N,N'-ethylenebis[N-acetylacetamide]

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is in accordance to the test guidelines in place at the time when the study was performed and GLP and therefore rated as reliable without restrictions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 12 May 1981

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-ethylenebis[N-acetylacetamide]

EC Number:

234-123-8

EC Name:

N,N'-ethylenebis[N-acetylacetamide]

Cas Number:

10543-57-4

Molecular formula:

C10H16N2O4

IUPAC Name:

N,N'-ethylenebis[N-acetylacetamide]

Details on test material:

- Name of test material (as cited in study report): TAED
- Analytical purity: 99.4%
- Impurities (identity and concentrations): TriAED 0.6%, DiAED < 0.1%, water < 0.1%
- Purity test date: 25 Jan 1993
- Lot/batch No.: 071292 (07 Dec 1992)
- Expiration date: 07 Dec 1995
- Storage condition of test material: roomtemperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Crl:CD (SD) Br rats from Charles River Italia S.p.A.
- Age at study initiation: 9-10 wks
- Weight at study initiation: 200-225 g
- Fasting period before study: -
- Housing: Makrolon cages, type 3D; after mating each gravid female was caged individualy
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: about 2 wks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2°C
- Humidity (%): 60% +/- 20%
- Air changes (per hr): 10-15 per hour
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 1993-02-08 To: 1993-03-22

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item was suspended whenever required in 1% CMC in bidistilled water. The formulates were kept under stirring and protected from light.


VEHICLE
- Justification for use and choice of vehicle (if other than water): 1% CMC was used to obtain a homogenous suspension of the test item.
- Concentration in vehicle: 4, 20 and 100 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the test item fromulates used were checked on the formulates perpared on Feb. 18, 1993. The results were found to conform to the expected values.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male/2 females
- Length of cohabitation: 16 hrs at a time
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

6th - 15th day of pregnancy

Frequency of treatment:

daily

Duration of test:

Beginning of mating: 1993-02-08
Beginning of dosing: 1993-02-15
Beginning of autopsies: 1993-03-01
Last autopsy day: 1993-03-22

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 40, 200, 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

25 copulated females were allocated to each of the four groups, in order to be sure to have 20 gravid dams per group.

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes, physical appearance, behavior and clinical signs. Any deviation from the norm was noted. During the treatment period the animals were observed for any possible reaction to the treatment.
- Time schedule: daily


MORTALITY / ABORTION: Yes
- Time schedule: daily


BODY WEIGHT: Yes
- Time schedule for examinations: on day 0, 6, 10, 15, 18, 20 of gestation


FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes


WATER CONSUMPTION: No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- gross pathology examination was performed

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: number and sex of viable and dead fetuses, individual fetus weight, individual placental weight

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:

To compare frequencies the heterogeneity test (CHI sqaure 2xN) and Fisher's exact test were applied. The Trend test was also applied. All tests were one-tailed.
All other data were compared using Bartiett's homogeneity of variance (homgeneous-/not homogeneous data), Anova and Dunnett's multiple comp. test (on homogeneous data) and Kruskal-Wallis non parametric Anova and Mann Whitney's U-test (on non homogeneous data).

Historical control data:

included

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
No clinical signs, behavioral changes, death or abortion were noted in any group.

The mean BODY WEIGHT VALUES and the mean BODY WEIGHT GAIN of the 1000 mg/kg/day
group were significantly lower than those of the control group from day 10 of gestation
to the end of pregnancy including the final body weight without gravid uterus.
In the 200 mg/kg/day group the mean body weight values were did not differ significantly from
those of the control group. Body weight gain was significantly lower from day 6 to
10 , from day 6 to 15, from day 6 to 18 of gestation and when the final body weight
excluding gravid uterus was compared to that of day 6 of gestation (1st day of
treatment).
In the 40 mg/kg/day group there was a significantly lower body weight gain only from
day 6 to day 10 of gestation. This lower body weight gain was followed by a recovery
period (significantly higher body weight gain from day 18 to day 20) in such a way no
differences were observed when the treatment period was considered as a whole (6-15 of
gestation). At this dose the effect on body weight, if any, was considered negligible.

The mean daily FOOD CONSUMPTION was significantly lower in the 200 and 1000
mg/kg/day groups during the treatment period (days 6-15) in comparison with the
control group.
No differences were observed in the 40 mg/kg/day treated group.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Effect level:

40 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
OBSERVATION ON GRAVID FEMALES
The number of dams with early resorptions, the frequency per group and the mean values per litter of early resorptions were similar in all experimental groups.
One dam with late resorptions was present in the 40 and 1000 mg/kg/day groups respectively.
No dam with only resorptions was present in any group.
One dam with dead fetuses was present in the 1000 mg/kg/day group; consequently the frequency per group of dead fetuses reached the significant level in this group, being however represented by a normal value.
The frequency per group and the mean values per litter of live fetuses was similar in all experimental groups; the significantly higher mean values per litter of live fetuses observed in the 40 mg/kg/day group compared to the controls was incidental since fell within the range of normal values for this strain of rats.
The post-implantation losses were similar in all experimental groups.
The frequency per group and the mean values per litter of implantations were significantly higher in the 40 mg/kg/day group compared to controls; the significant higher value was reached also in the 200 mg/kg/day group as frequency per group. This finding was considered incidental since not present at the highest dose and since fell within the normal range for this strain of rats.
Despite a significantly lower mean fetal weight at 200 and 1000 mg/kg/day only the value of the 1000 mg/kg/day was out of the historical control values, while that one of the 200 mg/kg/day fell well within the range as well as that one of the 40 mg/kg/day.
Moreover, a higher mean fetal weight was observed in the control group (3.96 g) as compared to the historical controls (3.68 g).
A slightly lower mean litter weight was found in the 1000 mg/kg/day group compared to the controls, without reaching the significant level.
The mean placental weight was significantly lower in the 1000 mg/kg/day group compared to the control group.

MALFORMATIONS, ANOMALIES AND VARIANTS
The following malformed fetuses without any significant increase with the dose were found:
- 40 mg/kg/day: one plurimalformed fetus was found in litter no. 48 showing kinked tail, anasarca, monolateral anotia and ectrodactyly of fore limbs
- 200 mg/kg/day : in litter no. 55 one fetus showed vascular ring and bilateral anophthalmia
- 1000 mg/kg/day: in litter no. 81 one fetus showed flashy tab and in litter no. 100 one showed anasarca and umbilical hernia.
No skeletal malformed fetuses were found in any group.

The following anomalies were found at the external and visceral examination:
- 200 mg/kg/day: in litter no. 54 one fetus showed monolateral subcutaneous hematoma near the hear
- in litter no. 62 one fetus showed monolateral renal papilla
- 1000 mg/kg/day: in litter no. 78 one fetus showed monolateral subcutaneous cranial hematoma
- in litter no. 88one fetus showed monolateral limb hematoma
- in litter no. 95 one fetus showed absence of monolateral renal papilla.
No important differences were found in the skeletal anomalies.

Concerning the skeletal variants the same pattern observed in the mean fetal weight was seen, i.e. a lower number of skeletal variants in the control group (78.05%) and a higher value in the high dose group (98.86%) as compared to the historical controls (87.20%). Both, the low and mid-dose groups, however were well within the historical controls.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

No clinical signs, behavioral changes or death were noted in any group. The body weight gain and the mean daily food consumption were lower in the 200 and 1000 mg/kg/day groups with a clear dose-relationship.In the females given the lowest dose of 40 mg/kg/day the effect on the body weight gain of the first days of treatment was mild and considered negligible.

No embryotoxic effects were found in any group.The few malformations found in the treated groups were not specific and not uncommon in the control population, moreover no dose-related significant increase was found.

In the control group there was a higher weight of fetuses (3.96g ± 0.69) compared to the historical data (3.68g ± 0.33). Also the percentage of skeletal variants was unusually low in the control group (78.05) when compared to the historical data (87.22). Even though at 40 and 200 mg/kg there were statistically lower mean fetal weight and higher percentage of skeletal variants, these values fell well within the range of historical control values and considered devoid of any toxicological relevance. At the high dose (1000 mg/kg/day) the parameters mentioned above were out of the historical data. Based on the above findings and considerations, it may be concluded that the NOEL for dams was 40 mg/kg/day and for fetuses 200 mg/kg/day.

Applicant's summary and conclusion

Conclusions:

Martenal toxicity was expressed as a dose-related lower mean body weight gain and mean daily food consumption at 200 and 1000 mg/kg bw/d.
Mean fetal and mean placental weight were significantly decreased and the percentage of skeletal variants was significantly increased at the high dose.These changes were only observed in the presence of maternal toxicity and not at lower dose levels, where maternal effects were transient (200 mg/kg bw/d) or did not occur (40 mg/kg bw/d). Thus, the changes can be regarded as a non-specific secondary consequence of the effects obeserved in dams and do not indicate specific developmental toxic effects caused by the test item.
The NOEL for maternal toxicity was 40 mg/kg bw/d. For fetuses the NOEL and NOAEL was 200 mg/kg bw/d for developmental toxicity and the NOAEL for teratogenicity was 1000 mg/kg bw/d.

Executive summary:

Teratogenicity was evaluated in groups of 25 pregnant Sprague-Dawley rats treated with TAED by gavage at dose levels of 0, 40, 200 and 1000 mg/kg BW/day from day 6 to 15 of pregnancy (RBM, 1994). The dams were caesarean-sectioned on day 20 of gestation and subjected to post-mortem examination. No clinical signs, behavioral changes, death or abortion were noted in any group. A dose-related lower mean body weight gain and mean daily food consumption was observed at 200 and 1000 mg/kg BW/day. Visceral and skeletal malformations or anomalies were not significantly increased at all dose levels in comparison to the controls. Mean fetal and mean placental weight were significantly decreased and the percentage of skeletal variants was significantly increased at the high dose.

The NOEL for maternal toxicity was 40 mg/kg bw/d (based on dose dependent reduction in body weight/body weight gain and food consumption at 200 and 1000 mg/kg bw/d). For fetuses the NOEL and NOAEL was 200 mg/kg bw/d for developmental toxicity (based on reduced fetal weight at 1000 mg/kg bw/d) and the NOAEL for teratogenicity was 1000 mg/kg bw/d (based on the absence of visceral and skeletal malformations or anomalies at all dose levels).