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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2001-03-15 to 2001-11-02
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese MAFF guidelines of 2000, amended in 2001
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC Commission Directive 88/302/EEC, 1988
Deviations:
no
GLP compliance:
yes
Remarks:
OECD Principles of Good Laboratory Practice as revised in 1997 (ENV/MC/CHEM(98)17) and the German Principles of Good Laboratory Practice (Bundesgesetzblatt Part I, No. 21 of May 14, 2001).
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: water solution
Details on test material:
- Name of test material (as cited in study report): IDS, Na-Salz (IDS, Na-Salt)
- Molecular formula (if other than submission substance): C8H7NNa408
- Molecular weight (if other than submission substance): 337 g/mol
- Smiles notation (if other than submission substance): C(C(C(=O)[O-])NC(CC(=O)[O-])C(=O)[O-])C(=O)[O-].[Na+].[Na+].[Na+].[Na+]
- InChl (if other than submission substance): InChI=1S/C8H11NO8.4Na/c10-5(11)1-3(7(14)15)9-4(8(16)17)2-6(12)13;;;;/h3-4,9H,1-2H2,(H,10,11)(H,12,13)(H,14,15)(H,16,17);;;;/q;4*+1/p-4
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: chelating agent
- Physical state: white powder
- Analytical purity: 72.1% IDS, Na-Salz (until April 11, 2001); 73.4% IDS, Na-Salz (from April 11, 2001)
- Purity test date: 2000-04-25; 2001-03-19 (check of expiry date)
- Lot/batch No.: FA36610
- Expiration date of the lot/batch: until March 14, 2002
- Stability under test conditions:
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
SPF rats of strain: Hsd Cpb:WU
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Age at study initiation: between 14 to 16 weeks
- Weight at study initiation: males > 300 g; females: between 208 and 248 g on day 0 p.c.
- Fasting period before study: not reported
- Housing: during the adaptation period in groups; starting from gestation day 0 individually
- Diet (e.g. ad libitum): ad libitum (a standard rat diet (NAFAG Nr. 9441 W10, supplied by EBERLE NAFAG AG, 9200-Gossau, Switzerland)
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: at least seven days before mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): approximately 50%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: March 30, 2001To: May 17, 2001

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
demineralized
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For treatment of the animals, administration formulations were prepared using demineralized water as the vehicle, the latter having no toxicological relevant effect on the parameters investigated at the volume administered (10 mL/kg bw). Fresh solutions for each concentration were prepared after 2 to at maximum 7 days of use. The administration formulations were stored for the duration of their use at room temperature.

VEHICLE
- Concentration in vehicle: 0, 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Purity: demineralized water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Investigations on the stability of the test compound in samples of 1.0 mg/ml and 100 mg/ml performed before the first substance administration and covering the concentrations used in this study, revealed no significant deviations from the content determined on the day of preparation after 8-day storage:
- Stability/ Homogeneity (Study No. N 00/0054/03 LEV, on 2001-03-06)
- Content of test substance in formulations (Study No. N 00/0054/04 LEV, on 2001-04-26 and Study No. N 00/0054/06 LEV, on 2001-05-11)
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
between 06:00 and 12:30 C.E.T.
Frequency of treatment:
once daily
Duration of test:
from day 6 to 19 p.c.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels used were selected according to a preceding dose-range finding study in rats with dose levels of 0, 50, 200 and 1000 mg/kg bw/day (study no T7068562).
8 inseminated female Wistar rats each were daily treated orally by gavage with IDS, Na-Salz solved in demineralized water from day 6 to day 19 p.c. with doses of 0, 50, 200 and 1000 mg/kg body weight (bw)/day (dose volume 10 ml/kg bw). The fetuses were delivered by cesarean section on day 20 p.c. Investigations were performed on general tolerance of the test compound by the females as well as on its effect on intrauterine development with external evaluation of fetuses.
The only signs of maternal toxicity for which treatment relationship could not be excluded consisted of marginally reduced feed intake during treatment and slightly impaired body weight development (body weight gain during gestation and corrected body weight gain marginally to slightly reduced) at the 1000 mg/kg dose level. Further on increased incidence of light colored feces was observed at a dose level of 200 mg/kg and above.
Reproductive parameters i.e. gestation rate, postimplantation loss, number of live fetuses per litter, fetal sex distribution and placental weight and appearance were not affected by treatment at a dose level up to and including 1000 mg/kg. Marginally reduced mean fetal weight was seen at the 1000 mg/kg dose level (3.62 g versus 3.74 g in the control), however due to comparability with historical control data, treatment relationship was not assumed for these findings. No external findings were seen in dosed fetuses up to and including 1000 mg/kg.
Summarizing all findings, treatment relationship could not be excluded for marginally to slightly reduced feed intake and body weight gain at a dose level of 1000 mg/kg and for light colored feces at a dose level of 200 mg/kg and above. An effect on intrauterine development was not evident at a dose level up to and including 1000 mg/kg.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (only once daily on weekends, on public holidays and on day 20 p.c.)
Attention was paid to disturbances in the general condition of the rats (appearance, behavior), and any alterations concerning their excretory products.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of the animals were determined on day 0 p.c. and daily from day 6 to day 20 p.c. Corrected body weight gain was calculated by substracting the weight of the uterus on day 20 p.c. from the body weight gain over the period from day 0 to day 20 p.c.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
The feed consumption of the animals on gestation days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20 was determined based on the differences in weight of feed provided and feed which remained unconsumed.


WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: once daily by visual estimation of the remaining quantities in the water bottles.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: 20
The females were subjected to gross pathological evaluation at the time of cesarean section on day 20 p.c.


OTHER:
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
- Other:
- Individual weight and appearance of the placentas
- dead fetuses (fetuses without signs of life, but without maceration)
- Number of live fetuses
- Sex of live fetuses
- Individual weights of live fetuses
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter.
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
Statistics:
see "Any other information on materials and methods"
Indices:
Fertility index (%) = (No. of females with implantation/ No. of inseminated females) x 100

Gestation index (%) = (No. of females with viable fetuses / No. of females with implantation) x 100
Historical control data:
Animals of this strain have been used at BAYER AG in developmental toxicity studies for years and historical data on the test parameters are available (attached to the study report).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Neither mortality nor clinical signs were observed during the study in dosed females at a dose level up to and including 1000 mg/kg bw/day. Water intake and excretions were not affected by treatment of the test substance. Food intake was slightly decreased at the 1000 mg/kg level from day 6 to 12 p.c., correlating with marginally reduced body weight gain (without statistical significance). Gross pathological findings at the 1000 mg/kg dose level were regarded as incidental and treatment relationship was not assumed. The number of inseminated females with implantation sites as well as the number of corpora lutea, preimplantation losses and implantations did not differ to a meaningful extent from the corresponding control values in the three dose groups. The gestation rate, postimplantation loss and number of live foetuses were unaffected by treatment with the test substance up to and including the dose of 1000 mg/kg bw/day.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Number of live fetuses, fetal sex distribution, fetal weight and placental weight and appearance were not affected by treatment. Neither the type nor the overall number of malformations on a fetal and litter basis indicated a treatment related teratogenic effect of IDS, Na-Salz at all dose level. The lowest incidence of malformations was found in the 1000 mg/kg dose group. The fetal external, visceral and skeletal including cartilaginous tissue evaluation revealed no toxicologically relevant findings up to and including a dose level of 1000 mg/kg bw/day.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Mortality and clinical signs

Neither mortality nor clinical signs were observed during the study in dosed females at a dose level up to and including 1000 mg/kg bw/day.

Feed intake

A toxicologically relevant treatment related effect on feed intake (Table 1) was not evident up to and including a dose level of 300 mg/kg bw/day. Marginally impaired feed intake in comparison with the control group and pretreatment values was observed at the 1000 mg/kg level from day 6 to 12 p.c. Although feed intake in this dose group was as well marginally lower before start of treatment, treatment relationship was assumed for reduced feed intake during treatment due to statistical significance (days 9 to 12 p.c.) and lack of body weight gain after start of treatment (Table 2).

Water intake

Water intake and excretions were not affected by treatment with IDS, Na-Salz up to and including 1000 mg/kg bw/day.

Body weight

Table 2 below provides an overview on the mean body weight gain of the females with viable fetuses during the treatment and gestation period.

Body weight gain was marginally impaired after start of treatment (days 6 to 7 p.a: no body weight gain) at the 1000 mg/kg dose level and related to as well marginal effects on feed intake. Further on, mean carcass weights and mean corrected body weight gain of the 1000 mg/kg dose group was marginally reduced in comparison to the current control. Although statistical significance was not evident, a treatment related effect could not be excluded due to marginally impaired body weight gain after start of treatment and decreased feed intake. Toxicologically relevant effects on body weight, body weight gain and carcass weights were not evident at a dose level up to and including 300 mg/kg bw/day.

Pathological Findings

No test-substance related gross pathological findings were ascertained in the females of the dosed groups at dose levels up to and including 300 mg/kg bw/day.

At the 1000 mg/kg dose level, 4 females revealed gross pathological alterations, including one female with blackish discolored mucosa of the caecum (female no. 84), another female with alteration of the kidneys (female no. 92: right kidney reduced in size, left kidney enlarged) and 2 females without implantation sites which showed either muddy or clear fluid in a thickened cervix. The caecal finding was a single finding and not related to other signs of impaired health in the affected female. Based on the type of the kidney findings of female no. 92 these alterations existed most probably already before start of treatment. The alterations of the cervix occurred in females without implantation sites and the incidence lay in the range of historical control data. Therefore all gross pathological findings at the 1000 mg/kg dose level were regarded as incidental and treatment relationship was not assumed.

General Reproduction Data

The number of inseminated females with implantation sites as well as the number of corpora lutea, preimplantation losses and implantations did not differ to a meaningful extent from the corresponding control values in the three dose groups (Table 3).

The number of inseminated females with implantation sites was slightly lower in the 1000 mg/kg dose group. However, fertility index lay within the normal range of variation of the rat strain used, statistical signficance was not evident for this finding and treatment started on day 6 post coitum when implantation generally already has occurred, so that treatment related unobserved early postimplantation loss was unlikely. Further on 2 females of the 1000 mg/kg dose group showed either muddy or clear fluid in a thickened cervix. These incidental findings interfered most probably with successful insemination/implantation. As 21 females with implantations were available in the 1000 mg/kg dose group, an impact on the outcome of the study is excluded due to the incidentally slightly lower fertility rate at the 1000 mg/kg dose level.

Gestation rate

Total resorption of a single implant occurred twice at the 100 mg/kg dose level (females nos. 24 and 38). At the 300 mg/kg dose level female no. 17 resorbed 7 implants. These findings were regarded as incidental since dose relationship was not evident, total resorptions do occur spontaneously in the rat strain used (cf. incidence in historical controls and different dose groups without dose relation) and mean number of postimplantation loss of all females with implantation sites was not affected. Further on at the 100 mg/kg dose level only litters with single implants were resorbed. Thus it is concluded, that the gestation rate (number of females with viable fetuses as a percentage of the number of females with implantations) was unaffected by treatment with IDS, Na-Salz up to and including the dose of 1000 mg/kg bw/day (see Table 4). The mean values for the parameters of intrauterine development are presented in Table 5. Appearance and weight of placentas were unaffected by treatment with IDS, Na-Salz up to and including the dose level of 1000 mg/kg bw/day.

Postimplantation Loss, Number of Fetuses

A treatment related effect on postimplantation loss and number of live fetuses in the dosed groups up to and including the dose level of 1000 mg/kg bw/day was not evident. As already discussed above two females of the 100 mg/kg group and one female of the 300 mg/kg group resorbed the complete litters comprising one implant each or seven implants, respectively. These resorptions did not influence mean number of postimplantation losses in the 100 mg/kg and 300 mg/kg dose groups and treatment relationship was not assumed.

Sex of fetuses

A treatment related effect on fetal sex distribution was not evident at a dose level up to and including 1000 mg/kg bw/day.

Fetal weight

Fetal weight was not affected by treatment with IDS, Na-Salz at a dose level up to and including 1000 mg/kg bw/day.

Fetal malformation

The number of fetuses and litters with malformations was not affected by treatment with IDS, Na-Salz at a dose level up to and including 1000 mg/kg bw/day with the lowest number of malformed fetuses (n = 2) in the highest dose group (Table 6). Both fetuses showed common findings: One of the 2 malformed fetuses at the 1000 mg/kg dose level had a defect in the membraneous part of the ventricular septum (fetus 675 of female no. 49). This fetus was a small fetus (3.03 grams) which revealed as well slight dilation of renal pelvis, indicative of retarded development, so that the ventricular septal defect (VSD) in the membraneous part of the ventricular cardial septum may as well be related to incidentally retarded fetal development. Based on this discussion, on the fact, that only a single fetus was affected and due to comparability with historical control data, the VSD was regarded as incidental and treatment relationship was not assumed. The other malformation in the 1000 mg/kg group (missing lumbar and sacral vertebral arches) occurred as well in the control group so that a treatment related effect was not evident. The malformations in the 300 mg/kg and 100 mg/kg dose groups were as well either single (common) findings without dose relationship and/or comparable to the findings in the current or historical control groups and treatment relationship was not evident for these findings. Thus neither the type nor the overall number of malformations on a fetal and litter basis indicated a treatment related teratogenic effect of IDS, Na-Salz at a dose level up to and including 1000 mg/kg bw/day. The lowest incidence of malformations was found in the 1000 mg/kg dose group.

Fetal External and Visceral Deviations

External deviations were not evident in dosed groups up to and including the 1000 mg/kg dose level. All visceral deviations observed during the evaluation of the study in dosed groups were of a common type, i.e. comparable to spontaneous findings in the current or historical control groups, thus characterizing the range of scattering of this rat strain (Table 7). Further on, dose relationship on a fetal and/or litter basis or statistical significance for individual deviations was not evident. The incidence of (slightly) undescended testicles in dosed groups lay in the range of historical control data and was comparable to the control group of a study performed few months after the current study at the Laboratory of Reproductive Toxicology of BAYER AG (not reported yet; study no. T6062800; live animal phase from August to September 2001; incidence in the control group: 9 fetuses within 8 litters affected). The statistically significantly increased overall number of fetuses with deviations at the 300 mg/kg dose level was not dose related so that treatment relationship was not assumed. Therefore it was concluded, that meaningful effects of the test-substance on external and visceral deviations of fetuses could not be observed at a dose level up to and including 1000 mg/kg bw/day.

Fetal Skeletal Deviations Including Cartilaginous Deviations

All skeletal including cartilaginous tissue findings, treatment related effects on degree of ossification and incidence of skeletal or cartilaginous deviations were not assumed at a dose level up to and including 1000 mg/kg bw/day.

Applicant's summary and conclusion

Conclusions:
No-observed-effect levels (NOEL) were determined:
- Maternal toxicity: 300 mg/kg bw/day
- Developmental toxicity: 1000 mg/kg bw/day
Executive summary:

Groups of 25 inseminated female Wistar rats each were treated daily orally (by gavage) with IDS, Na-salt (73.4%) solved in demineralized water from day 6 to day 19 p.c. in doses of 0, 100, 300 and 1000 mg/kg body weight (bw)/day, respectively (OECD 414; Klaus, 2002, Report No. PH-32141). The fetuses were delivered by cesarean section on day 20 of gestation. Investigations were performed on general tolerance of the test compound by the females. Findings of maternal toxicity consisted of transiently marginally impaired feed consumption and body weight gain at the 1000 mg/kg dose level. Further on treatment relationship could not be excluded for marginally reduced carcass weight and corrected body weight gain. All other parameters evaluated, i.e. mortality and clinical signs, water intake and excreta, overall body weight development including final body weight and gross pathological observations were not affected by treatment at a dose level up to and including 1000 mg/kg bw/day.With respect to intrauterine development, gestation rate, postimplantation loss and accordingly the number of fetuses, fetal sex distribution, placental weight and appearance and fetal weight were not affected by treatment with IDS, Na-Salz at a dose level up to and including 1000 mg/kg bw/day. External, skeletal and visceral evaluation of fetuses revealed neither toxicologically relevant treatment related effects at a dose level up to and including 1000 mg/kg bw/day. A teratogenic potential of IDS, Na-Salz was not evident at a dose level up to and including 1000 mg/kg bw/day.

Summarizing and evaluating all data investigated the following no-observed-effect levels (NOEL) were determined:

Maternal toxicity: 300 mg/kg bw/day

Developmental toxicity: 1000 mg/kg bw/day