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Toxicological information

Repeated dose toxicity: dermal

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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:
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 in the report.
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

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 sodium or potassium cation and the pyrophosphate anion as common functional groups.
All members of the group will ultimately dissociate into the common breakdown products of sodium / potassium cations and the pyrophosphate anions.
Both the Na+ and the K+ cation have a similar biological function and therefore pyrophosphate salts of these types are considered to have a systemic toxicity profile. Differences arise in their local effects profile due to the increasing or decreasing acidity/alkalinity and buffering capacities of the substances. The pyrophosphate ion is the simplest form of a condensed phosphate group. 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 states. 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.

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:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity 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 and as such GLP compliance is not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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.

Data source

Materials and methods

Results and discussion

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion