Acetic acid, oxo-, sodium salt, reaction products with cresol and ethylenediamine, iron sodium salts

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March - July 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Data on batch no. given; no full info on the composition of the test compound. Relatively well conducted and reported study according to guideline/standard. There were limitations, however, in the duration fo the treatment period (in comparsion to current OECD guideline) and details were missing such as acceptance criteria for the analyses

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

dosing from day 6 up to and including day 16 rather than from day 6 to day prior to planned sacrifice

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Acetic acid, oxo-, sodium salt, reaction products with cresol and ethylenediamine, iron sodium salts

EC Number:

283-041-9

EC Name:

Acetic acid, oxo-, sodium salt, reaction products with cresol and ethylenediamine, iron sodium salts

Cas Number:

84539-53-7

Molecular formula:

non specified (UVCB substance)

IUPAC Name:

non specified (UVCB substance)

Constituent 2

Reference substance name:

EDDHMA-Fe

IUPAC Name:

EDDHMA-Fe

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name of test compound: FeEDDHMANa
Trade name: Bolikel FE
Chemical name: N,N'-Bis(2-hydroxy-4-methylphenyl)ethylenediamine diacetic acid, ferric-sodium complex
Appearance: red brown powder
Batch no: 954243 d.d. 19 October 1995
Storage: at room T in the dark

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: ca. 8 weeks (not indicated whether this was at delivery or at start)
- Weight at study initiation: 219-300 g
- Fasting period before study: no
- Housing: in groups during acclimitisation; 3 females to 1 male during mating; thereafter individually
- Diet (e.g. ad libitum): ad lib
- Water (e.g. ad libitum): ad lib
- Acclimation period: at least 5 days prior to mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 22 (with fluctuations; no further info)
- Humidity (%): ca. 55% (with fluctuations; no further info)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 31 March (day 0 = day of mating) To: ca. 3 weeks later (day 21 = day of gestation)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: daily immediately prior to dosing. Formulations were shaken vigorously and stirred prior to and during dosing procedures


VEHICLE: distilled water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the first week (9 April 1996) and the last week (18 April 1996) samples were analysed to check stability and homogeneity
(low and high conc) and accuracy (all levels). Samples were analysed using HPLC. No acceptance criteria were indicated.
Accuracy was within 90-110% except for one high conc sample (84%); homogeneity was within 90-110%; and stability
measurements did not differ by more than 10%.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:3
- Length of cohabitation: until mating had occurred (no further info), and until 96 mated females had been obtained (out of 120)
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility (no info).
- Further matings after two unsuccessful attempts: no info
- Verification of same strain and source of both sexes: males from stock were used that had been succesfull in previous studies
(no further info)
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

From day 6 to day 16 (inclusive) of pregnancy

Frequency of treatment:

Daily gavage

Duration of test:

Animals were killed on day 21 of gestation

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 200 and 1000 mg/kg bw day
Basis:
actual ingested

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on preliminary study at same dose levels (reported separately)
- Rationale for animal assignment (if not random): at random (no further info)

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily (including cage debris to detect abortion or premature birth)

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6-17, and 21of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined: On days 0, 3, 6, 9, 13, 17 and 21 of gestation

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: thoracic and abdominal examinations, including ovary and uterine horn

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:

- External examinations: Yes: all per litter, including sex
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter (visceral); half per litter (skeletal)

Statistics:

If the variables can be assumed to follow a normal distribution, the Dunnett test (many-to-one t - t e s t ) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups. The Steel-test (many-to-one rank test) was applied instead of the Dunnett-test if the data can not be assumed to follow a normal distribution. The exact Fisher-test for 2x2
tables was applied if the variables could be dichotomized without loss of information. All tests were two-sided and in all cases
p<0.05 was accepted as the lowest level of significance. Macroscopic, skeletal and visceral findings were not subjected to
statistical analysis as this was considered not to provide any additional information.

Indices:

Pregnancy rate: (number of pregnant females/number of mated females) x 100
Pre-implantation loss: (number of corpora lutea - number of implantations) / (number of corpora lutea) X 100
Post-implantation loss: (number of implantations - number of live implants) / (number of implantations) X 100

Historical control data:

No info

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Number of mated females: 24 24 24 24 for the control, low, mid and high concentration group, respectively
Number of pregnant females: 21 24 23 22, respectively
Pregnancy rate (%): 88 100 96 92, respectively
Number of females with spontaneous deliveries: 1 1 0 1, respectively

MORTALITY: no

CLINICAL SIGNS: only 1 pregnant female receiving 1000 mg/kg/day showed a poor condition during the last week of pregnancy. There were no other clinical signs of toxicity or behavioural changes noted in this study.

BODY WEIGHT: following exclusion of non-pregnant females and females with spontaneous deliveries, body weight gain was reduced in the 1000 mglkg dose group from day 13 of pregnancy onwards.

FOOD CONSUMPTION: food consumption (excluding the non-pregnant females and females with spontaneous deliveries) in the 1000 mglkg group was reduced to a statistically significant level during days 9 to 13 and 13 to 17 of pregnancy. Food consumption was back to normal from days 17 to 21 of pregnancy, when treatment had ceased.

REPRODUCTION DATA: treatment of pregnant females with FeEDDHMANa did not reveal any adverse effect on the reproduction parameters recorded in this study. The number of corpora lutea, implantations and pre-and post implantation losses recorded in treated females were in the same range as control females. The statistically significant decrease in post-implantation loss recorded in the 1000 rng/kg group when compared to the control group, was considered not to be of toxicological relevance.

MACROSCOPIC EXAMINATION: Macroscopic observations at necropsy did not reveal test substance-related abnormalities.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
FOETAL MORTALITY: two foetuses of the 200 mg/kg/day dose group were found dead at necropsy. No test substance related mortality occurred. The incidence of embryonic and foetal deaths was comparable between the dose groups.

EXTERNAL EXAMINATIONS: macroscopic examination revealed no treatment-related abnormalities.

SEX RATIOS: sex ratios were comparable in treated and control groups.

FOETAL BODY WEIGHT: Statistically significant reductions in weights of live foetuses were seen on an individual basis in animals of the high concentration group. All foetuses of female 96 (1000 mglkg dose group) were smaller compared to the foetuses of other females in that group and in the control group, and was related to the poor physical condition and growth reduction noted in this female. This was considered to have influenced the slightly lower mean foetal weight in this dose group.

VISCERAL EXAMINATIONS: a number of anomalies was recorded in all groups, the types and group distribution of which did not suggest any association with treatment.

SKELETAL EXAMINATIONS: there was some intergroup variation in the incidences of the ossification parameters, but there were no consistent intergroup differences that were considered to represent toxicologically relevant adverse responses to treatment. Small numbers of foetuses with minor morphological changes were recorded in all groups, but their types and incidences did not suggest any dose-response relationship to treatment. The proportion of foetuses with supernumary (14th) rib(s) at the thoracolumbar border was unaffected by treatment.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The NOEL for maternal toxicity was 200 mg/kg bw based on reductions in BW gain and food intake; the NOAEL for developmental toxicity was 1000 mg/kg bw. Although a reduction in mean foetal BW was noted in animals of the high dose group, this reduction was very slight and considered to be mainly (but not exclusively) due to the poor condition of one female animal in this group.

Executive summary:

120 virgin females were mated with 40 males in order to obtain 4 groups of 24 pregnant females for the main study. From day 6 to day 16 of gestation inclusive, females of the treatment groups received daily oral administration of FeEDDHMANa at dose levels of 50, 200 or 1000 mg/kg body weight per day and females of the control group received daily oral administration of distilled water. Body weights and food consumption of females were determined at periodic intervals during the pregnancy period. On day 21 of gestation, all females were subjected to post-mortem examination and external, thoracic and abdominal macroscopic findings were recorded. The ovaries and uterine horn were dissected and examined for the number of corpora lutea, the weight of the gravid uterus, the number and distribution of live foetuses or still births , the weight and

sex of each live foetus and externally visible foetal macroscopic abnormalities. Alternate live foetuses of each litter were preserved in industrial methylated spirit or Bouin's fluid for examination of skeletal and visceral anomalies, respectively.

MATERNAL DATA: only 1 pregnant female of the 1000 mg/kg dose group showed signs of ill health during the last week of pregnancy. There were no other clinical signs of toxicity or behavioural changes noted in this study. Body weight gain was reduced in the 1000 mg/kg dose group from day 13 -17 of pregnancy. After correction for the uterus weight, body weight gain remained low on day 21 of pregnancy. Food consumption in the 1000 mg//g group was reduced to a statistically significant level during days 9 to 13 and 13 to 17 of pregnancy. Food consumption was back to normal from days 17 to 21. Post-mortem examinations of females of the treated groups (50, 200 and 1000 mg/kg/day), did not reveal any test substance- related effects.

REPRODUCTION PARAMETERS: treatment of pregnant females with FeEDDHMANa did not induce any adverse effect on the reproduction parameters recorded in this study.

FOETAL DATA: external, skeletal and visceral examinations of foetuses of dams treated at 50, 200 or 1000 mg/kg/day did not reveal any adverse

effect of the test substance. Sex ratios were comparable in treated and control groups. A slight reduction in mean foetal weights on an individual basis was seen in animals of the high dose group.

CONCLUSION: oral dosing of mated female Wistar rats with FeEDDHMANa at 50, 200 and 1000 mg/kg body weight revealed a slight reduction in maternal body weight gain and food consumption and in one pregnant female signs of ill health, towards the end of the treatment period, in the high dose group only. The effect on food consumption had disappeared by the end of the study. No adverse effects were recorded on any of the reproduction or foetal parameters recorded in this study. Visceral and skeletal examinations of foetuses did not indicate any teratogenic potential of FeEDDHMANa. From the results of this study, the No-Observed-Effect-Level (NOEL) based on maternal toxicity was 200 mg/kg body weight/day. The No-Observed-Adverse-Effect- Level (NOAEL) based on embryo-foetal parameters was 1000 mg/kg body weight/day.