REAKTIV-GELB F-68 072 FW

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 June to 20 October 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

lithium sodium 2-amino-4-{[4-(cyanoazanidyl)-6-[(3-sulfonatophenyl)amino]-1,3,5-triazin-2-yl]amino}-5-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)benzene-1-sulfonate

EC Number:

413-090-5

Cas Number:

189574-45-6

Molecular formula:

Hill formula: C24H18LixN10NayO12S4

IUPAC Name:

lithium sodium 2-amino-4-{[4-(cyanoazanidyl)-6-[(3-sulfonatophenyl)amino]-1,3,5-triazin-2-yl]amino}-5-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)benzene-1-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

See below

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species and strain: Crl:(Wi) BR-Wistar Rats
- Source: males: CHARLES RIVER EUROPE LABORATORIES INC., Germany
females: CHARLES RIVERlEUROPElLABORATORIES INC., TOXI-COOP 1103 Budapest, Hungary
- Age at study initiation: Males: from stock of 'experienced' males
Females: 45-55 days
- Weight at study initiation: 179 and 290 g (females)
- Housing: pre-mating and mating period : 1-3 animals/cage
during mating hours : 1 male with 1- 3 females
during pregnancy : 1-4 sperm positive females/cage
Three dams were caged individually
- Diet: Ssniff SM RIM-Z+H "Autoclavable complete feed for rats and mice - breeding and maintenance" ad libitum
- Water: tap water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 June To: 24 July 2007

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

The test item was administered at appropriate concentrations prepared with the vehicle. Preparation of test item solution was made using magnetic stirrer. Dose formulations were freshly prepared daily except for five cases when the formulations were prepared 24 or 48 hours before treatment and were kept in refrigerator.
Sampling for analytical control of dosing solutions was made during the first and last week of the treatment period by the Analytical Laboratory of LAB Hungary.

PREPARATION OF DOSING SOLUTIONS:
- Concentration in vehicle: 7.5, 20, 60 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): 9151006

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

PRINCIPLE OF THE ANALYTICAL METHOD
Test item content and homogeneity were determined in dosing formulations at two analytical occasions.
6 samples were taken from each test concentration and one from the control.
The samples were properly diluted and analysed by HPLC using UV detection. Concentration of the active ingredient was determined.

EQUIPMENT AND CHEMICALS
APPARATUS
HPLC system: Merck-Hitachi LaChrom HPLC system
D-7000 Interface, No. : 1442- 122
L-7100 HPLC pump, No.: 1516-030
L-7200 Autosampler, No.: 1406-005
L-7400 UV Detector, No.: 1502-017
L-7360 Column oven, No.: 00107295
L-7614 Degasser, No.: 144 12YA0500
Balances: L 2200P Sartorius, Germany, No.:38 100037
BP221 S Sartorius, Germany, No.: 1 18091 17
Ultrasonic bath: Elmasonic S 300 H, Elma No.: 01 08901 05
Water purification system: MILLIPORE, DIRECT 43, FOMNO 73341
Chromatographic conditions:
Detector: UV at 270 nm
Column: LiChrospher 60 RP Select B 5pm 250x4 mm,
No.: 437724
Column temperature: 30 deg C
Mobil Phase: 2.5 g TBAHS was dissolved in 620 ml water and then 280 ml acetonitrile and 100 ml isopropanol were added
Flow: 1.0 ml/min
Injection volume: 20 µl

all concentrations were between 94% and 101% of nominal concentrations

Details on mating procedure:

The oestrus cycle of female animals were examined before pairing. After acclimatisation, the females were paired according to their oestrus cycle to males in the morning for three hours (one male: one to three females) until the number of sperm positive females I group achieved twenty two. Vaginal smears were prepared from each female, stained with 1 % aqueous methylene blue solution and examined for presence of sperm. The day of mating was regarded as day 0 of pregnancy (vaginal plug and/or sperm in the vaginal smear). Sperm positive females were separated and caged in groups of 2 to 4 animals. Three dams were caged individually.

Duration of treatment / exposure:

day 5 to 19 of gestation

Frequency of treatment:

daily

Duration of test:

START OF THE EXPERIMENT: 1 8 June 2007
END OF IN-LIFE PHASE: 24 July 2007

No. of animals per sex per dose:

22 sperm-positive females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose level was selected on the basis of the results of the studies
- Reaktiv Gelb F-68 072 FW Testing for subacute oral toxicity (28 applications within 29 days) in male and female Wistar rats (Hoechst AG report No. 93.0537)
- Draft report 90-Day oral toxicity study of Reaktiv F68072 FW in rats. LAB Report dated 3. Jan. 2007

In the 28 day study with Reaktiv Gelb the daily administration of 600 mg/kg caused adverse clinical symptoms and increased water consumption Furthermore, decreases in serum-chloride and serum-sodium levels were observed. Increases in GPT-activities were evaluated as indicative for an adverse effect on the liver. Additionally, decreases in erythrocyte counts, haemoglobin and haematocrit values as well as increases of liver weight were observed. After administration of 120 mg/kg/day water consumption was still increased in both sexes and GPT acivities were higher compared to controls. The "no observed effect level" was therefore 24 mg/kg body weight.

In the 90 day study conducted in 2006 dose levels of 1000, 250 and 62.5 mg/kg/day were administered.
Polyuria was observed at all dose levels tested. At 1000 mg/kg/day, numbers of erythrocytes, haemoglobin and heamotocrit values and platelet counts were lower than in controls.
In summary a dose level of 250 mg/kg/day was considered to represent a "no toxic effect level".

Therefore, a dose level of 600 mgkg is determined to represent the highest dose level to be tested in the rat embryotoxicity study. A dose level of 600 mg/kg/day is by a factor of
25 higher than the "no toxic effect level" in the 28 day study and by a factor of 2.4 higher than the "no toxic effect level" in the 90 day study.

Examinations

Maternal examinations:

Clinical Observations
A careful clinical observation was made after dosing at least once a day and a cage side clinical observation was made in the afternoon. Individual observation included the check of behavior and general condition.
Checking for death was made at least once daily.

Body Weight
The body weight of the male animals was not measured.
The body weight of the female rats was measured at least once in the pre-mating period, but was not statistically evaluated. Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g).
Corrected body weight was calculated on the day 20 of pregnancy (body weight on day 20 minus the weight of the gravid uterus).

Food Consumption
The food consumption was measured between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet (accuracy: 1 g).

Water consumption
The water consumption was measured each day from gestation day 0 to 20 with reweighing the non-consumed water with a glass-measure (accuracy: 1 ml).

Examination for Sign of Implantation
On gestation days 13 and/or 14 the sperm positive females were checked for the presence of vaginal bleeding which indicates the implantation of conceptuses.

NECROPSY
All sperm positive females were sacrificed by CO2 gas anesthesia followed by cervical dislocation on day 20 of gestation. The abdomen was opened, the uterus with cervix and the left ovary were removed and weighed. The right ovary was placed into a Petri dish after removal. After removing the uterus and its content gross pathology of dams' viscera was performed.
Organs and tissues with undiagnosed macroscopic findings were examined histologically. All these organs were fixed in 10 % buffered formalin solution after necropsy. For histological examinations organs and tissues were embedded into paraffin after dehydration.
The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live fetuses, early and late embryonic death and fetal death were counted. Animals with unambiguous implantation sites, but not fetuses have been found, were considered as pregnant.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

Fetuses were removed from the opened uterus, viability assessments were performed and after the umbilicus was cut euthanasia of the viable fetuses was made by CO2 gas.
Each live fetus was weighed individually (accuracy 0.01 g), and was subjected to external examination. The placentas were examined externally. The gender of fetuses was determined according to the anogenital distance.
Thereafter the fetuses were individually identified and about the half of each litter was subjected to visceral examination and the other half for skeletal examination. In case of fetuses subjected to visceral examination the abdominal and thoracic region was opened and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in Sanomiya mixture then after fixation the body was micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. In case of fetuses subjected to skeletal examination the abdominal region was opened and the viscera and skin of fetuses were removed and the cadaver was fixed in alcian-blue - acetic acid – ethanol mixture. After fixation in isopropanol the skeletons were stained by KOH-Alizarin red-S method and the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

Statistics:

The statistical evaluation of data was performed with the program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett's homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett's test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. A foetus was considered as retarded in body weight, when its weight was below the average minus twofold standard deviation of all control foetuses.

Dams were excluded from statistical analysis as follows:
- non pregnant females:
Control: No.: 139, 146,214
75 mglkg: No.: 114,209
200 mg/kg: No.: 181,208
600 mg/kg: No.: 122

- female with no implantation, with corpora lutea: Control: 168
- females with 5 or less implantation: 75 mg : No.: 207; 600 mg : No.: 182
- females with placental inflammation or diffuse necrosis: 75 mg : No.: 184,103; 600 mg : No.: 204
- female excluded due to technical error (error in determining the day 0 of pregnancy): 200 mg : 137

The following data were evaluated by an appropriate statistical method (Bartlett, ANOVA - Duncan, Kruskal-Wallis - Mann-Whitney U test, chi2):

PREGNANT FEMALES
- Mortality
- (Corrected) body weight, body weight gains, gravid uterine weight, food consumption, water consumption: mean +/- S.D.
- Gross pathology observation

NECROPSY AND CESAREAN SECTION DATA
- Number of corpora lutea, number of implantations, number and percent of live fetuses, fetal weight, number and percent of intrauterine mortality: mean +/- S.D.

Indices:

- Pre-implantation loss: Number of corpora lutea - Number of implantations x 100 / Number of corpora lutea
- Post-implantation loss: Number of implantations - Number of live fetuses - x 100 /Number of implantations

FETUSES
- Sex distribution: Number of Male (Female) fetuses x 100 / Number of fetuses
- External abnormalities/litter: Number of fetuses with abnormality x 100 / Number of fetuses
- Visceral abnormalities/litter: Number of fetuses with abnormality - x 100 / Number of fetuses
- Skeletal abnormalities/litter: Number of fetuses with abnormality x 100 / Number of fetuses

Historical control data:

Yes, within LAB Research Inc, Hungary's historical range.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Observation of polyuria (wet bedding) was recorded for the dams in all cages in the 600 mg/kg dose group during the treatment period. It was considered to be a secondary maternal toxicity resulting from the high lithium content in the test material, as polydipsia and polyurea are a common side effect of lithium.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

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

effects observed, treatment-related

Description (incidence and severity):

The water consumption of dams was significantly higher than controls in the 600 mg/kg dose group during the treatment period (between gestation days 5- 1 1, 1 1 - 17 and 1 7-20). This was considered to be an effect resulting from the high lithium content in the test material, as polydipsia and polyurea are a common side effect of lithium.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Early death and consequential postimplation loss was statistically significant higher in the 75 mg/kg and 200 mg/kg dose groups compared to the control group. A statistically significant higher total intrauterine mortality in the 200 mg/kg dose group was also a consequence of the high postimplation loss level, however, early embryonic death, postimplantation loss and total intrauterine mortality were below the control level in the 600 mg/kg dose group.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

CLINICAL SYMPTOMS, MORTALITY
There were no dead animal in any group. Observation of polyuria (wet bedding) was recorded for the dams in all cages in the 600 mg/kg dose group during the treatment period. It was considered to be a sign of treatment related maternal toxicity. Piloerection was recorded for one single dam (No.: 184) in the 75 mg/kg dose group, this was not attributed to the administration of the test item.

BODY WEIGHT, BODY WEIGHT GAIN, GRAVID UTERINE WEIGHT, AND CORRECTED BODY WEIGHT
The body weight, body weight gain, gravid uterine weight, corrected body weight, corrected body weight gain of dams were at the same level in the control and in the Reaktiv Gelb F68072 FW treated groups.

FOOD CONSUMPTION
There was no difference in the food consumption of dams in the experimental groups.

WATER CONSUMPTION
The water consumption of dams was significantly higher than controls in the 600 mg/kg dose group during the treatment period (between gestation days 5- 1 1, 1 1 - 17 and 1 7-20). The 75 mg/kg and 200 mg/kg dose groups were unaffected.

NECROPSY
At necropsy there were no treatment related gross pathology alterations seen. Reddish-mottled lungs observed in one dam (No.: 198) in the control group, one dam (No.: 120) in the 75 mg/kg dose group, point-like haemorrhages on the lungs for one dam (No.: 143) in the 600 mg/kg dose group were related to euthanasia and exsanguination. Pale liver and dark coloured fluid in the uterus were recorded for the same dam (No.: 184) in the 75 mg/kg dose group and was not attributed to the treatment. A slightly enlarged spleen was recorded for one dam (No.: 204) in the 600
mg/kg dose group, this was not considered to be treatment related. Orange coloured content in the stomach recorded for 9 females (No.: 160, 1 12,2 19, 107, 1 15, 143, 149, 166, 190, 122) in the 600 mg/kg dose group was related to the orange colour of the test item Reaktiv Gelb F68072 FW.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

>= 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

All effects seen were considered to be secondary effects due to the high lithium content in the test material, as polydipsia and polyurea are a common side effect of lithium.

Results (fetuses)

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Two malformations out of 232 foetuses were found in the control group, one hydrops fetalis and one foetus with absent tail and with filum terminale hanging out.
One malformed foetus out of 235 was found in the 600 mg/kg dose group also with
the malformation mentioned above. These malformations are regarded as spontaneous and unrelated to treatment. There were no other malformations found during the external examination.

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

REPRODUCTION DATA, INTRAUTERINE MORTALITY, VIABLE FOETUSES AND THEIR SEX DISTRIBUTION

The intrauterine mortality or sex distribution of foetuses was most probably not influenced by the treatment. There was no difference between the experimental groups in the number of corpora lutea or implantations.

Early death and consequential postimplation loss was statistically significant higher in the 75 mg/kg and 200 mg/kg dose groups compared to the control group. A statistically significant higher total intrauterine mortality in the 200 mg/kg dose group was also a consequence of the high postimplation loss level, however, early embryonic death, postimplantation loss and total intrauterine mortality were below the control
level in the 600 mg/kg dose group. There were statistically significant fewer male foetuses and more female foetuses in the 75 mg/kg dose group compared with the other experimental groups, however, the sex distribution was within the historical background range in this dose group.

In summary, there were statistical differences in some parameters, but there was no dose-response and all values were considered to be normal. There were no effects of treatment on these parameters.

BODY WEIGHT OF FOETUSES
The average body weight of foetuses was similar in all groups.

FOETAL EXAMINATION
External Examination:
Two malformations out of 232 foetuses were found in the control group, one hydropsfetalis and one foetus with absent tail and with filum terminale hanging out. One malformed foetus out of 235 was found in the 600 mg/kg dose group also with the malformation mentioned above. These malformations are regarded as spontaneous and unrelated to treatment. There were no other malformations found during the external examination.
The incidence of body weight retarded foetuses was significantly higher in the 200 mg/kg group but the majority were all in one litter. There was a consequent higher incidence of foetuses with external abnormalities in this group. The incidence of external abnormalities in the 600 mg/kg dose group was lower than the control level. The type and incidence of external foetal observations was within the historical background range, there were no treatment related effects seen at external examination.

The incidence of placentas with fibrinoid degeneration (an inflammatory reaction) was significantly higher in the 600 mg/kg dose group compared with the other experimental groups. In 11 of 15 cases the change was seen in two litters the other 4 cases appeared sporadically. Small placentas were found in 6 out of 235 foetuses in the 600 mg/kg dose group. The differences in incidence of placental observations is not considered to be a foetal effect of treatment, it is equivocal as to being an effect of treatment, it may reflect maternal toxicity.
There was a sporadic incidence of fused, pale, oedemateous, dark brown discoloured placentas found in the experimental groups, without relationship to treatment.

Visceral Examination:
At visceral examination the incidence of abnormalities was higher in the control group than in the Reaktiv Gelb F68072 FW treated groups.
Four foetuses with malformations out of 1 15 were found in the control group. Three of these foetuses were multiple malformed and one foetus had situs inversus cordis. One of the three multiple malformed foetuses had one lung lobe in each side, stomach and intestines on the right side (situs inversus abdominalis).

The second of the three multiple malformed foetuses had one lung lobe on each side, aorta turned to the right, truncus pulmonalis turned to the left, right subclavian artery, right carotid artery and left carotid artery originated from the top of aortic arch, left subclavian artery originated from truncus pulmonalis, aorta turned behind oesophagus to the left, septum membraneceum was absent, (VSD), aorta was overriding the septal
defect, stomach and intestines were on the right side (situs inversus abdominalis), duodenum was narrowed by vena portae.The third multiple malformed foetus (one of the two foetuses with caudal regression syndrome) had small and empty stomach, absent gonads, kidneys close to each other and fused adrenals.
There was one malformed foetus with a malpositioned kidney out of 101 in the 75 mg/kg dose group. One malformed foetus with yellowish discolouration in the lens (histopathologically central cataracta) out of 108 foetuses was found in the 200 mg/kg dose group. Two malformations were recorded out of 120 foetuses in the 600 mg/kg dose group. One foetus had a malpositioned right kidney and the other foetus was multiple malformed with absent gonades and fused kidneys. This multiple malformed foetus also had caudal regression syndrome indicated by the observations of external and skeletal examinations (there was a similar foetus in the control group).
Variations unrelated to treatment observed included short and extremely short brachocephalic trunk, left common carotid artery originated close to brachiocephalic trunk, dilated renal pelvis and dilated third and lateral brain. The incidence of dilated renal pelvis was the highest in the 600 mg/kg dose group, but it was within the historical control range.
All types of visceral abnormalities and variations described are present in the historical database of our Laboratory or are described in teratology literature as spontaneous changes. There were no effects of treatment on the incidence of any visceral variations.

Skeletal Examination:
There were more skeletal malformations found in the control group than in the test item treated groups. There were no types of malformation present with a significantly higher incidence in the 600 mg/kg dose group than in the control group. There was one foetus out 1 19 with malformed vertebrae in the control group (the one of the two foetuses found during the study with caudal regression syndrome). This foetus had four cervical vertebrae, the other vertebrae and the ribs were absent. There were no foetus with malformed vertebrae in the 75 mg/kg or the 200 mg/kg dose
groups. Malformed vertebrae were recorded for one out of 1 16 foetuses in the 600 mg/kg dose group also with caudal regression syndrome. This foetus had similar skeletal abnormalities as the above mentioned one, 4 cervical vertebrae were present, fused bones instead of thoracic III and lumbar VI vertebrae and only the first and second ribs were present.

Short and/or bent clavicula and/or scapula were recorded for 4 foetuses in the control group out of 119, for 3 out of 98 in the 75 mg/kg dose group, for 2 out of 109 in the 200 mg/kg dose group and for 3 out of 1 16 in the 600 mg/kg dose group. Short and/or thickened humerus was observed in 2 foetuses in the control group, none in the 75 mglkg dose group, in 3 in the 200 mg/kg dose group and in 2 in the 600 mg/kg dose group.

Short and/or bent radius and/or ulna were found only in two foetuses in the control group.
Short and/or bent osilei was found only in one foetus in the 200 mg/kg dose group.
Short and/or bent femur and fibula were recorded for one control foetus.

At skeletal examination there were a range of variations in examined foetuses, but these variations showed no dose response and were unrelated to treatment. These variations included incomplete ossification of the skull, 2 or less than 2 sternal bodies, 13'h rib short, markedly wavy ribs with or without incomplete ossification, bipartite thoracic vertebrae, irregular calcification or not ossified thoracic, lumbar or sacral vertebral arches, 14 thoracic, 5 lumbar vertebrae, not ossified or incompletely ossified sacral vertebra 11, 2 or less or asymmetric metacarpus, 3 or less or asymmetric metatarsus, incomplete ossification of clavicula and slightly short clavicula and scapula, irregular periosteal ossification on humerus and/or ulna or femur. Incomplete ossification of clavicula, slightly short or bent clavicula or scapula. Incomplete or unossified os pubis.
Incomplete ossification or non-ossified os ischii was not present in the control group but it was recorded in the Reaktiv Gelb F68072 FW treated groups. However, in the most recent comparable study in this laboratory, the control incidence was 6/121 foetuses (and for reduced ossification of the os pubis it was 18/121 foetuses). Hence, taking into account the historic control data for incomplete ossification or non-ossified
os ischii, the statistical difference is not considered to be treatment related.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

>= 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

All effects seen were considered to be secondary effects due to maternal toxicity resulting from the high lithium content in the test material, as polydipsia and polyurea are a common side effect of lithium.

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Slight maternal toxicity was observed at 600 mg/kg bw/day only resulting from a high content of lithium salt. There were no effects of treatment on the early or late embryonic mortality or in fetal deaths. No fetal effects of treatment were observed in the type or incidence of external, visceral or skeletal observations.

Executive summary:

Groups of 22 sperm-positive female Crl:(Wi)BR-Wistar rats were treated with REAKTIV GELB F68072 FW orally in one control group and three dose levels of 75, 200, and 600 mg/kg/day with 10 ml/kg treatment volume from day 5 up to and including day 19 post coitum. Controls were given vehicle alone, the vehicle was distilled water.

Rats were examined daily for viability and clinical signs. Body weight was recorded between Days 0, 3, 5, 8, 11, 14, 17 and 20 of gestation. Food consumption was determined on Days 0-3, 3-5, 5-8, 8-1 1, 11-14, 14-17 and 17-20 of gestation, water consumption was measured each day from gestation day 0 to 20. Caesarean section and gross-pathology were performed on day 20 of pregnancy. Implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea in each ovary were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were examined externally. About half of each litter was subjected to visceral examination and the other half to skeletal examination (with double staining). All abnormalities found during the fetal examinations were recorded.

There was no maternal mortality in any group. Observation of high water intake and polyuria (wet bedding) was recorded for the dams in each cage in the 600 mg/kg dose group during the treatment-period as a treatment related maternal clinical sign. There were equivocal statistical differences in the incidence of placentas with fibrinoid degeneration at 600 mg/kg/day, which may be related to maternal toxicity but was without any fetal developmental effects. There were no treatment related clinical signs in the other experimental groups.

The test item had no effect on the body weight, body weight gain, gravid uterine weight, corrected body weight, corrected body weight gain or food consumption of dams.

At necropsy no treatment related gross pathology alterations were seen. The intrauterine mortality or sex distribution of fetuses was not influenced by the treatment. There was no difference between groups in the number of corpora lutea or implantations.

The body weight of fetuses was similar in all experimental groups. There were no abnormalities attributable to the treatment at external, visceral or skeletal examinations.

In Conclusion:

Slight maternal toxicity was observed at 600 mg/kg/day only. There were no effects of treatment on the early or late embryonic mortality or in fetal deaths.

No fetal effects of treatment were observed in the type or incidence of external, visceral or skeletal observations.

Under the conditions applied in this study the observations made in the dams treated with Reaktiv Gelb F68072 FW, and their fetuses the following no observable-adverse-effect levels were derived:

NOAEL maternal toxicity: 200 mg/kg bw/day

NOAEL developmental toxicity: 600 mg/kg bw/day

NOAEL teratogenicity: 600 mg/kg bw/day