Spinflam MF 83 PP-25

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: carcinogenesis bioassay of melamine

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

From August 1978 to September 1980

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.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

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:

Test item was administered in the diet to F344 rats for 103 weeks (chronic) to determine its carcinogenic potential. The dose levels of melamine were 2250 or 4500 ppm for male rats and 4500 or 9000 ppm for female rats.

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: Charles River Breeding Laboratories, Portage, MI.
- Age at study initiation: 6 weeks old.
- Housing: five per Polycarbonate cage, changed twice per week.
- Bedding: Absorb-Dri@ hardwood chips (Lab Products, Inc.); changed twice per week.
- 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 %
- Air changes: 12-1 5 times per hour
- Photoperiod: 12 hours of fluorescent light per day

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Preparation: test diets were prepared by first mixing a small amount of PurinaB Lab Chow and the required amount of test item 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.
- Maximum Storage Time: 3 weeks
- Storage Conditions: -20 °C
- Analysis: dosed feed samples were analyzed periodically by ultraviolet spectroscopy. The results obtained indicate that only one of the formulations analyzed was slightly (+10.6 %) out of specifications (> ± 10 %). Results from three separate referee analyses at MRI verified the accuracy of the formulations.

Duration of treatment / exposure:

103 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Female rats: 0, 4500 and 9000 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Male rats: 0, 2250 and 4500 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

50 males and 50 females, except for only 49 control male rats

Control animals:

yes, plain diet

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATION
Observed twice daily for mortality and signs of morbidity.
Clinical signs were recorded monthly.

BODY WEIGHT
Body weight data collected weekly for the first 13 weeks, monthly until week 91, and every 2 weeks thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE
Feed consumption data collected every 4 weeks.

Postmortem examinations (parental animals):

All animals necropsied and examined histologically.

GROSS PATHOLOGY: Yes.
Tissues examined: gross lesions, skin (with mammary gland), mandibular lymph nodes, salivary gland, sternum (with bone marrow), larynx or anterior trachea, oesophagus, thyroid, parathyroid, lungs with mainstream bronchi, heart, stomach (glandular and nonglandular), duodenum, large intestine, liver, pancreas, spleen, kidneys, adrenal glands, urinary bladder, gonads, prostate or uterus, brain, and pituitary.

Tissue preservation: tissues were preserved in 10 % neutral buffered formalin embedded in paraffin, sectioned, and stained with haematoxylin and eosin.

HISTOPATHOLOGY: Yes

Statistics:

Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone's (1975) extensions of Cox's meth ods for testing for a dose-related trend.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

no compound-related clinical signs were observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

after week 20 lower than those of the controls

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

after week 20 lower than those of the controls

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

statistically significant between bladder stones and bladder tumours in male

Other effects:

no effects observed

Description (incidence and severity):

Test substance intake: 97 % and 99 % that of the controls for males and 99 % and 99 % for females

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
Survival of high-dose male rats was significantly lower (P < 0.05) than that of the controls. Survival of all other dosed rat groups was comparable with that of the respective controls.
No compound-related clinical signs were observed.

BODY WEIGHT AND WEIGHT GAIN
After week 20, mean body weights of dosed rats of each sex were lower than those of the controls.

FOOD CONSUMPTION AND COMPOUND INTAKE
The average daily feed consumption per rat by low-and high-dose rats was 97 % and 99 % that of the controls for males and 99 % and 99 % for females.

HISTOPATHOLOGY: NEOPLASTIC
Urinary Bladder: transitional-cell carcinomas in the urinary bladder of male rats occurred with a statistically significant (P ≤ 0.002) positive trend (controls, 0/45; low-dose, 0/50; high- dose, 8/49, 16 %) and the incidence in the high-dose group was significantly higher (P ≤ 0.016) than that in the controls. The combined incidence of transitional-cell carcinomas and papillomas showed a statistically significant (P < 0.001) positive trend (controls, 0/45; low-dose, 0/50; high-dose, 9/49, 18 %) and the incidence in high dose rats was significantly higher (P ≤ 0.008) than that in the controls. These tumours were not observed in statistically significant proportions in female rats (0/49, 1/49, 1/47).
The transitional-cell carcinomas were visualized grossly as 1- to 1.5-cm masses attached to the mucosal surface of the urinary bladder. Seven of the eight high-dose male rats with transitional-cell carcinomas also had bladder stones (calculi). Microscopically, most of the carcinomas had transitional-like cells that formed protrusions into the bladder lumens. Some had papillary areas. The carcinomas had moderate numbers of mitoses and some nuclear pleomorphism. Discrete invasions through the bladder wall occurred in one male rat, but no metastases were evident in the lungs or other tissues.

Kidney: chronic inflammation was observed in significantly (P ≤ 0.01) increased incidence in dosed female rats . The dose relationship and intensity of the increased interstitial lymphoplasmocytic infiltrates and cortical fibrosis clearly set these changes apart from the minor inflammatory component that may accompany the progressive nephropathy normally encountered in aging rats. The changes in the high-dose females were often observed grossly as pitted or roughened renal cortical surfaces. Chronic inflammation of the kidney was not significant in dosed male rats. In those animals in which this lesion was observed, there was no correlation with urinary bladder stones.

Pancreas: pancreatic islet-cell carcinomas in male rats occurred with a statistically significant (P = 0.034) negative trend (control, 3/44, 7 %; low-dose, 0/48; high-dose, 0/45) by the Cochran-Armitage test. The incidences were not significant in pair-wise comparisons between the dosed groups and the controls, and these tumours were not observed in female rats. Total pancreatic islet-cell tumours (adenomas or carcinomas) were not significantly different for either sex of rats.

Thyroid: C-cell carcinoma in the thyroid was observed in female rats with a statistically significant (P ≤.0.038) positive trend (controls, 0/50; low-dose, 0149; high-dose, 3/ 50, 6 %). Neither the pair-wise comparisons of the high-dose group with the controls nor the combination of C-cell adenomas or carcinomas was statistically significant in any of the tests. These tumours were not observed in statistically significant proportions in male rats.

Uterus: endometrial stromal polyps were observed in statistically significant (P ≤ 0.017) negative trend (controls, 11/50, 22 %; low-dose, 7/50, 14 %; high-dose, 2/50, 4 %) and in decreased incidence (P ≤ 0.022) in the high-dose group in a pair-wise comparison with the controls. The combined incidence of endometrial stromal polyps and sarcomas was statistically significant (P ≤ 0.017) in the negative direction (controls, 14/50, 28 %; low-dose, 7/50, 14 %; high-dose, 4/50, 8 %). A significantly lower (P ≤ 0.029) incidence in the high-dose group was observed in the pair-wise comparisons with the controls. The combined incidence of endometrial stromal polyps and sarcomas in the high-dose group was not significantly different from the historical rate of this tumour in untreated female at the same laboratory (117/759, 15.4 %).

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Male rats: Nonneoplastic lesions. Reproductive system

Effect		Control	Low dose	High dose
*Mammary gland		(49)	(50)	(50)
	Dilatation/ducts	8 (16 %)	6 (12 %)	6 (12 %)
	Galactocele	1 (2 %)	1 (2 %)
	Calcification, NOS	1 (2 %)
	Hyperplasia, NOS		1 (2 %)	1 (2 %)
*Mammary lobule hyperplasia, NOS		(49)	(50)	(50)
		1 (2 %)	2 (4 %)
*Preputial gland abscess, NOS		(49)	(50)	(50)
				2 (4 %)
#Prostate		(49)	(48)	(46)
	Haemorrhage			1 (2 %)
	Inflammation, suppurative	2 (4 %)	4 (8 %)	1 (2 %)
	Inflammation, acute			1 (2 %)
	Inflammation, active chronic	1 (2 %)	2 (4 %)
	Inflammation, chronic	1 (2 %)	1 (2 %)	2 (4 %)
	Inflammation, chronic suppurativ	1 (2 %)	1 (2 %)
*Seminal vesicle		(49)	(50)	(50)
	Haemorrhage			1 (2 %)
	Inflammation, acute			1 (2 %)
#Testis		(49)	(50)	(50)
	Mineralization		1 (2 %)
	Athrophy, NOS	13 (27 %)	8 (16 %)	7 (14 %)
	Hyperplasia, interstitial cell	1 (2 %)	1 (2 %)	5 (10 %)
#Testis/tubule		(49)	(50)	(50)
	Calcification, NOS			1 (2 %)
#Spermatid		(49)	(50)	(50)
	Cytomegaly		1 (2 %)	1 (2 %)
*Epididymis		(49)	(50)	(50)
	Inflammation, granulomatous	1 (2 %)
	Granuloma, spermatic			1 (2 %)

*Number of animals necrospied

#Number of animals with tissue examined microscopically

Male rats: Neoplasms. Reproductive system

Effect		Control	Low dose	High dose
*Mammary gland		(49)	(50)	(50)
	Fibroadenoma		3 (6 %)
	Adjusted (a) Terminal (b)	0.0 % 0/30 (0 %)	8.9 % 1/30 (3 %)	0.0 % 0/19 (0 %)
Statistical test (c)	Life table Incidental tumour test Cochran-Armitage Trend, Fisher Exact test	P = 0.526 P = 0.514 N P = 0.634 N	P = 0.135 P = 0.196 P = 0.125	(d) (d) (d)
*Preputial gland		(49)	(50)	(50)
	Carcinoma, NOS	1 (2 %)
	Adenoma, NOS	1 (2 %)	1 (2 %)	1 (2 %)
	Adenocarcinoma, NOS	2 (4 %)	1 (2 %)
	Adjusted (a) Terminal (b)	12.2 % 3/30 (17 %)	5.7 % 1/30 (3 %)	5.3 % 1/19 (5 %)
Statistical test (c)	Life table Incidental tumour test Cochran-Armitage Trend, Fisher Exact test	P = 0.191 N P = 0.152 N P = 0.113 N	P = 0.325 N P = 0.310 N P = 0.329 N	P = 0.309 N P = 0.312 N P = 0.175 N
#Prostate		(49)	(48)	(46)
	Adenoma, NOS	5 (10 %)	3 (6 %)	3 (7 %)
	Adjusted (a) Terminal (b)	16.7 % 5/30 (17 %)	8.8 % 2/29 (7 %)	15.7 % 2/16 (13 %)
Statistical test (c)	Life table Incidental tumour test Cochran-Armitage Trend, Fisher Exact test	P = 0.541 N P = 0.482 N P = 0.312 N	P = 0.367 N P = 0.388 N P = 0.369 N	P = 0.617 N P = 0.606 N P = 0.393 N
*Seminal vescicle		(49)	(50)	(50)
	Carcinosarcoma	1 (2 %)
#Testis		(49)	(50)	(50)
	Interstitial-cell tumour	42 (86 %)	44 (88 %)	42 (84 %)
	Adjusted (a) Terminal (b)	95.4 % 28/30 (93 %)	100.0 % 30/30 (100 %)	97.6 % 18/19 (95 %)
Statistical test (c)	Incidental tumour test Cochran-Armitage Trend, Fisher Exact test	P = 0.561 N P = 0.463 N	P = 0.555 N P = 0.484	P = 0.607 N P = 0.517 N
*Epididymis		(49)	(50)	(50)
	Mesothelioma, NOS		1 (2 %)

*Number of animals necrospied

#Number of animals with tissue examined microscopically

(a) Kaplan-Meier estimated lifetime tumor incidence after adjusting for intercurrent mortality.

(b) Observed tumor incidence at terminal kill.

(c) Beneath the control incidence are the P-values associated with the trend test. Beneath the dosed group incidences are the P-values corresponding to pairwise comparisons between that dosed group and the controls. The life table analysis regards tumors in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The incidental tumor test regards these lesions as non-fatal. The Cochran- Armitage and Fisher’s exact tests compare directly the overall incidence rates. A negative trend or lower incidence in a dose group is indicated by (N).

(d) Not statistically significant; no tumors observed in dosed or control groups.

Female rats: Nonneoplastic lesions - Reproductive system

Effect		Control	Low dose	High dose
*Mammary gland		(50)	(50)	(50)
	Dilatation/ducts	17 (34 %)	19 (38 %)	23 (46 %)
	Galactocele	9 (18 %)	6 (12 %)
	Hyperplasia, NOS	2 (4 %)	2 (4 %)	1 (2 %)
*Mammary lobule hyperplasia, NOS		(50)	(50)	(50)
		6 (12 %)	6 (12 %)	7 (14 %)
*Preputial gland abscess, NOS		(50)	(50)	(50)
	Dilatation/ducts			1 (2 %)
	Abscenss, NOS		1 (2 %)
#Uterus		(50)	(50)	(50)
	Hydrometra			1 (2 %)
	Cyst, NOS			1 (2 %)
	Haematoma, NOS	1 (2 %)
	Inflammation, suppurative		1 (2 %)
	Abscenss, NOS			1 (2 %)
	Haemosiderosis		1 (2 %)
#Cervix uteri		(50)	(50)	(50)
	Inflammation, acute			1 (2 %)
	Abscenss, NOS		1 (2 %)
#Uterus/endometrium		(50)	(50)	(50)
	Cyst, NOS	6 (12 %)	2 (4 %)	1 (2 %)
	Inflammation, suppurative			1 (2 %)
	Haemosiderosis	1 (2 %)
	Hyperplasia, NOS	1 (2 %)
	Hyperplasia, cystic	4 (8 %)	5 (10 %)	3 (6 %)
#Ovary		(50)	(50)	(50)
	Cyst, NOS	3 (6 %)	2 (4 %)	1 (2 %)
	Inflammation, granulomatous			1 (2 %)

*Number of animals necrospied

#Number of animals with tissue examined microscopically

Female rats: Neoplasms. Reproductive system

Effect		Control	Low dose	High dose
*Mammary gland		(50)	(50)	(50)
	Adenocarcinoma, NOS			1 (2 %)
	Cystadenoma, NOS	1 (2 %)
	Papillary cystadenoma, NOS			1 (2 %)
	Fibroma	1 (2 %)
	Fibrosarcoma	1 (2 %)
	Fibroadenoma	11 (22 %)	11 (22 %)	6 (12 %)
	Adjusted (a) Terminal (b)	28.5 % 7/34 (21 %)	31.4 % 7/30 (23 %)	20.7 % 4/27 (15 %)
Statistical test (c)	Life table Incidental tumour test Cochran-Armitage Trend, Fisher Exact test	P = 0.251 N P = 0.129 N P = 0.124 N	P = 0.474 P = 0.508 P = 0.595	P = 0.276 N P = 0.135 N P = 0.143 N
*Preputial gland		(50)	(50)	(50)
	Carcinoma, NOS		2 (4 %)	1 (2 %)
	Adenoma, NOS			1 (2 %)
	Cystadenoma, NOS		1 (2 %)
	Adjusted (a) Terminal (b)	0.0 % 0/34 (0 %)	9.6 % 2/30 (7 %)	5.9 % 1/27 (4 %)
Statistical test (c)	Life table Incidental tumour test Cochran-Armitage Trend, Fisher Exact test	P = 0.164 P = 0.225 P = 0.202	P = 0.102 P = 0.102 P = 0.121	P = 0.215 P = 0.298 P = 0.274
#Uterus		(50)	(50)	(50)
	Endometral stromal polyp	11 (22 %)	7 (14 %)	2 (4 %)
	Adjusted (a) Terminal (b)	31.2 % 10/34 (29 %)	19.3 % 4/30 (13 %)	7.4 % 2/27 (7 %)
Statistical test (c)	Life table Incidental tumour test Cochran-Armitage Trend, Fisher Exact test	P = 0.017 N P = 0.007 N P = 0.006 N	P = 0.304 N P = 0.179 N P = 0.218 N	P = 0.022 N P = 0.018 N P = 0.007 N
	Endometral stromal sarcoma	3 (6 %)		2 (4 %)
	Adjusted (a) Terminal (b)	8.8 % 3/34 (9 %)	0.0 % 0/30 (0 %)	6.1 % 1/27 (4 %)
Statistical test (c)	Life table Incidental tumour test Cochran-Armitage Trend, Fisher Exact test	P = 0.461 N P = 0.423 N P = 0.390 N	P = 0.143 N P = 0.143 N P = 0.121 N	P = 0.588 N P = 0.544 N P = 0.500 N
#Uterus/endometrium		(50)	(50)	(50)
	Adenocarcinoma, NOS		1 (2 %)
#Ovary		(50)	(50)	(50)
	Cystadenoma, NOS			1 (2 %)

*Number of animals necrospied

#Number of animals with tissue examined microscopically

(a) Kaplan-Meier estimated lifetime tumor incidence after adjusting for intercurrent mortality.

(b) Observed tumor incidence at terminal kill.

(c) Beneath the control incidence are the P-values associated with the trend test. Beneath the dosed group incidences are the P-values corresponding to pairwise comparisons between that dosed group and the controls. The life table analysis regards tumors in animals dying prior to terminal kill as being (directly or indirectly) the cause of death. The incidental tumor test regards these lesions as non-fatal. The Cochran- Armitage and Fisher’s exact tests compare directly the overall incidence rates. A negative trend or lower incidence in a dose group is indicated by (N).

Applicant's summary and conclusion

Conclusions:

No indication of an effect to the reproductive organs was obtained from the repeated dose and chronic toxicity studies: mammary glands, ovaries, prostate, seminal vesicles, testes and uterus were examined macroscopically and microscopically and were found to be unaffected by test item at each of the doses used.

Executive summary:

Test item was administered in the diet to F344 rats for 103 weeks (chronic) to determine its carcinogenic potential. The dose levels of melamine were 2250 or 4500 ppm for male rats and 4500 or 9000 ppm for female rats. 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. In the female rat chronic inflammation of the kidney was observed at an increased incidence (relative to controls) in both the low (4500 ppm) and high (9000 ppm) dose groups.

Conclusion

No indication of an effect to the reproductive organs was obtained from the repeated dose and chronic toxicity studies: mammary glands, ovaries, prostate, seminal vesicles, testes and uterus were examined macroscopically and microscopically and were found to be unaffected by test item at each of the doses used.