Spinflam MF 83 PP-25

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test procedure is not following a guideline, nevertheless the report is scientifically acceptable. Justification for read across approach is explained in the endpoint summary.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1984

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

1983

Materials and methods

Principles of method if other than guideline:

Two separate 13 -week studies were conducted in male and female F344 rats: the first study included 12 animals in male and female, while the second included 10 animals per group. The dose levels ranged from 6000 to 18000 ppm in the first study and from 750 to 12000 ppm in the second one. Animals were observed daily for mortality and signs of morbidity. Body weights and feed consumption data were collected weekly. Necropsies were performed on all animals.

GLP compliance:

not specified

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Frederick Cancer Research Center (Frederick, MD).
- Age at study initiation: 5-6 weeks old.
- Housing: four per Polycarbonate cage, changed twice per week.
- Bedding: Absorb-Dri@ heat- treated hardwood chips (Lab Products, Inc.).
- Diet: ad libitum, Purina Laboratory Chow.
- Water: ad libitum, acidified with hydrochloric acid to pH 2.5.
- Acclimation period: approximately 2 weeks before the test began.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 - 26 °C
- Humidity: 30 - 70 %

Administration / exposure

Route of administration:

oral: feed

Details on oral exposure:

DIET PREPARATION
Test diets were prepared by first mixing a small amount of PurinaB Lab Chow and the required amount of melamine with a mortar and pestle and then adding this premix to the required amount of animal meal and mixing for 10 to 30 minutes in a Patterson-Kelly@ twin- shell blender equipped with an intensifier bar. Prepared diets containing 100000 ppm melamine were analyzed at Midwest Research Institute and were found to be stable for 2 weeks at temperatures up to 45 °C.
Test diets were stored in the freezer for no longer than 3 weeks.

Duration of treatment / exposure:

91 days

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
First Study: 0, 6000, 9000, 12000, 15000 and 18000 ppm (0, 560, 850, 1100, 1400 and 1700 mg/kg/day for male rats; 0, 560, 880, 1200, 1400 and 1600 mg/kg/day for female rats)
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Second Study: 0, 750, 1500, 3000, 6000 and 12000 ppm (0, 72, 150, 300, 590 and 1300 mg/kg/day for male rats and 0, 84, 150, 300, 600 and 1300 mg/kg/day for female rats)
Basis:
nominal in diet

No. of animals per sex per dose:

First Study: 12 male and 12 female rats
Second Study: 10 male and 10 female rats

Control animals:

yes, plain diet

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATION
Observed daily for mortality and signs of morbidity.

BODY WEIGHT
Body weight data collected weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE
Feed consumption data collected weekly.

Sacrifice and pathology:

Those animals that were judged moribund were killed and necropsied.
At the end of the 91-day studies, survivors were killed with carbon dioxide, and necropsies were performed on animals that survived to the end of the studies and on all animals found dead, unless precluded in whole or in part by autolysis or cannibalization.

GROSS PATHOLOGY: Yes.
In the first 13-week study, the following tissues were examined for control and high-dose groups: gross lesions, tissue masses, abnormal lymph nodes, skin, mandibular lymph nodes, mammary gland, salivary gland, thigh muscle, sciatic nerve, bone marrow, costochondral junction (rib), thymus, larynx, trachea, lungs and bronchi, heart, thyroid, parathyroid, oesophagus, stomach, duodenum, jejunum, ileum, colon, mesenteric lymph nodes, liver, gallbladder (mice), pancreas, spleen, kidneys, adrenals, urinary bladder, seminal vesicles/ prostate/ testes or ovaries/ uterus, nasal cavity, brain, and pituitary gland.
In the second study, the kidneys and urinary bladders of all animals were examined microscopically.

Tissues were preserved in 10 % neutral buffered formalin, embedded in paraffin, sectioned, and stained with haematoxylin and eosin. Only the kidney and urinary bladder of lowest-dose animals were examined microscopically.

HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

first suty: one male at 18000 ppm and two males at 6000 ppm died. Second study: none of the rats died

Mortality:

mortality observed, treatment-related

Description (incidence):

first suty: one male at 18000 ppm and two males at 6000 ppm died. Second study: none of the rats died

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

first study: depressed by more than 8 % in males and females at 12000 ppm. Second study: depressed by more than 10 % at 6000 and 12000 ppm

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

first study: at 18000 ppm 80-90 % that of the control. Second study: not affected

Urinalysis findings:

no effects observed

Description (incidence and severity):

examined only in the second study

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

both first and second studies stones found in the urinary bladders

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

both first and second studies epithelial hyperplasia of the urinary bladder

Details on results:

CLINICAL SIGNS AND MORTALITY
_First study: one male receiving 18000 ppm and two males receiving 6000 ppm died.
_Second study: none of the rats died

BODY WEIGHT AND WEIGHT GAIN
_First study: mean body weight gain in males and females receiving 12000 ppm or more was depressed by more than 8 % when compared with controls.
_Second study: mean body weight gain was depressed by more than 10 % when compared with controls for male rats receiving 6000 and 12000 ppm, but no depression was observed in any group of dosed females.

FOOD CONSUMPTION AND COMPOUND INTAKE
_First study: feed consumption by rats receiving 18000 ppm was approximately 80 - 90 % that of controls.
_Second study: feed consumption was not affected by incorporation of melamine in the feed.

GROSS PATHOLOGY
_First study: stones were found in the urinary bladders of most dosed male rats, and the incidence was dose related; 25 % or more in the two highest dosed groups has stones.
_Second study: other than stones in the bladder of dosed male rats, no compound-related effects were observed at necropsy. The incidence of stones in the urinary bladder of male rats was dose related. Stones were present even in the male group receiving 750 ppm.

HISTOPATHOLOGY
_First study: histopathologic evaluations were performed on 10 animals of either sex from the high-dose (18000 ppm), low-dose(6000 ppm) and control groups. Diffuse epithelial hyperplasia of the urinary bladder was found in 8/ 10 females receiving 18000 ppm melamine, while in animals receiving 6000 ppm melamine, focal ppm epithelial hyperplasia was observed in only 1/ 10 males and in none of the females. The urinary bladders of animals from other dosed groups were not examined microscopically. No other compound-related histopathologic effects were observed.
_Second study: hyperplasia of the transitional epithelium of the bladder was present in 1/10 male rats receiving 3000 ppm, in 3/10 receiving 6000 ppm, and in 9/9 receiving 12000 ppm melamine. The hyperplastic epithelial changes, which were found only in male rats that had bladder stones, were accompanied by prominent capillaries and occasional oedema and scattered mast cells in the submucosa. Kidney changes in male rats were minimal. There was no evidence of urinary bladder stones or hyperplasia of the bladder epithelium in any groups of dosed female rats, but dose-related calcareous deposits were observed in the straight segments of the proximal tubules in female rats (2/10 controls, 3/ 10 receiving 750 ppm, 4/ 10 receiving 1500 ppm, 10/10 receiving 3000 ppm, 8/10 receiving 6000 ppm and 10/10 receiving 12000 ppm).

URINALYSIS
_Second study: rine samples were analyzed from male and female rats receiving 750 ppm melamine and compared with urine samples from control rats. There were no differences in the urine samples that could be attributed to the presence of melamine in the feed. Microscopic examination of the urine did not provide any evidence of melamine crystalluria.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

750 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: (estimated melamine consumption for each animal was 72 mg/kg/day)

2

Dose descriptor:

NOAEL

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: (estimated melamine consumption for each animal was 1200 mg/kg/day)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

FIRST STUDY RESULTS

Survival, mean body weight and feed consumption of rats in the first study

Dpse (ppm)	Survival* (week of death)	Mean Body Weights [g]			Weight Change relative to respective controls** [%]	Feed consumption (% of control)
		Initial	Final	Gain
Males
0	12/12	88	297	209
6000	10/12 (8, 10)	87	287	200	-4	96
9000	12/12	90	288	198	-5	92
12000	12/12	81	276	185	-11	94
15000	12/12	92	259	167	-20	90
18000	11/12 (7)	87	255	168	-20	88
Females
0	12/12	87	191	104
6000	12/12	79	181	102	-2	87
9000	12/12	83	187	104	0	94
12000	12/12	83	179	96	-8	94
15000	12/12	82	174	92	-12	84
18000	12/12	81	165	84	-19	78

* Number surviving/number per group

** Weight Change Relative to Controls = [(Weight Change (Dosed Group) -Weight Change (Control Group)) / Weight Change (Control Group)] * 100

Incidence of rats with urinary bladder stones or granular material

Dpse (ppm)	Number of Rats with Urinary Bladder Stones
Males
0	0/12
6000	6/12
9000	8/12
12000	12/12
15000	10/12
18000	12/12
Females
0	0/12
6000	0/12
9000	0/12
12000	0/12
15000	3/12
18000	5/12

SECOND STUDY RESULTS

Survival, mean body weight and feed consumption of rats in the second study

Dpse (ppm)	Survival* (week of death)	Mean Body Weights [g]			Weight Change relative to respective controls*** [%]	Feed consumption (% of control)
		Initial	Final **	Gain
Males
0	10/10	120	312	192
750	10/10	120	302	182	-5	96
1500	10/10	119	302	183	-5	92
3000	10/10	120	299	179	-7	94
6000	10/10	119	290	171	-11	90
12000	10/10	119	276	157	-18	88
Females
0	10/10	95	176	81
750	10/10	94	179	85	+5	100
1500	10/10	95	179	84	+4	91
3000	10/10	93	175	82	+1	87
6000	10/10	95	176	81	0	89
12000	10/10	93	173	80	1	92

* Number surviving/number per group

**Weight on day 84

*** Weight Change Relative to Controls = [(Weight Change (Dosed Group) -Weight Change (Control Group)) / Weight Change (Control Group)] * 100

Incidence of rats with urinary bladder stones or granular material

Dpse (ppm)	Number of Rats with Urinary Bladder Stones	Hyperplasia of the Bladder Epithelium
Males
0	1/10	0/10
750	2/10	0/10
1500	5/10	0/10
3000	7/10	1/10
6000	9/10	3/10
12000	9/9	9/9
Females
0	0/10	0/10
750	0/10	0/10
1500	0/10	0/10
3000	0/10	0/10
6000	0/10	0/10
12000	0/10	0/10

Applicant's summary and conclusion

Conclusions:

NOAEL male: 750 ppm
NOAEL female: 12000 ppm

Executive summary:

test item was administered in the diet to F344 rats for 13 weeks (subchronic) to determine its toxicological profile.

Two separate 13 -week studies were conducted in male and female F344 rats: the first study included 12 animals in male and female, while the second included 10 animals per group. The dose levels ranged from 6000 to 18000 ppm in the first study and from 750 to 12000 ppm in the second one.

Animals were observed daily for mortality and signs of morbidity. Body weights and feed consumption data were collected weekly. Moribund animals and animals that survived to the end of the 13-week study were killed by CO₂ asphyxiation. Necropsies were performed on all animals, unless precluded by autolysis or cannibalization.

Compound-related lesions were observed in the urinary tract. Most noticeable was the development of uroliths (urinary bladder stones), which occurred at a greater frequency in males than females. Increased incidences of urinary bladder stones and hyperplasia of the bladder epithelium were observed.

Conclusion

NOAEL male: 750 ppm

NOAEL female: 12000 ppm