted and no treatment-related effects were noted in female dose groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A significant decrease was noted in the top dose males during weeks 8 and 9 and a significant increase in food consumption was noted for females dosed with 1,000 mg/kg bw/day during week 4.

Food efficiency:

not examined

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):

See 'details on results' for a description of the effects noted.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See 'details on results' for a description of the effects noted.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The relative weight of the liver in both male and females dosed with 500 and 1,000 mg/kg bw/day was significantly increased.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no grossly visible findings or lesions in any dose group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Effects were noted in the kidneys in the high dose groups (see ' details on results' for a full description)

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality and clinical signs
There was no treatment-related mortality in the animals treated with the test substance during the study. Female rats treated with 500 and 1,000 mg/kg of test substance showed hair loss. No significant clinical signs were observed in any other group.

Body weight
The terminal bodyweights of male rats treated with 1,000 mg/kg of test substance were lower than that of the control group, while there were no differences in body weight between the 250, 500 mg/kg, and control group. There was no treatment-related change in body weight in female rats

Food consumption
A significant decrease in food consumption was observed in the 1,000 mg/kg group of males at week 8 and 9. Food consumption in females increased significantly in the 1,000 mg/kg group at week 4. There were no significant changes in food consumption in the other groups .

Urinalysis and haematology
There were no specific symptoms in urinalysis at the doses of 250, 500, and 1,000 mg/kg. There were no significant changes in haematology in the 250 mg/kg group. WBC and neutrophil counts were significantly increased in males and females of the 1,000 mg/kg group, whereas the lymphocyte count was significantly lower in males and females of the 1,000 mg/kg group. RBC, HB, HCT, PT, and
APTT were significantly reduced in the males of the 1,000 mg/kg group, and PT was significantly reduced in males of the 500 mg/kg group.

Biochemistry
There were no significant changes in the serum biochemistry of males and females in the 250 mg/kg group. Total protein was significantly reduced in both sexes in the 500 and 1,000 mg/kg group. Albumin was significantly reduced in males of the 500 and 1,000 mg/kg group, and in females of the 1,000 mg/kg group. The A/G ratio was significantly increased in females of the 500 and 1,000 mg/kg group. T-BIL was significantly increased in females of the 1,000 mg/kg group, and ALP was significantly reduced in females of the 1,000 mg/kg group. AST was significantly increased in males of the 1,000 mg/kg group, but ALT was significantly reduced in females of the 1,000 mg/kg group. TG was significantly increased in both sexes in the 1,000 mg/kg group, while Ca, IP, Na, K, and Cl were significantly reduced in both sexes in this group. IP was also reduced in males of the 500 mg/kg group.


Necropsy findings and organ weight
There were no grossly visible findings or lesions in any group. There were no significant changes in organ weight in males and females of the 250 mg/kg group. The relative weight of the liver in the males of the 500 mg/kg and 1,000 mg/kg groups showed a significant increase. The absolute and relative weight of the liver in females of the 500 mg/kg and 1,000 mg/kg groups were also significantly increased

Histopathology
No exposure-related histopathological changes were observed in any of the organs examined in SD rats, with the exception of kidney lesions. Cortical tubular basophilia of the renal tubule was more evident in males of the 1,000 mg/kg group and mineralization of the kidney was evident in females of the 1,000 mg/kg group. Other background lesions were observed in the 0, 250, 500, and 1,000 mg/kg groups in both sexes.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Haematological parameters were altered in the rats receiving 500 and 1000 mg/kg bw/day. These changes were not considered to be of toxicological relevance.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Tubular basophilia was noted at 1000 mg/kg bw/day in both male and female rats.

Critical effects observed:

not specified

Table 3. Food consumption

Week	Male				Female
	Control	250 (mg/kg bw/day)	500 (mg/kg bw/day)	1000 (mg/kg bw/day)	Control	250 (mg/kg bw/day)	500 (mg/kg bw/day)	1000 (mg/kg  bw/day)
1	27.1± 1.0	27.0± 1.6	26.7± 0.9	27.1± 1.8	19.1± 1.4	20.6± 1.7	19.9± 1.5	20.2± 1.0
2	30.4± 0.9	28.5± 6.4	30.7± 1.7	29.2± 1.4	21.1± 2.6	21.3± 3.9	21.4± 2.1	20.3± 2.1
3	33.8± 1.9	34.1± 2.2	32.9± 3.1	30.4± 3.6	22.6± 2.2	22.6± 2.7	21.8± 2.1	21.7± 1.0
4	30.9± 1.2	31.6± 2.5	31.8± 6.6	28.5± 3.2	19.7± 2.5	20.5± 1.4	21.3± 1.5	22.7± 1.3*
5	33.4± 2.8	30.8± 1.2	31.8± 2.7	30.5± 2.1	21.7± 0.6	20.3± 3.3	19.3± 2.5	22.9± 2.7
6	29.3± 3.9	27.9± 2.6	29.2± 1.9	27.9± 2.9	18.1± 2.0	17.1± 3.7	18.2± 1.8	19.2± 1.3
7	33.1± 2.7	33.3± 2.6	33.8± 1.2	32.9± 3.2	24.3± 2.0	21.9± 2.0	23.2± 2.8	23.9± 2.9
8	31.5± 2.1	31.3± 2.1	33.0± 1.6	28.3± 0.5**	20.8± 2.0	20.3± 2.9	21.7± 2.8	21.0± 2.2
9	31.8± 2.1	31.2± 1.7	30.5± 1.1	28.2±2.5*	20.6± 3.8	18.8± 2.7	19.9± 1.4	19.8± 2.1
10	32.7± 2.0	31.6± 2.3	31.9± 1.1	28.8± 1.8	21.2± 1.3	18.9± 3.8	21.2± 2.4	19.4± 1.7
11	29.8± 2.7	30.3± 3.0	29.6± 2.0	27.8± 1.6	20.5± 3.5	19.5± 2.3	21.1± 3.7	21.0± 3.9
12	33.8± 0.5	33.5± 0.5	33.2± 0.6	33.4± 0.5	20.2± 1.8	19.7± 3.1	21.5± 2.0	20.3± 2.9
13	30.1± 2.2	29.4± 2.3	31.2± 1.8	28.5± 1.7	20.1± 1.5	18.6± 1.9	20.4± 3.6	21.1± 2.5

*p<0.05, **p<0.01.

Table 4. Haematology

Parameter measured	Male				Female
	Control	250 (mg/kg bw/day)	500 (mg/kg bw/day)	1000 (mg/kg bw/day)	Control	250 (mg/kg bw/day)	500 (mg/kg bw/day)	1000 (mg/kg  bw/day)
WBC (K)	7.20± 1.830	6.62± 0.71	7.76± 1.580	9.35± 1.31*	3.72± 1.860	6.84± 2.090	4.44± 0.660	6.39± 1.83**
Neutrophil (%)	14.2± 4.600	18.6± 6.20	20.1± 6.600	24.8± 4.900	12.2± 3.700	17.7± 5.000	17.3± 7.300	20.9± 6.3*0
Lymphocyte (%)	80.1± 4.500	74.8± 6.20	73.6± 6.900	68.7± 6.000	81.4± 5.000	74.4± 6.100	75.4± 7.400	73.2± 7.0*0
Monocyte (%)	3.6± 1.100	3.9± 1.60	3.4± 1.100	4.1± 1.800	3.6± 1.500	3.7± 0.200	3.8± 1.700	3.3± 0.900
Eosinophil (%)	1.4± 0.500	1.9± 0.50	2.3± 0.7*0	1.8± 0.600	2.1± 0.900	1.9± 0.300	2.6± 1.100	2.1± 0.600
Basophil (%)	0.2± 0.100	0.2± 0.10	0.2± 0.100	0.3± 0.100	0.2± 0.100	0.2± 0.000	0.2± 0.100	0.2± 0.100
RBC (M)	8.56± 0.190	8.43± 0.34	8.55± 0.420	8.19± 0.19**	7.82± 0.280	8.16± 0.400	7.96± 0.230	7.82± 0.250
Hb (g/dl)	15.3± 0.500	14.9± 0.70	14.8± 0.800	14.4± 0.3**	14.4± 0.500	14.5± 0.600	14.6± 0.400	14.1± 0.400
Hct (%)	45.5± 1.400	44.3± 1.80	44.4± 2.200	43.1± 0.8**	42.3± 1.500	43.1± 2.000	42.8± 1.300	42.0± 1.200
MCV (fL)	53.1± 1.100	52.5± 1.30	52.0± 2.700	52.7± 0.900	54.1± 1.200	52.0± 2.400	53.8± 1.300	53.7± 0.900
MCH (pg)	17.9± 0.400	17.6± 0.50	17.3± 1.000	17.6± 0.400	18.5± 0.500	17.5± 0.900	18.3± 0.500	18.1± 0.200
MCHC (g/dL)	33.8± 0.500	33.5± 0.50	33.2± 0.600	33.4± 0.500	34.1± 0.400	33.0± 1.400	34.1± 0.200	33.7± 0.400
Reticulocyte (%)	2.04± 0.340	2.1± 0.30	2.38± 0.520	2.37± 0.380	1.79± 0.410	2.11± 0.260	2.02± 0.490	2.27± 0.420
PLT (K)	1148.2± 124.4	1071.6± 98.4	1213.9± 123.4	1264.5± 110.5	1153.7± 171.4	1153.2± 105.1	1169.5± 427.3	1441.4± 210.2
PT (sec)	17.7± 0.500	17.6± 0.60	16.7± 0.6**0	16.5± 0.6**0	16.9± 0.800	17.1± 0.600	17.1± 1.000	16.4± 0.900
APTT (sec)	20.3± 1.200	19.5± 1.90	19.4± 1.200	18.4± 1.1*0	16.4± 0.800	18.8± 1.500	16.5± 0.900	16.7± 0.8

WBC: white blood cell, RBC: red blood cell, Hb: hemoglobin, Hct: hematocrit, MCV: mean corpuscular volume,

MCH: mean corpuscular haemoglobin, MCHC: mean corpuscular hemoglobin concentration, PLT: platelet. PT: prothrombin time,

APTT: activated partial thromboplastin time.

*p<0.05,**p<0.01

See attachment for the following tables:

- Table 5. Biochemistry

- Table 6. Absolute organ weights

- Table 7. Relative organ weights

See Figures for information on changes in bodyweight.

Conclusions:

The NOAEL was determined to be 500 mg/kg bw/day on the basis of changes observed in the kidneys of the rats in the high dose group.
Rats in general and particularly female rats are known to be susceptible to nephrocalcinosis when administered high doses of phosphates (typically starting at about 0.5 – 1.0 % in the diet).The effects are only seen in high dose animals (well above the recommended classification limits for STOT RE as defined in the Guidance on the Application of Regulation (EC) No 1272/2008) and therefore classification for STOT RE is not justified and no classification is proposed.

Results and discussion

Target system / organ toxicity

Critical effects observed:

not specified