Diphosphoric acid, compound with 1,3,5-triazine-2,4,6-triamine

Endpoint summary

Description of key information

For the oral route, a gavage 28-day repeat dose toxicity study (OECD 407, GLP) was performed with MPP.  Based on histopathological changes in the kidney (tubular basophilia/dilation) the No Observed Adverse Effect Level (NOAEL) in the 28-day repeat dose toxicity study was established as 15 mg/kg bw/day. 

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 August 2010 to 28 February 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in accordance with an internationally recognised method

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050, Repeated dose 28-Day Oral Toxicity Study in Rodents

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: reputable laboratory animal supplier
- Age at study initiation: 29 - 35 days
- Weight at study initiation: 108 - 135 g
- Fasting period before study:
- Housing: five of one sex per cage. The cages were made of a polycarbonate body with a stainless steel mesh lid. Wood based material was used as bedding and was sterilised by autoclaving and changed at appropriate intervals each week.
- Diet: standard rodent diet, ad libitum
- Water: potable water from the public supply, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 40 - 70
- Air changes (per hr): none, continuous supply of filtered fresh air
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

IN-LIFE DATES: From: To: 18 October to 15 November 2010

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle:
0 mg/ml (control)
3 mg/ml (15 mg/kg/day)
30 mg/ml (150 mg/kg/day)
200 mg/ml (1000 mg/kg/day)

- Amount of vehicle (if gavage): 5 ml

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of MPP in the doses was quantified by high performance liquid chromatography using UV detection.

The analytical procedure used was successfully validated with respect to linearity of detector response, precision of injection, specificity of chromatographic analysis, limit of detection, accuracy and precision.

The homogeneity and stability was confirmed for MPP in corn oil formulations at nominal concentrations of 1 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, ambient temperature storage for 2 days and refrigerated storage for up to 15 days.
The mean concentrations of MPP in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
15 mg/kg/day 3mg/l
Basis:
actual ingested

Remarks:

Doses / Concentrations:
150 mg/kg/day 30 mg/ml
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg/day 200 mg/ml
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Dose selection rationale: The doses used in this study (0, 15, 150 and 1000 mg/kg/day) were with reference to a preliminary study (Huntingdon Life Sciences Report Number CVJ0069). The preliminary study showed that MPP was generally well tolerated at doses of 100, 300 or 1000 mg/kg/day; with no clinical signs or post dose observations considered to be related to treatment. Animals treated at 300 or 1000 mg/kg/day had low overall bodyweight gain and low food consumption and animals at 1000 mg/kg/day had high water consumption and some had pale areas on the kidneys. It was therefore considered that a high dose of 1000 mg/kg/day would be appropriate for the present study, with low and intermediate doses at 15 and 150 mg/kg/day, respectively.

- Rationale for animal assignment: random

- Rationale for selecting satellite groups: no satellite groups used


- Post-exposure recovery period in satellite groups:


- Section schedule rationale (if not random):

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: one week before treatment then each week throughout the treatment period

FOOD CONSUMPTION: Yes
- Time schedule for examinations: one week before treatment then each week throughout the treatment period


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necroscopy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Haematocrit, Haemoglobin concentration, Erythrocyte count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Total leucocyte count, Differential leucocyte count, Platelet count, Morphological changes, Prothrombin time, Activated partial thromboplastin time.

BLOOD CHEMISTRY: Yes
- Time schedule for collection of blood: at necroscopy
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Alanine aminotransferase, Aspartate aminotransferase, Total bile acids, Urea, Creatinine, Glucose, Total cholesterol, Sodium, Potassium, Total protein, Albumin


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4, prior to dosing
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy

HISTOPATHOLOGY: Yes
Adrenals, Brain, Femur with joint, Heart, Kidneys, Liver, Lungs, Seminal vesicle, Spinal cord, Sternum, Stomach, Thyroid, Uterus

Other examinations:

Organ weights: Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Uterus with cervix, Ovaries

Statistics:

Grip strength, motor activity, bodyweight, haematology, blood chemistry and organ weight data:
Bartlett's test for variance homogeneity (Bartlett 1937)
Williams’ test (Williams 1971, 1972)
Dunnett's test (Dunnett 1955, 1964)
Shirley's test (Shirley 1977)
Steel's test (Steel 1959)
Fisher’s Exact tests (Fisher 1973)
Angervall and Carlstrom, 1963

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

low in males and females

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

low in males

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

high in males and females

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

high urea and creatinine concentrations, marginally high total protein concentration and high alanine aminotransferase activity in both sexes.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

high kidney weight

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

change in appearance of kidneys

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

effects seen in kidneys in both males and females

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no deaths and detailed physical and arena observations revealed no signs that could be attributed to treatment.
There were no toxicologically significant signs after dose administration.


BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain in animals receiving 1000 mg/kg/day was comparable to Control during the first week of treatment. From the second week of treatment to the end of the study, weight gain was markedly low (males 0.57; females 0.73X Control). Overall weight gain in these animals was low (males 0.70; females 0.78X), when compared with Control.

Bodyweight gain at 15 or 150 mg/kg/day was not affected by treatment.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Overall food consumption was low for males treated at 1000 mg/kg/day (0.86X Control); food consumption in females was lower than Control during three out of four weeks of treatment; however overall food consumption for these animals (0.92X) was comparable to Control.

Food consumption in both sexes at 15 or 150 mg/kg/day was not affected by treatment, when compared with Control.

FOOD EFFICIENCY
Not examined

WATER CONSUMPTION:
Measured water consumption during Week 4 revealed slightly high values in males receiving 150 mg/kg/day (1.23X Control) and markedly high in animals receiving 1000 mg/kg/day (males 1.87; females 1.88X Control).

Daily visual water consumption appeared high on some days in both sexes receiving 150 mg/kg/day and appeared high throughout the study period in animals treated at 1000 mg/kg/day.

When compared with Control, measured water consumption during Week 4 in both sexes at 15 mg/kg/day and females receiving 150 mg/kg/day was not affected by treatment.

OPHTHALMOSCOPIC EXAMINATION
Not examined

HAEMATOLOGY
Haematological parameters were not affected by treatment.

All differences from Control were present in a single sex, were not statistically significant or considered not to be biologically or toxicologically significant or adverse.

BLOOD CHEMISTRY
Animals receiving 1000 mg/kg/day had markedly high concentrations of urea (males 5.59; females 4.99X Control) and creatinine (males 5.36; females 3.92X Control), total protein concentration was marginally high (males 1.07; females 1.08X Control). Alanine aminotransferase activity was significantly high in males and females (1.46 or 1.64X Control respectively).

Cholesterol concentration was high in both sexes (males 1.21; females 1.14X Control) with statistical significance in males. Aspartate aminotransferase activity was high in males only (1.25X Control). The ratio of albumin to globulin was low in females (0.86X Control). Bile acid concentration appeared low in males; however this may have been the result of high individual variation in the Control. It is considered that these changes were not toxicologically important, as they were either not statistically significant in both sexes, were evident in a single sex or were considered only marginally different from Control.

Electrolyte concentrations were considered not to be affected by treatment.

Blood chemical parameters were not affected by treatment at 15 or 150 mg/kg/day.


URINALYSIS
Not examined


NEUROBEHAVIOUR
Sensory reactivity and grip strength - Sensory reactivity findings showed considerable inter-group variation, with higher than expected numbers of animals showing weak or absent responses. There was, however, no indication of an association with treatment as performance of treated animals was generally similar to that of Control or, where performance was inferior, this was seen in animals at 15 or 150 mg/kg/day. Forelimb and hindlimb grip strength values were unaffected.

Motor activity - Motor activity scores were considered to be unaffected by treatment.

ORGAN WEIGHTS
Bodyweight adjusted mean kidney weight was high in males and females receiving 1000 mg/kg/day (1.45 and 1.86X Control, respectively).

Organ weights in animals receiving 15 or 150 mg/kg/day were not affected by treatment.

GROSS PATHOLOGY
The kidneys of some males and females treated with 1000 mg/kg/day of MPP were enlarged, granular, pale or showed pale areas.


HISTOPATHOLOGY: NON-NEOPLASTIC
The kidneys of males treated with 1000 mg/kg/day of MPP exhibited moderate tubular basophilia and dilatation, slight tubular casts, minimal tubular necrosis/degeneration, moderate interstitial inflammation of the papilla, minimal to slight mineralisation of the medulla, minimal mineralisation of the cortex and slight transitional cell hyperplasia in the pelvis. These findings were found bilaterally.

Moderate to marked tubular basophilia and dilatation, minimal to slight tubular casts, minimal to slight tubular necrosis/degeneration, slight to marked interstitial inflammation of the papilla, slight to marked mineralisation of the medulla and minimal to slight transitional cell hyperplasia in the pelvis were evident in the kidneys of all females treated with 1000 mg/kg/day of MPP. These findings were evident in both kidneys.

Minimal tubular basophilia/dilatation and minimal tubular casts were present in some males given 150 mg/kg/day of MPP.

Although minimal tubular casts were present in one male and one female control and one male given 15 mg/kg/day of MPP, there was an increased incidence of tubular casts in males and females treated with 1000 mg/kg/day of MPP and an increase in severity in males given 1000 mg/kg/day of MPP.

Minimal focal mineralisation in the medulla was present in one female given 150 mg/kg/day. This was one unilateral focal lesion and in the absence of any other treatment related findings, is not considered to be test article related.


Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

All other histological changes were considered to be unrelated to treatment.

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related changes at the low dose level (15 mg/kg/day)

Critical effects observed:

not specified

Conclusions:

The results indicated the kidney as a target organ for toxicity. There were adverse histopathological changes in the kidney in both sexes at 1000 mg/kg/day and males at 150 mg/kg/day. Additionally, at 1000 mg/kg/day there were correlating macroscopic and blood chemistry changes, higher water consumption, and lower bodyweight gain and food consumption values. There were no treatment related changes at the low dose level
(15 mg/kg/day) and this level is considered to be the no-observed-effect-level (NOEL) and by default the no-observed-adverse-effect-level (NOAEL).

Reference 2

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Part of OECD 422 study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 February 2016 -

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 22 March 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Males 70 to 77 days old; Females 63 to 70 days old.
- Weight at study initiation: Males 338 to 397 g; Females 214 to 261 g.
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid bottom cages were used during the acclimatization,pre-pairing, gestation, littering and lactation periods, contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Diet (e.g. ad libitum): Powdered diet. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent, non-restricted.
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals, non-restricted.
- Acclimation period: Five days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20-24ºC.
- Humidity (%): Monitored and maintained within the range of 40-70%.
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

Route of administration:

oral: feed

Details on route of administration:

The dietary route of administration was chosen to simulate the conditions of potential human exposure.

Vehicle:

other: Standard diet

Details on oral exposure:

Via diet. Treated at constant dietary concentrations (ppm) for each group. No significant discrepancy was found.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of a homogeneous formulation has been confirmedin both frozen and ambient conditions for 32 days at 100 ppm and 22 days at 20000 ppm.

Duration of treatment / exposure:

Males were treated continuously for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks.
Females were treated continuously for two weeks before pairing, throughout pairing, gestation and until Day 6 of lactation.

Frequency of treatment:

Continuously.

Dose / conc.:

0 ppm

Remarks:

Control

Dose / conc.:

250 ppm

Dose / conc.:

2 000 ppm

Dose / conc.:

15 000 ppm

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

The study consisted of one control and three treated groups. The F0 generation of the study was identified as follows:
Group Treatment Level (ppm) Number of animals : Male Female
1 Control 0 10 10
2 MPP 250 10 10
3 MPP 2000 10 10
4 MPP 15000 10 10

Observations and examinations performed and frequency:

Clinical and Behavioral Observations: at least twice daily
Cages were inspected daily
Detailed physical examination and arena observations: Before treatment commenced and during each week of treatment and on Days 0, 6, 13 and 20 after mating and Days 1 and 6 of lactation
Sensory reactivity and grip strength: during Week 5 on males, at Days 4-6 of lactation on females
Motor activity: During Week 5 of treatment for males and at Days 4-6 of lactation for females
Body Weight: males: Before treatment commenced (Day 1) and weekly thereafter. On the day of necropsy.
Body Weight: females: Before treatment commenced (Day 1) and weekly before pairing. Days 0, 6, 13 and 20 after mating. Day 1, 4, and 7 of lactation. On the day of necropsy.
Food Consumption: Weekly, from the day that treatment commenced. Males: Week 1, 2 and 4. Females: Days 0-5, 6-12 and 13-19 after mating, Days 1-3 and 4-6 of lactation.
Hematology (peripheral blood): Week 2 before pairing
Blood Chemistry: Week 2 before pairing
Estrus Cycles: After pairing until mating
Organ weight and macroscopic pathology and histopathology investigations were undertaken.

Statistics:

Bartlett's test for variance homogeneity.
- If not significant at the 1% level: a parametric analysis.
For pretreatment data (group differences): analysis of variance.
Inter group comparisons: t-tests, with the error mean square from the one-way analysis of variance.
For all other comparisons: F1 approximate test.
- If significant at the 1% level: a non-parametric analysis.
For pretreatment data: Kruskal-Wallis’ test.
Inter group comparisons: Wilcoxon rank sum tests.
For all other comparisons: H1 approximate test, non-parametric equivalent of the F1 test.
For grip strength, motor activity, clinical pathology, litter size and survival indices, if 75% of the data (across all groups) were the same value: Fisher’s exact tests.
Treatment groups: pairwise comparisons of each dose group against the control.
Treated group, compared to control: Wald chi-square test.
Organ weight data: analysis of covariance using terminal body weight as covariate.
For some parameters, including pre-coital interval and mating performance the similarity of the data was such that analyses were not considered to be necessary.
Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs that were considered to indicate an immediate or delayed reaction to treatment.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no adverse effect of body weight gain in males throughout or females before pairing, during gestation and lactation.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no indication of a food consumption effect on either males throughout or females before pairing, during gestation or lactation.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no indication of a food consumption effect on either males throughout or females before pairing, during gestation or lactation.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At haematology evaluation during Week 2 of study there were no statistical differences from the Control for males.
For females receiving 15000 ppm: - mean cell haemoglobin and mean cell haemoglobin concentration: statistically lower than Control
- red blood cell distribution width: statistically higher than Control
- No other parameters attained statistical differences to the Control.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Urea concentration: males receiving MPP at 15000 ppm : statistically higher than Control
females receiving MPP at 15000 ppm : statistically lower than Control
Potassium concentration: males receiving MPP at 15000 ppm: statistically higher than Control
Calcium and phosphorus concentration: female receiving 15000 ppm or 2000 and 15000 ppm respectively: statistically lower than Control
Total protein concentration: males receiving 15000 ppm: statistically lower than Control
Glucose concentration: all groups of treated females: statistically significantly higher than Control
All other differences from controls were attributed to normal biological variation

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no effects on behavioral parameters.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Kidney weights (at 15000 ppm only): statistically higher
Testes weight (at all dose levels of MPP): statistically higher than control, but there was no dose response and detailed microscopic examination of the testes did not reveal any treatment related changes.
No other organ weights were significantly different to the control.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Majority of males and two females at 15000 ppm: Granular kidneys (Table 1)
Majority of females at 15000 ppm: Pale areas (Table 1)
Occasional males at 15000 ppm and 250 ppm: depressions (Table 1)
The incidence and distribution of all other findings were considered to be unrelated to treatment.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no indications of any adverse effects of MPP on sensory reactivity or grip strength assessments.
Motor activity was considered not to be adversely affected by the test material.
An isolated statistical significance of lower than control hind limb grip strength occurred for males receiving 250 ppm during week 5 of study, however, in the absence of any dose related trend or similar effect in females this was considered to have arisen by chance.
Males during Week 5 of treatment receiving 15000 ppm and females at Day 4-6 of lactation receiving 15000 ppm displayed statistically lower activity than Control low beam and for males only high beam activity during a single 6 minute interval this was not part of an extended trend and overall values were no significantly different to the control therefore this difference is not attributed to treatment. Also, females receiving 2000 ppm displayed statistically higher low beam activity than Control for a single 6 minute period this was not part of a dose related trend and no other period were significantly different to the control.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological changes related to treatment with MPP were seen in males and in females administered 15000 ppm but not at 250 or 2000 ppm.
Effects of MPP were detected in the kidney of all males and in the majority of females at 15000 ppm (Table 2). The changes seen in the kidneys at 15000 ppm were considered to be adverse.
All other histological changes were considered to be unrelated to treatment.

Histopathological findings: neoplastic:

not specified

Key result

Dose descriptor:

NOAEL

Remarks:

for systemic toxicity

Effect level:

2 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

15 000 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Table 1: Summary of findings in the kidneys for animals killed after 5 weeks of treatment

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Level (ppm)	0	250	2000	15000	0	250	2000	15000
Granular	0	0	0	8	0	0	0	2
Pale area(s)	0	0	0	0	0	0	0	8
Depression(s)	0	1	0	2	0	0	0	0
Number of tissues examined	10	10	10	10	10	10	10	10

Table 2: Summary of treatment related findings in the kidney for males killed after 5 weeks of treatment and females on Day 7 of lactation

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Level (ppm)	0	250	2000	15000	0	250	2000	15000
Cortical Tubular Basophilia/Dilatation
Minimal	0	0	0	1	0	0	0	3
Slight	0	0	0	4	0	0	0	3
Moderate	0	0	0	5	0	0	0	1
Total	0	0	0	10	0	0	0	7
Inflammation/Fibrosis, Interstitial
Minimal	0	0	0	3	0	0	0	3
Slight	0	0	0	4	0	0	0	1
Moderate	0	0	0	2	0	0	0	0
Total	0	0	0	9	0	0	0	4
Papilla - Tubular Dilatation
Minimal	0	0	0	7	0	0	0	5
Slight	0	0	0	2	0	0	0	0
Moderate	0	0	0	1	0	0	0	0
Total	0	0	0	10	0	0	0	5
Papilla - Tubular Degeneration/Regeneration
Minimal	0	0	0	7	0	0	0	5
Slight	0	0	0	3	0	0	0	2
Total	0	0	0	10	0	0	0	7
Papilla - Inflammation
Minimal	0	0	0	4	0	0	0	2
Slight	0	0	0	1	0	0	0	0
Total	0	0	0	5	0	0	0	2
Inflammatory Cell Debris/Eosinophilic Material, Intratubular
Minimal	0	0	0	8	0	0	0	5
Slight	0	0	0	0	0	0	0	1
Total	0	0	0	8	0	0	0	6
Hyperplasia, Papillary Epithelium
Minimal	0	0	0	1	0	0	0	2
Slight	0	0	0	0	0	0	0	1
Total	0	0	0	1	0	0	0	3
Hyperplasia, Pelvic Epithelium
Minimal	0	0	0	0	0	0	0	1
Total	0	0	0	0	0	0	0	1
Number of tissues examined	5	6	5	10	5	5	5	9

Conclusions:

In conclusion dietary administration of MPP to Sprague-Dawley rats receiving doses 250, 2000 or 15000 ppm for 5 weeks to males and females for 2 weeks before pairing, throughout gestation up to Day 7 of lactation was associated with adverse changes to the kidney of the adult rats receiving 15000 ppm. The no-observed-adverse-effect-level (NOAEL) for systemic toxicity was considered to be 2000 ppm.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

The effects seen in the kidneys with MPP and melamine (literature data) are similar therefore this mode of action could be relevant to human. In 2008 Chinese infants consumed formula derived from raw milk adulterated with a very pure preparation of melamine and suffered renal damage. The formation of melamine–uric acid crystals was thought to be the cause of the primary renal damage observed (EFSA, 2010; WHO, 2009a, 2009b). Moreover 700 mg/kg/day melamine by nasal-gastric gavage to monkeys resulted in test article-related clinical signs including turbid and whitish urine, urine crystals, red blood cell changes, increased serum alanine aminotransferase and kidney and/or liver weights, and microscopic findings including nephrotoxicity, pericarditis, and increased hematopoiesis. Nephrotoxicity was also noted at 200 mg/kg/day (Early et, 2013).

 

Early RJ, Yu H, Mu XP, Xu H, Guo L, Kong Q, Zhou J, He B, Yang X, Huang H, Hu E, Jiang Y.Repeat oral dose toxicity studies of melamine in rats and monkeys.Arch Toxicol. 2013 Mar;87(3):517-27. doi: 10.1007/s00204-012-0939-7. Epub 2012 Sep 28.

 

EFSA Journal 2010.Scientific Opinion on Melamine in Food and Feed. 8(4):1573 [145 pp.].

 

WHO, 2009a. World Health Organization, Toxicological and Health Aspects of Melamine and Cyanuric Acid, Ottawa Canada, 1 - 4 December 2008, http://whqlibdoc.who.int/publications/2009/9789241597951_eng.pdf

 

WHO, 2009b. World Health Organization; Expert Meeting to Review Toxicological Aspects of Melamine and Cyanuric Acid, 1-4 December 2008; Last reviewed/updated 16 April 2009; accessed 19 June 2009;http://www.who.int/foodsafety/fs_management/infosan_events/en/print.html

Justification for classification or non-classification

MPP is classified as STOT-RE category 2 according to CLP criteria (see table below)

 

Type of study - effects	NOAEL ppm (mg/kg/d)	LOAEL ppm (mg/kg/d)	CLP repeat exposure (STOT) classification
MPP Rat (Crl:CD(SD)) male/female Subacute (oral: gavage) 15, 150 and 1000 mg/kg/day   Kidney findings	15 mg/kg/d	150 mg/kg/d	Category 2: 4 week guidance values 30<x≤300 mg/kg/d
MPP Rat (Crl:CD(SD)   Subacute (oral: feed) and repro screening study   0, 250, 2000 and 15000 ppm (nominal in diet) (0, 14.8, 120, 854 mg/kg/day for males and 0, 17.9, 144 and 1049 mg/kg/day for females)   Kidney findings	2000ppm (120mg/kg/d for males and 144mg/kg/d for females)		Category 2: 4 week guidance values 30<x≤300 mg/kg/d