Spinflam MF 83 PP-25

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study is well described and scientifically acceptable. Justification for read across approach is explained in the endpoint summary.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2010

Materials and methods

Principles of method if other than guideline:

Test item was respectively administered at 0, 40 and 400 mg/kg body weight by daily gavage from gestation day (GD) 13 to GD 20 to control (C), low dose (LM) and high dose (HM) groups of pregnant female F344 rats. Rats were sacrificed 30 min after the last gavage.

GLP compliance:

not specified

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test animals

Species:

rat

Strain:

Fischer 344

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: purchased from Japan SLC Inc., Shizuoka, Japan.
- Age at study initiation: ten- to twelve-week-old.
- Weight at study initiation: 150–200 g.
- Housing: rats were housed in plastic cages.
- Diet: ad libitum.
- Water: ad libitum.
- Acclimation period: 1 week in the test facilities.

ENVIRONMENTAL CONDITIONS
- Photoperiod: 12 h light/dark cycle.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1 %

Details on mating procedure:

After the adaptation period, one or two female rats were housed with one male for mating.
Vaginal plug check and vaginal smear observation by microscope were carried out each morning.
Day 0 of gestation (GD 0) was determined by the presence of a vaginal plug and/or spermatozoids in the vaginal smear. The female rat was then housed separately from the male and body weight (BW) was recorded daily.

Duration of treatment / exposure:

From gestation day 13 to gestation day 20.

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 40 and 400 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

Pregnant dams were randomly divided into three groups (six rats per group): control (C), low dose (LM), and high dose (HM).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the doses were based on the doses used by Dobson et al. (2008).
- Necropsy: at GD 20, rats were sacrificed after isoflurane anesthetization, 30 min after the last gavage.

Examinations

Maternal examinations:

POST-MORTEM EXAMINATIONS
- Blood was withdrawn from the inferior vena cava in a heparinized tube, centrifuged at 3000 g for 15 min, and the plasma was stored at -80 °C until analysis.
- Amniotic fluid was sampled.

PLASMA BIOCHEMICAL PARAMETERS
Plasma gamma-glutamyltransferase (GGT), blood urea nitrogen (BUN), creatinine (CRE), uric acid (UA), glutamic pyruvic transaminase/alanine aminotransferase (GPT/ALT), and glutamic oxalacetic transaminase/aspartate aminotransferase (GOT/ AST) were measured.

Fetal examinations:

Pups were weighed.
Fetuses and placentas were frozen in liquid nitrogen and stored at -80 °C.

HYSTOPATHOLOGY
The kidneys of dams and fetuses were removed and fixed in 10 % formalin for histopathological analyses. Samples were then stained with haematoxylin–eosin, following by standard techniques for histopathological analysis, and were observed by light microscopy.

Statistics:

Statistical analysis was carried out using SPSS 11.0 software. Data are expressed as mean ± SD. Pair-wise comparisons were made between melamine groups and the control group. The minimum level of probability accepted for significance was P < 0.05.

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Dose descriptor:

dose level:

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
No significant differences were observed for dam body weight, number of fetuses, or fetal and placental weights between the melamine groups and the control (P > 0.05).

HISTOPATHOLOGY
The glomeruli and tubules of dams in the HM group did not differ from those of control specimen; however, spotted lesions with cellular aggregations of mesenchymal origin around blood vessels in the cortico-medullar transitional area were observed. No increment in renal stones was observed in any of the kidney samples from dams and fetuses. In the fetal kidney, differentiation of the kidney glomerular apparatus is still proceeding. In the middle of the cortex, some mature glomeruli are evident but many immature glomeruli still exist in the peripheral cortical area, surrounded by many immature cells beneath the cortical capsule.
There were no significant histopathological differences in the fetal kidneys of control and experimental groups.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Dam final body weight (BW), number of fetuses, and fetal and placental weights.

Groups	Dams BW (g)	Number of fetuses	Fetus weight (g)	Placenta weight (g)
Control	221.1 ± 20.5	6.5 ± 2.7	3.4 ± 0.3	0.5 ± 0.1
40 mg/kg bw	226.4 ± 12.9	7.2 ± 1.6	3.3 ± 0.1	0.5 ± 0.1
400 mg/kg bw	226.2 ± 7.8	7.5 ± 2.4	3.2 ± 0.2	0.5 ± 0.1

Values are expressed as mean ± SD, n = 6 rats/group.

PLASMA, AMNIOTIC FLUID, PLACENTAL and FETAL CONCENTRATIONS

Test item was not detected in the control samples.

Test item was present in both the low treated group (239.1 ± 39.6 µg/ml) and high treated group (1413.7 ± 146.8 µg/ml) plasma samples; however, the concentration in the high dose group was significantly higher than in the lower group (P < 0.01).

For amniotic fluid, melamine was detected only in the high dose group (192.8 ± 30.0 µg/ml).

Test item was present in the fetuses of both the low and high dose groups; however, the concentration was significantly higher in the high dose group (570.0 ± 107.7 µg/g) than in the low dose group (327.7 ± 117.0 µg/g) (P < 0.01).

Test item was detected only in the placenta of the high dose group. It appears that melamine can cross the placental barrier and its transport is dose-dependent.

concentration in plasma, amniotic fluid, fetus and placenta.

Groups	Plasma (µg/ml)	Amniotic fluid (µg/ml)	Fetus (µg/g bw)	Placenta (µg/g)
Control	ND	ND	ND	ND
40 mg/kg bw	239.1 ± 39.6	ND	327.7 ± 117.0	ND
400 mg/kg bw	1413.7 ± 146.8*	192.8 ± 30.0	570.0 ± 107.7*	1117.7 ± 304.4

Values are expressed as mean ± SD, n = 6.

ND: not detected.

* P < 0.01.

LIVER FUNCTION PARAMETERS: PLASMA GGT, GOT/AST and GPT/ALT

No significant differences were found among the three groups for GGT, GPT, and GOT. Melamine administration did not affect liver function as determined by these three enzymes.

Concentration of plasma gamma-glutamyltransferase, glutamic pyruvic transaminase/alanine aminotransferase, and glutamic oxalacetic transaminase/aspartate aminotransferase.

Groups	GOT/AST (U/L)	GPT/ALT (U/L)	GGT (U/L)
Control	54.4 ± 9.6	34.4 ± 1.9	2.5 ± 1.4
40 mg/kg bw	54.3 ± 10.3	36.8 ± 3.7	2.5 ± 0.8
400 mg/kg bw	58.5 ± 6.1	33.5 ± 5.1	3.8 ± 3.1

Values are mean ± SD, n = 6.

KIDNEYS FUNCTION PARAMETERS: PLASMA CRE, UA and BUN

The plasma concentrations of UA and CRE were significantly higher in the high dose group than in the control (P < 0.01). BUN was significantly higher in the high dose group than in the control (P < 0.05). A tendency toward a dose-dependent increase of plasma UA, CRE and BUN was noted. These data show that renal functions were affected by the administration of melamine.

Concentration of plasma uric acid (UA), plasma creatinine (CRE), and blood urea nitrogen (BUN).

Groups	CRE (mg/dl)	BUN (mg/dl)	UA (mg/dl)
Control	0.6 ± 0.1	10.8 ± 1.5	0.4 ± 0.1
40 mg/kg bw	0.6 ± 0.1	14.7 ± 2.5	0.5 ± 0.1
400 mg/kg bw	0.7 ± 0.1**	15.9 ± 3.5*	1.0 ± 0.3**

Values are mean ± SD, n = 6.

* P < 0.05.

** P < 0.01.

DISCUSSION and CONCLUSION

the dose employed did not significantly influence the body weight of dams, number of fetuses, or fetal and placental weights. These findings suggest that the dose alone showed no apparent effects on health.

The plasma concentration increased in a dose-dependant manner 30 min after the last gavage. The results showed that administration to dams reached the fetus via the placenta and that the fetal melamine concentration was dependent on the dose administered to the dams. Thus, if pregnant dams are continuously exposed for long periods, or to high doses, the fetuses are expected to be indirectly exposed. Consequently, consumption of contaminated foods should be avoided by expectant mothers to prevent indirect fetal melamine exposure.

It was unclear why test item was detected in the amniotic fluid of the high dose group and not low dose group. This may be the result of a dose-dependent effect or secretion back into the amniotic fluid from the fetuses in the high dose group. Test item was detected in the placental of the high dose group. These results may explain the lack of retention in the placental compartment for small doses or indicate the rapid transfer through this organ, or both.

The data recorded showed that plasma concentrations of CRE, UA and BUN were increased by melamine exposure, suggesting that kidney function was affected in the melamine groups, especially in the high dose group.

Some lesions with cellar aggregations were noted in the cortico-medullar area of the kidney in the high dose group; there were no histological differences in fetal kidneys between the control and high dose groups. However, the immature state of fetal kidney development must be taken into consideration.

Plasma concentrations of GGT, GPT/ALT, and GOT/AST were not affected, suggesting no melamine effect on liver function in these rats.

During the study the whole fetus was homogenized for analysis. It is possible that determining distribution in specific fetal tissues could provide more information. Additionally, identification of which fetal organ has the highest concentration and whether test item affects the renal function of newborn rats requires further study.

In conclusion, the data recorded clarifies that test item ingested by pregnant female rats can pass through the placenta, reaching the fetus in a dose-dependent manner.

Applicant's summary and conclusion

Conclusions:

Ingested test item affects renal function in dams and dose-dependently passes the placental barrier to reach the fetus.

Executive summary:

In this study, the possibility of placental transfer with consequent effect to fetuses and pregnant dams were determined. Test item was respectively administered at 0, 40 and 400 mg/kg body weight by daily gavage from gestation day (GD) 13 to GD 20 to control (C), low (LM) and high (HM) groups of pregnant female F344 rats. Rats were sacrificed 30 min after the last gavage.

Test item was not detected in any of the control and placental samples, or in amniotic fluid from the LM group. Plasma and fetal concentrations in the HM group were significantly higher than in the LM group (P < 0.01). Liver enzyme determination revealed no differences among the three groups. However, plasma creatinine, plasma uric acid and blood urea nitrogen concentrations in dams were significantly increased (P < 0.05). These results show that ingested test item affects renal function in dams and dose-dependently passes the placental barrier to reach the fetus.

Conclusion

Ingested test affects renal function in dams and dose-dependently passes the placental barrier to reach the fetus.