Orthophosphoric acid

Repeated dose toxicity: oral

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

Test material information

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 / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.3, 1.0 and 3.0%
Basis:
nominal in diet

Doses / concentrations 2

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

1

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

2

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/mm^3) Day:		Erythrocyte count (millions/mm^3) 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.