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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
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 chemical and orthophosphoric acid have the following similarities:

(1) All substances are ionic and share the PO43-anion as a common functional groups.
(2) All members of the group will ultimately dissociate into the common breakdown products of the Na+or K+cations (for sodium and potassium orthophosphates respectively) and the PO43-anion (all). Thus phosphoric acid is systemically bioavailable as phosphate.
3) Sodium aluminium phosphate is essentially a sodium orthophosphate that also contains an aluminium ion. Although aluminium is known to have toxic effects, no evidence of aluminium toxicity was observed in any of the studies. The addition of aluminium in the phosphate compound is unlikely to have an impact on the use of this data for the sodium and potassium phosphates as any toxicity observed is considered to be due to the phosphate content of the test material; renal effects (nephrocalcinosis) indicative of high phosphate intake were the only toxic effects noted in the studies. Therefore the results of the tests performed with sodium aluminium phosphate can reliably be read across to orthophosphoric acid. This conclusion is further supported by the data on a number of analogous substances which suggests potassium and sodium orthophosphates and orthophosphoric acid itself are not considered to be systemically toxic.


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’.
Cross-reference
Reason / purpose for cross-reference:
read-across: supporting information

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1972
Report date:
1972

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
A 90-day oral toxicity study was conducted with groups of albino rats fed Levn-Lite at dietary levels of 0.3, 1.0 and 3.0%.
GLP compliance:
no
Remarks:
Study predates GLP
Limit test:
no

Test material

Constituent 1
Reference substance name:
Phosphoric acid, aluminium sodium salt
EC Number:
232-090-4
EC Name:
Phosphoric acid, aluminium sodium salt
Cas Number:
7785-88-8
Constituent 2
Reference substance name:
sodium aluminium phosphate (3:2:8)
IUPAC Name:
sodium aluminium phosphate (3:2:8)
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): Levn-Lite

Test animals

Species:
rat
Strain:
not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., North Wilmington, Mass.
- Age at study initiation: No data
- Weight at study initiation: Males: 185-187 g; Females: 149-150 g
- Housing: Animals were housed individually in standard, wire-bottomed steel rat cages.
- Diet: standard rat ration available ad libitum
- Water: No data
- Acclimation period: No data


ENVIRONMENTAL CONDITIONS
No data


IN-LIFE DATES: No data

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: No data


DIET PREPARATION
- Rate of preparation of diet: Fresh diets were prepared each week. Each rat was offered an amount of diet sufficient for one weeks' ad libitum feeding. However, checks were made periodically to ensure that the food jars were not empty.
- Diet prepared by mixing appropriate amounts with standard rat ration in a Hobart Mixer.


VEHICLE
Not applicable
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
90 day exposure
Frequency of treatment:
Continuous exposure in feed
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.3, 1.0 and 3.0%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
Male: 155.36, 545.64 and 1796.95 mg/kg bw/day Female: 181.18, 701.65 and 2070.10 mg/kg bw/day
Basis:
other: Calculated using the mean of the weekly body weight and food consumption
No. of animals per sex per dose:
15 animals/sex/dose
Control animals:
yes, plain diet
Details on study design:
No data
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily


BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the first day of the test and at weekly intervals thereafter.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption data were collected individually for 5 rats of each sex in every group weekly during the study.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 45 and 84 days of feeding
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: collected individually from 10 rats of each sex from both the control and the high dose group
- Parameters: haematocrit value, erythrocyte count, haemolglobin concentration, total leukocyte count, differential leukocyte count


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 45 and 84 days of feeding
- Animals fasted: No data
- How many animals: collected individually from 10 rats of each sex from both the control and the high dose group
- Parameters: blood urea nitrogen (BUN) concentration, serum alkaline phosphatase (SAP) activity, serum glutamic-pyruvic transaminase (SGPT) activity, fasted blood glucose concentration


URINALYSIS: Yes - collected individually from 10 rats of each sex from both the control and the high dose group
- Time schedule for collection of urine: after 45 and 84 days of feeding
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters: glucose concentration, albumin concentration, microscopic elements examination, pH, specific gravity


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Following 90 days of feeding, all surviving rats were sacrificed by carbon dioxide asphyxiation and autopsied. At the time of gross examination a complete set of organs and other tissues were removed from each rat and preserved in formalin solution.

GROSS PATHOLOGY: Yes
Organ weights were determined for the liver, kidneys, spleen, gonads, heart and brain of each rat.

HISTOPATHOLOGY: Yes
Microscopic examination of tissues was performed for 10 rats of each sex from both the control and the high dose group. The following tissues were included: oesophagus, stomach (cardia, funuds and pylorus), small intestine (duodenum, jejunum and ileum), caecum, colon, liver, kidneys, spleen, pancreas, urinary bladder, pituitary gland, adrenal gland, testes, seminal vesicle, ovary, bone marrow, thyroid gland, parathyroid gland, salivary gland, prostate gland, heart, aorta, lung, lymph node (cervical and mesenteric), skeletal muscle, peripheral nerve, bone (femur), spinal cord, uterus, trachea, eye, optic nerve and brain (cerebrum, cerebellum and pons)
Other examinations:
None
Statistics:
Statistical analyses were conducted upon the absolute organ weights and their corresponding ratios to the weight of the body and brain. An analysis of variance was conducted first and any significant effects disclosed by that treatment were further studies by "t" tests.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Three deaths occurred during the study. All of these deaths resulted from trauma incurred during the collection of blood samples.
No untoward behavioural reactions were noted among any of the animals employed in the study.


BODY WEIGHT AND WEIGHT GAIN
Statistical comparisons of final body weights and total weight gains revealed no significant differences between test and control rats. The 90 day average total weight gains were:
Control: Male: 337 g/rat; Females: 181 g/rat
0.3%: Male: 395 g/rat; Females: 162 g/rat
1.0%: Male: 355 g/rat; Females: 157 g/rat
3.0%: Male: 355 g/rat; Females: 148 g/rat


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Test rats ate amounts of food comparable to that consumed by control rats. The 90 day average total food consumption was:
Control: Male: 2296 g/rat; Females: 1705 g/rat
0.3%: Male: 2318 g/rat; Females: 1566 g/rat
1.0%: Male: 2127 g/rat; Females: 1570 g/rat
3.0%: Male: 2194 g/rat; Females: 1680 g/rat


HAEMATOLOGY
No outstanding differences between test and control rats were noted with respect to any of the parameters investigated (see Tables 1 and 2).


CLINICAL CHEMISTRY
Values for test rats were not different from those of control rats (see Tables 3 and 4).


URINALYSIS
No significant differences between the urine of the test rats and control rats were observed (see Table 5).


ORGAN WEIGHTS (see Table 6)
The number of statistically significant intergroup differences which were noted was considered to be normal for a random population of albino rats. The lack of any consistent dose or sex related response and the absence of any deleterious histopathological changes further substantiate that none of the intergroup differences were related to the ingestion of Levn-Lite.
No statistically significant treatment effects were found in the liver, spleen
Statistically significant differences were seen at the 95% confidence level in the kidneys at the 3.0% dose level.
Statistically significant differences were seen at the 99% confidence level in the gonads at the 0.3% dose level.


GROSS PATHOLOGY
No outstanding differences were noted between test and control rats upon gross pathological examination.


HISTOPATHOLOGY (see Table 7)
There are microconcretions present in the renal tubules of the female rats from all three dose levels. These concretions are located in the tubules at the corticomedullary junction and they consist of an amorphous material which shattered on sectioning. They are blue in colour and probably calcified. These concretions are believed to be related to the test material since they are absent in the control animals and since the incidence and severity of this finding appear to be dose related.
The other lesions observed are those of spontaneous disease and they are not unusual for the albino rat.

Effect levels

open allclose all
Dose descriptor:
LOAEL
Effect level:
155 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Nephrocalcinosis
Dose descriptor:
NOAEL
Basis for effect level:
other: overall effects Histopathology: Microconcretions in the renal tubules of the female rats from all three dose levels
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Haematological data – summary of mean values

Dietary level (%)

Sex

Total Leukocyte count (thousands/mm3)

Day:

Erythrocyte count (millions/mm3)

Day

Haemoglobinconcentration (g/100mL)

Day

Haematocritvalue (%)

Day:

45

84

45

84

45

84

45

84

Control

M

17.1

15.9

7.72

8.08

15.7

15.9

39.3

40.5

-

F

14.7

9.9

7.70

7.52

15.5

15.7

38.4

37.9

3.0

M

15.0

14.2

7.75

8.23

15.9

16.2

39.4

41.0

-

F

14.6

9.2

7.78

7.77

15.9

15.7

39.1

39.3

 

 

Table 2: Haematological data – summary of mean values

Dietary level (%)

Sex

Differential leukocyte count (No. of cells per 100)

Lymphocytes

Day:

Neutrophils

Day

Monocytes

Day

Eosinophils

Day

Basophils

Day

45

84

45

84

45

84

45

84

45

84

Control

M

90.6

86.7

8.6

11.1

0.8

1.7

0.0

0.5

0.0

0.0

-

F

83.2

85.5

15.2

12.6

0.8

1.0

0.8

0.9

0.0

0.0

3.0

M

84.4

83.1

13.2

13.8

1.6

2.1

0.8

1.0

0.0

0.0

-

F

85.6

87.1

12.2

11.7

1.0

1.0

1.2

0.2

0.0

0.0

 

 

Table 3: Clinical blood chemistry data – summary of mean values

Dietary level (%)

Sex

Serum alkaline phosphatase activity (King-Armstrong units)

Day

Serum glutamic-pyruvic transaminase activity (Dade units)

Day

45

84

45

84

Control

M

31

24

30

27

-

F

20

12

24

28

3.0

M

33

23

26

32

-

F

18

12

26

21

 

 

Table 4: Clinical blood chemistry data – summary of mean values

Dietary level (%)

Sex

Blood urea nitrogen concentration (mg %)

Day

Fasted blood glucose concentration (mg %)

Day

45

84

45

84

Control

M

16

15

129

141

-

F

17

14

130

137

3.0

M

16

14

144

153

-

F

16

14

154

143

 

 

Table 5: Urine analysis data - summary of mean values

Dietary level (%)

Sex

Glucose

Day

Albumin

Day

Microscopic elements

Day

pH

Day

Specific gravity

Day

45

84

45

84

45

84

45

84

45

84

Control

M

N

N

T

N

+1

+2

7.0

6.8

1.025

1.039

-

F

N

N

N

N

+2

+1

7.8

6.8

1.027

1.036

3.0

M

N

N

N

T

+1

t

6.8

6.8

1.021

1.053

-

F

N

N

N

T

+1

+1

6.6

6.6

1.025

1.032

 

Glucose and albumin:

N = negative

T = trace; less than 30 mg/100mLurine

Microscopic elements:

t = minimal

+1 = slight amounts

+2 = moderate amounts

 

 

Table 6: Organ weight and ratio data – summary of mean results

Dietary level (%)

Organ weight (g)

Organ/ Body weight ratio (g/100g)

Organ/ brain weight ratio (g/g)

Males

Females

Males

Females

Males

Females

Liver

None

18.140

10.064

3.4790

3.2526

8.8872

5.0896

0.3

19.513

10.862

3.3432

3.3496

9.4149

5.7542

1.0

19.024

9.034

3.4991

2.9358

9.2539

4.8977

3.0

17.015

10.119

3.1883

3.3999

8.1248

5.2737

Kidneys

None

3.666

2.117

0.7019

0.6798

1.8015

1.0695

0.3

3.713

2.134

0.6364

0.6686

1.7939

1.1141

1.0

3.739

2.067

0.6930

0.6688

1.8148

1.1259

3.0

3.644

2.227

0.6812

0.7468*

1.7462

1.1639

Spleen

None

0.936

0.618

0.1805

0.2031

0.4599

0.3087

0.3

0.973

0.599

0.1681

0.1869

0.4684

0.3119

1.0

0.884

0.482

0.1641

0.1588

0.4315

0.2609

3.0

0.926

0.578

0.1737

0.1934

0.4445

0.3025

Gonads

None

3.611

0.077

0.6924

0.0253

1.7724

0.0392

0.3

3.537

0.066

0.6064**

0.0208

1.7317

0.0348

1.0

3.566

0.083

0.6653

0.0271

1.7115

0.0449

3.0

3.638

0.085

0.6823

0.0285

1.7724

0.0449

Heart

None

1.613

1.078

0.3095

0.3484

0.7937

0.5447

0.3

1.696

1.093

0.2904

0.3417

0.8199

0.5724

1.0

1.674

1.025

0.3096

0.3365

0.8163

0.5574

3.0

1.608

1.001

0.3015

0.3367

0.7707

0.5230

Brain

None

2.041

1.982

0.3914

0.6431

1.0000

1.0000

0.3

2.076

1.917

0.3575*

0.6038

1.0000

1.0000

1.0

2.063

1.838*

0.3841

0.6098

1.0000

1.0000

3.0

2.086

1.919

0.3914

0.6457

1.0000

1.0000

* Statistically significant difference at the 95% confidence level

** Statistically significant difference at the 99% confidence level

 

 

Table 7:Histopathological changes

Number of animals

Organ examined

Findings

Incidence

Average grade

Control

10 males

Trachea

Chronic tracheitis

6

1.0

-

Lung

Chronic murine pneumonia

3

1.0

-

Colon

Parasites

1

1.0

-

Kidney

Focal lymphoid infiltration

3

1.0

-

Urinary bladder

Mucoid plug

6

1.0

10 females

Lung

Chronicmurine pneumonia

1

1.0

-

Colon

Parasites

1

1.0

Dose level: 0.3%

15 females

Kidney

Microconcentrations

4

0.5

-

-

Hydronephrosis

1

2.0

-

-

Focal lymphoid infiltration

1

2.0

Dose level: 1.0%

10 males

Kidney

No findings

-

-

10 females

Kidney

Microconcretions

5

2.0

-

-

Focal lymphoid infiltration

5

2.0

Dose level: 3.0%

10 males

Trachea

Chronic tracheitis

4

1.0

-

Lung

Chronic murine pneumonia

5

1.0

-

Kidney

Focal lymphoid infiltration

1

1.0

-

Urinary bladder

Mucoid plug

1

1.0

10 females

Trachea

Chronic tracheitis

4

1.0

-

Colon

Parasites

1

1.0

-

Kidney

Microconcentrations

9

1.0

 

 

Focal lymphoid infiltration

1

1.0

All other tissues and organs were normalhistologically.

Grading system:

0.5 = minimal

1.0 = slight

2.0 = mild

3.0 = moderate

4.0 = severe

5.0 = extreme

Applicant's summary and conclusion

Conclusions:
Results obtained from microscopic examination of tissues and organs disclosed microconcretions (nephrocalcinosis) in the renal tubules of the female rats from all three test groups. These concretions are believed to be related to the test material since they are absent in the control animals and since the incidence and severity of this finding appear to be dose related. No abnormalities were observed in body weight gains, food consumption, haematologic studies, clinical blood chemistry studies, urine analyses, gross pathological studies and organ weights and ratios. As no NOAEL was determined, this study is not preferred for use as a key study to derive the DNELS for use in risk assessment however it is considered to be a worst-case option for the derivation on an oral DNEL for the general population.

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