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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1979
Report date:
1979
Reference Type:
publication
Title:
Teratology and multigeneration reproduction studies with maleic anhydride in rats
Author:
Short R.D. et al
Year:
1986
Bibliographic source:
Fundamental and Applied Toxicology 7, 359-66

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(administered volume in the control and high dose group is higher (1.4 ml/100 g bw) than the advised maximum volume in the guideline (0.4 ml/100 g bw))
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Maleic anhydride
EC Number:
203-571-6
EC Name:
Maleic anhydride
Cas Number:
108-31-6
Molecular formula:
C4H2O3
IUPAC Name:
furan-2,5-dione
Specific details on test material used for the study:
- Appearance: White briquettes
- Purity: > 99%.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING:
Briquettes of maleic anhydride were finely ground with a mortar and pestle and suspended in corn oil with the aid of a tissue homogenizer. All doses were prepared daily in corn oil in order to minimize problems with stability. A 1% (w:v) concentration was
used to administer all doses.

Test animals

Species:
rat
Strain:
other: Charles River CD rats
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Wilmington, Mass.)
- Age at study initiation: approximately 12 weeks
- Weight at study initiation: 242-244 g
- Fasting period before study: not applicable
- Housing: Rats were individually housed, except during mating and lactation, in wire mesh cages or plastic cages with corn-cob bedding. One male and one female were housed together for mating.
- Diet (e.g. ad libitum): Purina Rodent Chow (Ralston-Purina, St. Louis, MO.)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: All rats were acclimated to the laboratory for at least 10 days prior to initiating a study.

ENVIRONMENTAL CONDITIONS
All animals were maintained in environmentally controlled rooms
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Briquettes of maleic anhydride were finely ground with a mortar and pestle and suspended in corn oil with the aid of a tissue homogenizer. All doses were prepared daily in corn oil in order to minimize problems with stability. A 1% (w:v) concentration was used to administer all doses.

VEHICLE
- Concentration in vehicle: 1% (w/v)
- Amount of vehicle (if gavage): 0.3-1.4 ml/100g bw
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
from gestation day 6 through day 15
Frequency of treatment:
daily
Duration of test:
day 20 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
low dose
Dose / conc.:
90 mg/kg bw/day (nominal)
Remarks:
mid dose
Dose / conc.:
140 mg/kg bw/day (nominal)
Remarks:
high dose
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 6, 9, 12, 15, and 20
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: fetal swellings, number of viable and nonviable fetuses
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [1/3 per litter ]
- Skeletal examinations: Yes: [2/3 per litter ]
Statistics:
Several different statistical methods were used to compare measurements made on test animals to the corresponding values determined for controls. The methods and the measurements to which they were applied are analyis of variance and Dunnett’s test (Steel and Tonie, 1960) for adult body weights, litter size, and pup body weights; Fisher’s exact probability test (Siegel, 1956) for mortality and fertility data; Mann-Whitney U test (Siegel, 1956) for fetal body weights; and x2 test with Yates’ correction or Fisher’s exact probability test (Siegel, 1956) for litters with anomalies. In all instances, p < 0.05 was selected as the level of significance.
Indices:
Mean implants/dam, viable fetuses/dam, resorptions/dam were calculated.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
The general appearance and behavior of rats were not altered by treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
While one adult died in each of the experimental groups, the overall survival in these groups was 96%.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Dams in the experimental groups either failed to gain weight or lost weight between Days 6 and 9 of gestation. However, this effect was reversible, and there were no statistically significant effects on body weight at any of the times examined.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Dams from all test groups produced normal-sized litters, and there was no evidence of postimplantation loss
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The number of resorptions/dam was not altered after treatment.
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The number of viable fetuses/dam was comparable between treatment groups and control.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
There was a minimal reduction in the ratio of pregnant animals to the number of treated animals between control (23/25) and the low (24/25), mid (20/25) and high dose (21/25) treated animals.
Other effects:
not examined
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Respiratory involvement and red nasal discharge were observed in all dosage groups. The incidence of these was higher in the treated groups, though not in a dose-related pattern. No treatment-related deaths (one rat in each dosage group died during the first part of treatment; the cause of death was not determined) nor abnormal behavior was observed in any of the maleic anhydride treated groups. Mean body weight gain was reduced in the 30 mg/kg/day dosage group for the first three days of treatment. There was a slight mean body weight loss in the 90 and 140 mg/kg/day dosage groups for the first three days of treatment. These reductions in weight gains resulted in reduced mean body weight gains over the entire treatment period in all treatment groups compared to the control (however, mean weight of all groups was within 5% of control on days 15 and 20. No biologically meaningful differences in the mean number of viable fetuses, implantations, post implantation losses, corpora lutea, or in the male to female sex ratio between any of the maleic anhydride treated groups and the control group. The general appearance and behavior of rats were not affected by treatment.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
140 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Only slight effects on the body weight in the 90 and 140 mg/kg treatment groups. These effect were reversible, and there were no statistically significant effects on body weight at any of the times examined.

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Compared with concurrent controls, fetal body weights were slightly reduced for all test groups, but the reductions were statistically significant only in the low- and high-dose groups, However, this is not considered to be compound-related, because fetal weights for concurrent control and all treated groups were slightly greater than the values for historical controls.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of viable fetuses/dam was not affected by treatment.
Changes in sex ratio:
not examined
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Dams from all test groups produced normal-sized litters.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations were observed in one fetus (one litter) from the control group, two fetuses (two litters) from the low-dose group, and three fetuses (three litters) from the high-dose group. Since each malformation was a single occurrence and the malformations differed among the various groups, there was no evidence of a dose-related increase in any specific malformation.
The fetal variations were comparable both in type and frequency in the control and treated groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations were observed in one fetus (one litter) from the control group, two fetuses (two litters) from the low-dose group, and three fetuses (three litters) from the high-dose group. Since each malformation was a single occurrence and the malformations differed among the various groups, there was no evidence of a dose-related increase in any specific malformation.
The fetal variations were comparable both in type and frequency in the control and treated groups.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Malformations were observed in one fetus (one litter) from the control group, two fetuses (two litters) from the low-dose group, and three fetuses (three litters) from the high-dose group. Since each malformation was a single occurrence and the malformations differed among the various groups, there was no evidence of a dose-related increase in any specific malformation.
The fetal variations were comparable both in type and frequency in the control and treated groups.
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Mean fetal body weights were lower in the treatment groups than in the control group. This was not considered compound related due to the unusually high mean fetal body weight in this concurrent control group (mean: 4 g). External evaluation, internal examination, and skeletal observations of fetuses from all three treatment groups showed no anomalies in fetal development which could be attributed to maleic anhydride (slight increase in fetal malformations in the 30 (2/23 litters) and 140 mg/kg/day dosage group (3/21 litters) when compared to the control group (1/23 litters) is considered due to random occurrence due to the variety of abnomalities observed).

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
140 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEL
Effect level:
140 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In developmental toxicity study (equivalent to OECD 414) maleic anhydride (>99% purity) was administered to 25 female Wistar rats/dose in corn oil at dose levels of 0, 30, 90 or 140 mg/kg bw/day from days 6 through 15 of gestation. On day 20 of gestation the animals were sacrificed. Based on the results, the NOAEL for both maternal toxicity and fetal toxicity of maleic anhydride in this study is considered to be to 140 mg/kg bw/day.
Executive summary:

In a developmental toxicity study (equivalent to OECD 414), CD rats (25 mated females/group) were treated orally with 30, 90, or 140 mg/kg/day of maleic anhydride from Day 6 through Day 15 of gestation. Mated females in the control group were treated in a similar manner with 14 mL/kg of corn oil. All females were sacrificed with carbon dioxide on Day 20 of gestation, and the fetuses were delivered by cesarean section. All fetuses were weighed and examined for external abnormalities. Approximately one-third of the fetuses were placed in Bouin’s fixative and examined for soft tissue abnormalities . The remaining fetuses were fixed in alcohol, cleared with potassium hydroxide, stained with Alizarin Red S and examined for skeletal abnormalities.

There were no changes in appearance or behavior attributable to treatment with maleic Anhydride at dosage levels of 30, 90 and 140 mg/kg bw/day. Respiratory involvement and red nasal discharge were observed in all dosage groups. The incidence of these was higher in the treated groups, though not in a dose-related pattern. One rat in each dosage group dl.dd during the first part of treatment. The cause of death was not determined. There were reduced mean maternal body weight gains during the first three days of treatment in the 30 mg/kg bw/day dosage group when compared to the control group. In the 90 and 140 mg/kg bw/day dosage groups mean body weight losses were observed for the first 3 days of treatment. These reductions in weight gains resulted in reduced mean weight gains over the entire treatment period in all treatment groups when compared to the control group. There were no biologically meaningful differences in the mean number of viable fetuses, implantations, post implantation losses, corpora lutea or in the male to female sex ratio between any of the maleic anhydride treated groups and the control group. Based on the results, the NOAEL for maternal toxicity is considered to be 140mg/kg bw/day.

There were no treatment-related effects in developmental parameters. Therefore, based on the result the developmental NOAEL is also considered to be 140 mg/kg bw/day.

 

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rats.

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