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Carcinogenicity

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Description of key information

A weight-of-the-evidence approach is applied to support the lack of carcinogenicity of STMP. Inorganic phosphates are not mutagenic or genotoxic and carcinogenicity studies have not found an association of inorganic phosphate administration in the diet with increased tumor incidence of any kind. Taken together collectively, a rationale can be made that STMP is not likely to be carcinogenic using a weight-of evidence approach. Four studies are available to assess the carcinogenicity of trisodium trimetaphosphate and related substances. 
The test material is not considered to be carcinogenic and therefore further in vivo studies are not deemed to be necessary. Carcinogenicity is not a required endpoint for REACH.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
This study is considered to fulfil the data requirements as defined under Annex XI, section 1.2. of Regulation (EC) No. 1907/2006 (REACH) as part of a weight of evidence approach for the following reasons. As stated in Klimisch et al (Reg. Toxicol. Pharmacol. 25, 1-5, 1997): “If a complete report is available or if the test, although not performed according to national/international standard methods, is described sufficiently and carried out according to a scientifically acceptable standard, the studies may be assessed as ‘reliable’ as well. The basic data (test organisms, data on the method, and on the scope of the investigations) should be available and documented in the data set of the substance especially if a standard method was not used. Data on the purity of a substance are necessary particularly if impurities may have a substantial influence on the toxicity. This can be assessed only on a case-to-case basis. Information on dose/concentration is essential. Even if some criteria of an international standard are not met, the expert may decide that the study is ‘reliable with restrictions’ and may be used for a risk assessment.” The Hodge chronic toxicity/carcinogenicity study is a scientifically sound study that met the scientific standards at the time it was conducted. It included many parameters that meet the current OECD 453 guideline and one that exceeded it (bone physiology evaluation). Although it was not a GLP study, the report contains detailed information on the study conduct, individual animal data and summary tables. The deficiencies included the following: inadequate test material characterization, incomplete food and water consumption, inadequate clinical observations and ophthalmology, lack of clinical chemistry, some hematological parameters not included, not all required organs were weighed and not all required tissues were evaluated for histopathology. Survival generally met the criteria for survival after 24 months, but did not meet the criteria for the percentages of deaths due to infections and other causes. Survival in the study was poor due to respiratory infections and pericarditis-peritonitis which were not treatment-related and occurred in all groups. Nevertheless, the Hodge study was able to adequately identify potential target organs, evaluate hematological effects, body weight gain, and, indirectly, food consumption, as well as to provide histopathological evaluation of many organs and tumors. The lack of purity analysis, while a clear deficiency, is not considered to affect the reliability because there were no unexpected toxic effects due to the test material at a high dose of 10% in the diet which would lead one to believe that the test material had toxic impurities. The results of the study are appropriate for those expected of an inorganic phosphate and similar to those observed with other inorganic phosphates. Therefore, it is concluded that a Klimisch code of 2: reliable with restrictions applies to this study, using the criteria for reliability categories described by Klimisch et al (1997).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
yes
Remarks:
please see 'rationale for reliability'
GLP compliance:
no
Remarks:
study predates GLP
Species:
rat
Strain:
other: Rochester Strain (Ex-Wistar 1923)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: 21 days, post-weaning
- Weight at study initiation: 85-88g for the males, 81-82g for the females
- Fasting period before study: no data
- Housing: The rats were housed 5 to a cage in galvanized iron cages with screen doors; a metal pan containing wood shavings served as a cage floor.
- Diet (e.g. ad libitum): Purina Fox Chow Meal, ad libitum
- Water (e.g. ad libitum): Rochester tap water supply, ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
no data

IN-LIFE DATES: From: day 0 of study To: week 104
Route of administration:
oral: feed
Type of inhalation exposure (if applicable):
other: not applicable
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: no data

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly, freshly
- Mixing appropriate amounts with (Type of food): Purina Fox Chow Meal
- Storage temperature of food: no data
- other: the trimetaphosphate was mixed with a mechanical mixer into the basal ration of Purina Fox Chow Meal and stored in galvanized iron pails with covers.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
not applicable
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
daily, ad libitum
Post exposure period:
none, animals sacrificed at end of exposure period
Remarks:
Doses / Concentrations:
0.0% (controls), 0.1%, 1.0%, & 10.0%
Basis:
nominal in diet
No. of animals per sex per dose:
50/sex/group
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
Pilot studies.
The acute toxicity of the test material when given intra-peritoneally was estimated in young, adult, female albino rats. The LD50 was calculated to be 3650 ± 620 mg/kg.

In a pilot feeding study of one month's duration, three groups of 5 male rats were fed: 0.2%, 2.0% and 10.0% sodium trimetaphosphate. The rats were serially sacrificed on the 3rd, 7th, 15th and 28th days. The 10.0% group showed retardation in growth, slight increase in kidney weight, [two of three rats] exhibitied phosphate nephritis of kidney tubules by day 15, but by day 28 none of the 5 rats examined showed phosphate nephritis. None of the rats died.

Pilot study: Two dogs were dosed with trimetaphosphate as follows: 0.1g/kg/day for one month and 1.0g/kg/day rising incrementally to 4.0g/kg/day for one month. Insignificant weight changes occurred in the 0.1g/kg/day animal, the dog given a final dose of 4.0g/kg/day lost bodyweight from 12.7 kg to 11.3 kg. Urine samples showed no abnormalities for sugar or protein content, blood samples yielded normal hematological values, organ weights at the time of sacrifice were within the normal range, the heart of the dog given the greater dose was at the upper limit of the normal range of heart weights. Thorough histological studies showed no changes in the tissues of the dog given 0.1g/kg/day. Only the kidneys in the dog dose at 4.0g/kg/day showed any change, they exhibited phosphate-nephritis-like damage.

- Rationale for animal assignment (if not random): anaimals were grouped according to approximate weights and sex. There is no data for the choice of animals used, however, rats are described in the OECD guideline.

- Rationale for selecting satellite groups: no data

- Post-exposure recovery period in satellite groups: not applicable

- Section schedule rationale (if not random): no data
Positive control:
no positive control
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: yes (weekly for the first twelve weeks, biweekly thereafter )

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
yes - After one month of the dietary regimen, 5 male rats from the control and from the 10.0% groups were tested. After 4 months on the diet each animal was provided with a set amount of basal or treated diet for the daily recording of food consumption for the 1 month measurement. For the 4 month measurement, control animals were provided with the amount of diet consumed by the treated animal on the preceding day. Body weights were also monitored during this measurement period.

HAEMATOLOGY: Yes
Number of animals: 5 animals/sex/group
Time points: Study initiation and on study days 34-41, 65-71, 93, 125, 142, 181, 229, 310, 379, 457, 560, 664, 707 and 730.
Parameters: Haematocrit, haemoglobin concentration, erythrocyte count, erythrocyte characteristics, total and differential leukocyte count, plasma cell.
Parameters not evaluated: platelet count, prothrombin time, mean activated partial thromboplastin time, mean corpuscular volume, mean corpuscular haemoglobin and haemoglobin concentration.
Anaesthetic used for blood collection: No data
Animals fasted: No data

URINALYSIS: Yes
Number of animals: 5 animals/sex/group
Time points: Pre-test and After approximately 4, 8, 12, 16, 20 and 23 months on study.
Parameters: protein, glucose.
Parameters not evaluated: Appearance, volume, osmolality, specific gravity, pH, blood.
Metabolism cages used for collection of urine: No data
Animals fasted: No data
Sacrifice and pathology:
ORGAN WEIGHTS:
All surviving animals at terminal sacrifice. The numbers of animals/group examined for organ weights from the control males and females were 16 and 19 animals/ group, respectively. The numbers of animals/group examined from the low, mid and high dose males were 15, 18 and 20 animals/group, respectively. The numbers of animals/group from the low, mid and high dose females were 21, 17 and 6 animals/group, respectively.
Organs: Liver, kidneys, testes, spleen, brain, stomach, lungs, heart
Organs not evaluated: adrenals, epididymides, uterus, ovaries, thyroid.

GROSS PATHOLOGY:
Macroscopic examination of organs looking for treatment-related lesions.

HISTOPATHOLOGY:
Animals at terminal sacrifice were examined for histopathology. Ten animals per sex per group were examined, except for the high dose females. Only 6 high dose females survived to termination.
Organs: Brain, small and large intestines, stomach, liver, kidneys, adrenals, spleen, heart, trachea, lungs, gonads, urinary bladder, bone marrow, skeletal muscle and any abnormal tissue.
Other: skeletal muscle, bone.

Organs not evaluated: pituitary, thyroid, parathyroid, thymus, oesophagus, salivary glands, pancreas, adrenals, uterus, female mammary gland, prostate, lymph node, peripheral nerve, bone marrow, skin, eyes, aorta, cervix, coagulating gland, epididymides, Haderian gland, lacrimal gland, sseminal vesicles, trachea
Other examinations:
Bone analysis: 10 animals from all male groups and 9, 8, 10 and 6 females from the control, low, mid and high dose groups, respectively: length and weight of the femurs, composition of bone (% water, dry weight, ash weight, % organic material, % calcium, % phosphorus, calcium:phosphate ratio). Mortality was monitored throughout the study.
Statistics:
no data
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
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:
effects observed, treatment-related
Clinical biochemistry findings:
not examined
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:
no effects observed
Details on results:
MORTALITY
Mortality was due primarily to respiratory infections and pericarditis-peritonitis. Some animals, mainly control rats, died due to excessive heat due to a thermostat failure.
There was no increase in mortality with increasing dose of test material. Median life span was similar to controls for all groups except the high dose females. The high dose females showed a reduced life span, 489 days compared to 685 in the control females. There is a high percentage of deaths due to other causes (not specified) in the high dose female group.

CLININCAL SIGNS
Some animals fed the high dose diet had moderate to severe diarhea. Aside from a softness of the feces (a possible mild cathartic action) in the groups given the 10% diet, the rats were healthy and maintainted good condition throughout the study.

BODY WEIGHT AND WEIGHT GAIN
Growth retardation was observed in rats of both sexes at the high dose (10.0% in diet) during the study. At the mid-dose level (1.0% in diet), males exhibited growth retardation during the first 12 months, then the growth rate was compensated and body weights were similar to controls at 24 months. Females administered the mid-dose level did not show growth retardation. There were no effects on growth at the low dose in either sex (0.1% in diet).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
After one month, control and high dose males consumed similar amount of diet. After 4 months, control and high dose animals of both sexes exhibited similar food consumption in the pair-fed measurement: control males: 12.7-17.5 g/day; high dose males: 14.1-19.7 g/day; control females: 10.2-11.7 g/day; high dose females: 10.6-12.9 g/day. Animals fed the high dose diet had moderate to severe diarrhoea.

HAEMATOLOGY
Periodic haematology evaluations found that all values in the treated groups were within the normal ranges, except for lower red blood cell counts and haematocrit levels in the high dose female group.

URINALYSIS
No effects on glucose or protein in urine at any dose level at any evaluation time.

ORGAN WEIGHTS
The fresh organ weights are comparable from group to group with the exception of smaller livers in the male rats given the 10% diet. The high average lung weight in the male 0.1% group can be traced to a single anomolous rat. When organ weights are expressed on the basis of body weights the small size of the carcass is reflected in the larger ratios for several organs. The larger stomach, brain, testes weights can be attributed to reasonably normal weight organs in smaller carcasses.
The organ weights gave no evidence of any specific toxic effect of TMP when given in the diets over a period of 2 years. Liver weights were decreased among the high dose group animals at study termination. Evaluation of the organ weight:body weight ratio attenuated this finding. There were no other effects on organ weights or weight rations in the other organs examined in any dose group

GROSS PATHOLOGY
Gross necropsy examination found no treatment-related lesions associated with test material dose administration.

HISTOPATHOLOGY: NON-NEOPLASTIC
The kidneys displayed concretions across all dose groups, including control, low and mid dose females and high dose males and females. These calcifications are thought to be due to nematode infections and not related to treatment.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
The incidence of tumours among survivors at terminal sacrifice did not show any significant increase in any particular tumour type related to treatment. All other lesions were typical of old and ill rats.
Deaths related to tumors did not show a relationship to treatment. Up to 3 rats (/50) per group during the 2 year period
Time to tumours: Monthly counts of tumours were provided. No specific treatment-related trend in time-to-tumour development was observed.

BONE ANALYSIS:
The 10.0% dose animals of both sexes exhibited higher calcium and phosphorus percentages in ash and lower water content compared to controls.
Femur length was slightly reduced among high dose males and to a lesser extent among high dose females. Since both the femur weights and body weights were both reduced, the femur weight:body weight ratios were not reduced and even increased for the high dose females because the body weights were reduced more than the femur weight. The reduction in femur weight indicated that a true growth retardation occurred.

OTHER: In high dose animals of both sexes exhibited higher calcium and phosphorus percentages in ash and lower water content compared to controls. This hypercalcification can be considered treatment-related.
Relevance of carcinogenic effects / potential:
There was no apparent increase in tumour incidence to suggest a carcinogenic effect, though the number of survivors at termination was generally low due to infections or other causes in all dose groups.



Reduced median life span and reduced red blood cell count and hematocrit were noted in high dose females. Histopathological evaluation indicated kidney concretions which were considered to be due to infections and aging. Kidney calcification in the high dose group may have been related to treatment. There was no apparent increase in tumour incidence to suggest a carcinogenic effect, though the number of survivors at termination was generally low due to infections or other causes in all dose groups. The high dose (10% in the diet) is excessive and exceeds the current guidance for a limit dose of no more than 5% in the diet. The high dose exceeded the maximum tolerated dose as evidenced by the reduced median life span among females.

Dose descriptor:
NOAEL
Effect level:
> 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Effect type: carcinogenicity (migrated information)

Conversion from % in diet to mg/kg bw was calculated in accordance with Appendix F of the guidelines for the preparation of toxicological working papers for the joint FAO/WHO expert committee on food additives, December 2000.

Conclusions:
The test material showed no toxicity when administered in the diet of rats at a level of 10% for two years. The no-observed effect level for chronic toxicity in this study is 1.0% in the diet for females and 0.1% in the diet for males. At the high dose there were reduced body weight gain and liver weights. Reduced median life span and reduced red blood cell count and hematocrit were noted in high dose females. Histopathological evaluation indicated kidney concretions which were considered to be due to infections and aging. Kidney calcificationj in the high dose group may have been related to treatment. There was no apparent increase in tumour incidence to suggest a carcinogenic effect, though the number of survivors at termination was generally low due to infections or other causes in all dose groups. The high dose (10% in the diet) is excessive and exceeds the current guidance for a limit dose of no more than 5% in the diet. The high dose exceeded the maximum tolerated dose as evidenced by the reduced median life span among females.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The dataset has been reviewed by an external assessor (M Weiner, TOXpertise, LLC) and it is concluded that the data is adequate and reliable for use as a key study under REACH. Please see expert report attached in endpoint under 'background information'.

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

DISCUSSION:

The Hodge Report on STMP partially meets the requirements of the OECD 453 Guideline. The Hodge Report documents all procedures and parameters conducted. Since it was conducted prior to formal international guidelines, it did not have some of the specific requirements included in the OECD 453 guideline for chronic toxicity/carcinogenicity. Nevertheless, the study was well documented and scientifically acceptable. Restrictions exist due to lack of some information and due to a high rate of deaths due to infections and other causes. Specific areas of deficiency are discussed below.

Test Material Characterization: There was no analysis of the test material for purity and verification of test material. Specific lot numbers are included, indicating verification of test material and its origin. The results are consistent with other studies on STMP. Effects on toxicity parameters are consistent with those expected for the test article and do not indicate the presence of a toxic impurity. No unexpected toxicity was found which would suggest that the test material contained a toxic impurity.

Food Consumption: Food consumption was evaluated in control and high-dose animals only at one and four months only. Body weight was measured for the full 24 months; therefore, it is possible to conclude that the animals were growing and to evaluate the effects of the treated diets on growth. Lack of food consumption data after 4 months is not considered a critical deficiency since body weight data support the normal growth of the animals and the growth retardation at the highest dose tested.

Haematology: Haematological tests were conducted for all haematological parameters, except mean corpuscular volume, mean corpuscular haemoglobin and haemoglobin concentration, prothrombin time and activated partial thromboplastin time. Though the study is not robust in this respect, there were findings of lowered red blood cell counts and haematocrit percentages in the high-dose female rats (only). All other haematological values in the treated groups were within the normal range. These data support the sensitivity of the methods employed to detect haematological findings. Additional support can be gleaned from other subchronic and chronic studies for haematological evaluations on STMP and other inorganic phosphates.

Urinalysis: Urinalysis was evaluated approximately every four months, corresponding to intervals as per specified in the OECD guideline with some variation. No effects were noted for urinalysis parameters during the study and this endpoint was adequately addressed in the study for the parameters measured. The number of animals sampled is not stated. Appearance, volume, osmolality, specific gravity, pH, and blood were not evaluated.

Clinical Chemistry: No clinical chemistry parameters were evaluated. The clinical chemistry parameters often detect renal and hepatic toxicity; both organs were essentially normal in this study. This deficiency could possibly be corrected from studies on other inorganic phosphates or STMP.

Organ Weights: Organ weights on ovaries, uterus, epididymides, thyroid and adrenals were not included as per the current guideline. Adequate numbers of survivors were evaluated except for the high-dose female group (6 instead of 10 rats). There were no effects on any of the measured organ weights except for lower liver weights in high-dose males and females. Because the liver weight: body weight ratios were normal, it was concluded that STMP did not cause any specific toxic effect on the liver organ weights. Although ovary weights were not evaluated, histopathology was conducted on female gonads and no adverse findings were noted. This deficiency is not considered to be a major one.

Histopathology:The study did not evaluate every tissue required under the current guideline (list in Footnote A of Table 1 in attached document). The lack of data on the missing tissues constitutes a data gap for STMP. In addition, tumour incidence data seem to indicate fewer tumours than expected in normal, untreated rats of the same age. Nevertheless, there were no obvious increases in tumour incidence across treatment groups with generally adequate survival (>25%). No specific target organ effects were identified, including the typical kidney lesions often noted in rats fed diets high in phosphates. The pathologist specifically looked for “chronic tubular nephropathy”, but did not find any lesions in the high dose animals and found only one male in the mid-dose group with this lesion. Changes to the kidneys were attributed to infection with a nematode. Nevertheless, a weight-of-the-evidence approach can be used to support the lack of carcinogenicity of STMP with data from other inorganic phosphates. Some tissues may not have been evaluated, and lack of data on these tissues constitutes a data gap for STMP. This deficiency could possibly be corrected with data from studies on other inorganic phosphates or STMP.

Survival:The primary causes of death appeared to be due to respiratory infections and “other causes”. A thermostat failure during April, 1958 or approximately 13 months into the study caused an increase in the deaths due to “other causes”; however, the majority of these deaths are not explained. OECD Guideline 453 requires that a valid test must have no less than 25% survival in each group after 24 months in rats and no more than 10% of any group should be lost to autolysis, cannibalism or management problems, including infections. Table 2 in the attached report summarizes the survival and causes of death in the Hodge chronic toxicity/carcinogenicity test.

Although large numbers animals in each group died due to respiratory infections and “other causes”, the overall survival exceeded 25% for all groups except the high-dose females (24%); however, the percentages of deaths due to infections and other causes exceed the criteria. The histopathological findings were typical of aged rats. There were very few deaths due to tumours (2-6%) and no obvious relationship of these deaths to treatment. There did not appear to be an increase in tumours in the animals which survived to the end of the study. Deaths were primarily due to respiratory infections and pericarditis-peritonitis (See Table 2 in attached report).

The incidence of tumours at study termination for all animals is shown in Table 3 below. Not all tumours were considered the cause of death; thus, the numbers of tumours at termination (Table 2) do not match the number of tumours evaluated for the study (Table 3 in the attached report). A weight-of-the-evidence approach will be necessary to review the carcinogenicity studies of other inorganic phosphates to further support the lack of carcinogenicity of STMP. Inorganic phosphates are not mutagenic or genotoxic and carcinogenicity studies have not found an association of inorganic phosphate administration in the diet with increased tumour incidence of any kind. Taken together collectively, a rationale can be made that STMP is not likely to be carcinogenic using a weight-of evidence approach. The data in this study can be considered supportive, but not definitive for the carcinogenicity endpoint.


Justification for selection of carcinogenicity via oral route endpoint:
The dataset has been reviewed by an external assessor (M Weiner, TOXpertise, LLC) and it is concluded that the data is adequate and reliable for use as a key study under REACH. Please see expert report attached in endpoint under 'background information'. The endpoint selected is taken from a weight of evidence approach based on similar substances.

Justification for classification or non-classification

The chronic carcinogenicity studies of Hodge (1960) and Kitahori (1998) present reliable evidence that the substance should not be classified for carcinogenicity according to Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging (CLP) of substances. Read across data for similar (poly)phosphates are also provided to support this conclusion.