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Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
See attached justification
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
developmental toxicity
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Justification for Read Across is detailed in the report attached to the IUCLID section 13.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
The purpose of this study was to examine the impact of supplemental target substance on pregnancy course and outcome in rats. From day 0 until day 20 of pregnancy, rats received one of the following diets ad libitum: at 50, 100 or 500 % (i.e. 390, 780 and 3850 mg/kg bw/day), excess over controls (C). One group of rats received a control diet ad libitum and each treatment group had a matched pair-fed group receiving a control diet.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Tyler Laboratories.
- Housing: animals were housed individually in screen bottom cages in a university-approved vivarium.
- Water: water was available ad libitum and changed three times weekly.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 °C
- Photoperiod: 12 hours cycle dark/light was automatically controlled.
Route of administration:
oral: feed
Details on exposure:
BASIC DIET
Rats designated as controls or pair-fed controls were fed a modified American Institute of Nutrition AIN-76
Semipurified diet containing by percent weight:
cornstarch 35.0 % (an increase from 15.0 %)
sucrose 30.0% (a decrease from 50.0 %)
casein (vitamin-free) 20.0 %
corn oil 5.0 %
cellulose fiber 5.0 %
mineral mix (AIN Mineral Mixture) 3.5 %
vitamin mix (AIN Vitamin Mixture 76) 1.0 %
DL-methionine 0. 3%
choline bitartrate 0.2 %.
The modification was the substitution of cornstarch for part of the sucrose. The diet has been found to be satisfactory for reproduction and lactation.
The calorie density of the diet was 3.9 kcal/gram.

PREPARATION OF DIET
The powdered diet was mixed in large batches to minimize variation among groups. The diet was sifted several times, before and after the addition of oil, then separated into portions. Part of the diet was left as is to serve as the basic control diet.
For the experimental diets, test item was added in predetermined amounts to the basic diet. The diets were placed in double plastic bags with air excluded, labeled and frozen until use. The diets were refriger ated between feedings to retard rancidity. Each animal was provided with diet in a glass feeding jar which was washed as necessary.

PAIR-FED CONTROL
Seven pair-fed control animals in each group were fed the control diet, but at the level recorded for the experimental animals that they were paired with.
Details on mating procedure:
Virgin Sprague Dawley rats were time mated at night by the supplier.
Duration of treatment / exposure:
20 days
Dose / conc.:
7 800 mg/kg diet
Dose / conc.:
15 600 mg/kg diet
Dose / conc.:
77 900 mg/kg diet
No. of animals per sex per dose:
Control: 11 animals per group
Pair-fed control: 7 animals per group
Test group: 7 animals per group
Control animals:
yes, plain diet
Details on study design:
PILOT PROJECTS
Before beginning the main protocol, two pilot studies were completed. The intent of the first was to determine if diets supplemented with various levels of test substance would adequately support growth and health in non-pregnant female rats. The second pilot project was designed to determine if the chosen supplementation levels of subsatnce would support pregnant rats so that delivery of viable offspring would be possible.

FIRST PILOT
Forty adult non-pregnant rats were divided into eight groups and fed one of eight experimental diets containing various levels of excess substance (chosen arbitrarily).
Sucrose was reduced so that lysine or tryptophan could be increased. Four of the experimental groups received diets with excess substance (50, 100, 500, 1000). A final group of 8 animals served as controls.
After monitoring body weight, behavior and general appearance for four weeks, animals were sacrificed and evaluated with regard to the normality in appearance of internal organs and amount (or absence) of subcutaneous fat deposits.

SECOND PILOT
16 virgin Sprague Dawley rats were time mated by the supplier. With the appearance of a seminal plug, the following morning was designated as the beginning of day 0. The animals were delivered to the university vivarium on day 0, weighed and randomly assigned to groups.
6 animals were fed the standard control diet and the remaining ten animals were divided into five groups of two rats each. Each of these groups was fed one of the following experimental diets: 50, 100, 500.
Animals were fed ad libitum and food intake and spillage were monitored five times weekly from day 0 through day 20 of gestation.
Body weight was measured three times weekly. General appearance of the animals was carefully noted throughout the course of pregnancy. On day 20, the animals were sacrificed; the uteri and ovaries were removed and the numbers of corpora lutea and fetuses plus resorption sites were compared to verify the
number of resorptions. Placentas were weighed on a top loading balance after removal of membranes and a general examination for external malformations was completed. The fetuses were measured and sexed.
Maternal examinations:
FOOD CONSUMPTION AND COMPOUND INTAKE
Food intake was monitored daily from day 0 until day 20 of gestation.
Fetal examinations:
Approximately three-fourths of the fetuses from each litter were placed in Bouin's solution (75 % saturated picrie acid, 25 % formaldehyde, 5 % Glacial acetic acid) for subsequent examination of any structural variations. The remaining one-fourth of the fetuses from each litter were placed in 95 % ethanol for later processing with alizarin stain which facilitated skeletal examination.
After two weeks' fixation in Bouin's solution, the fetuses were razor sectioned. The head, neck and lower trunk were serially sectioned with a razor blade. After opening the thoracic cavity, sagittal sections were made of the heart. The fetuses stained for approximately 24 hours with alizarin red stain (1 part Alizarin Red S to 20000 parts of 1 % KOH) and subsequently placed in increasing concentrations of glycerin were examined generally for skeletal defects and variatiors in ossification.
The number of ribs and phalanges were counted.
Statistics:
In order to determine if variability in the data resulted mostly from variability within groups or variability between groups, one-way analysis of variance (ANOVA) was used to analyze selected data from the main study.
Duncan's multiple comparison procedure was used to ensure that too many false significant differences were not declared. Significance level was set at 95 % (p = 0.05).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Maternal weight gain values for the test item treatment groups, pair-fed controls and the ad libitum control group do not present significant differences.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The substance treatment groups and their pair-fed controls did not differ significantly from each other or the ad libitum control group in the amount of food consumed.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
The highest supplemented group, 500 %, had a significantly smaller food efficiency ratio than any other group.
Details on results:
FIRST PILOT PROJECT
The animals fed the higher substance supplemented diets were more active when compared to the control animals or those on the lower supplemented diets. Otherwise, all of these animals appeared reasonably healthy with the exception of those provided diets 1000 % who developed scaly tails and thinning hair. Based on these observations, the following supplementation levels were chosen for further investigation: 50, 100 and 500 %.

SECOND PILOT PROJECT
All animals appeared healthy and gained weight steadily throughout the course of pregnancy.
Necropsy revealed viable fetuses; 13 visible resorptions were present among the ten experimental animals and 1 visible resorption among the six control animals. The placentas of all groups appeared normal. These results supported the use of the chosen diets for the next step in the study.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The treated groups did not show any significant differences in the mean number of early resorptions. One pair-fed control group, 50 %, had a significantly higher number of late resorptions than any of the other groups (1.29 + 0.52).
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
No statistically significant differences within treated groups in the mean number of viable fetuses.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Within the treated groups, the pair-fed control group 500 % had significantly larger mean total placental weights than any of the other groups.
Dose descriptor:
NOAEL
Effect level:
15 600 mg/kg diet
Based on:
test mat.
Basis for effect level:
food efficiency
Abnormalities:
effects observed, treatment-related
Localisation:
placenta
Description (incidence and severity):
low incidence and severity
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
The total fetal weight in each litter did not differ significantly among the treated groups, their pair-fed controls or the control group.
500 % had significantly smaller average fetal weights than all other treated groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No significant differences were found in the ratio of males to females within treated groups.
External malformations:
no effects observed
Description (incidence and severity):
No obvious congenital malformations or abnormalities were observed in any of the fetus.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No skeletal variations were found consistently within any group although some variation in ossification of phalanges existed, particularly in the smaller fetuses.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Sectioning of fetuses preserved in Bouin's solution showed that the fetuses were structurally normal; the few exceptions included all fetuses from a litter in group 100 % with larger livers than those of any other group.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The rats receiving the highest amount of substance had fetuses that were significantly shorter than all other groups, with the exception of the pair-fed control 50 %.
Details on embryotoxic / teratogenic effects:
Gross examination of both fresh and preserved fetal specimens revealed no evidence that any level of substance supplementation.
If defects were present that were either too small to see through a dissecting microscope or characterized as biochemical in nature, these were not pursued in the present study. Therefore, although no major malformations were observed, it should not be concluded that test item produce no embryopathic effects when fed in large amounts. Since there was no trend toward increasing structural variations and/or fetal resorptions or decreasing litter size in higher supplemented groups, there would appear to be limited cause for alarm at this point about major fetal damage produced by maternal substance supplementation.

Food consumption and maternal weight gain are both continuous variables that provide an early indication of a substance's toxicity. While there were no significant differences in the amount of food consumed within the treated groups, the highest supplemented group had significantly decreased maternal weight gains.
With reduction in food intake and/or food efficiency in the highly supplemented animals, it is not surprising that total fetal weight and individual fetal size was affected.
The treated groups showed this trend in average, but not total, fetal weight. Because 500 % had a significantly decreased average fetal weight yet did not consume significantly less diet, it appears that the decrease in fetal weight was related to reduced food efficiency.
Dose descriptor:
NOAEL
Effect level:
15 600 mg/kg diet
Based on:
test mat.
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
effects observed, treatment-related
Localisation:
other: not specified
Description (incidence and severity):
low incidence and severity
Developmental effects observed:
yes
Lowest effective dose / conc.:
77 900 mg/kg diet
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
not specified
Relevant for humans:
not specified
Conclusions:
The NOAEL for maternal and foetal toxicity is equal to 15.6 g/kg diet.
Executive summary:

Method

The purpose of this study was to examine the impact of supplemental TS on pregnancy course and outcome in rats. From day 0 until day 20 of pregnancy, rats received one of the following diets ad libitum: TS at 50, 100 or 500 % excess over controls (C). One group of rats received a control diet ad libitum and each treatment group had a matched pair-fed group receiving a control diet.

Observations

No congenital malformations were observed in any of the fetuses. Lower supplementation levels of substance do not appear to have any negative impact on maternal weight gain, fetal weight or other fetal parameters that may signify toxicity. Diets supplemented with a high amount of substance (500 % excess) appear to be associated with a lower than expected maternal weight gain and a significantly reduced fetal weight.

Results

The NOAEL for maternal and foetal toxicity is equal to 15.6 g/kg diet.

Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
developmental toxicity
Remarks:
screening for reproductive / developmental toxicity
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Justification for Read Across is detailed in the report attached to the IUCLID section 13.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other:
Version / remarks:
OECD combined repeated dose and reproductive/developmental toxicity screening test
Principles of method if other than guideline:
combined repeated dose and reproductive/developmental toxicity screening test
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Route of administration:
oral: gavage
Details on mating procedure:
14 days
Duration of treatment / exposure:
Males: for 52 days
Females: from 14 days before mating to day 4 of lactation
Frequency of treatment:
Daily
Duration of test:
Males: for 53 days
Females: from 43 to 47 days
Dose / conc.:
0 mg/kg bw/day
Remarks:
vehicle
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
12 animals/sex/dose
Control animals:
yes, concurrent vehicle
Number of abortions:
no effects observed
Description (incidence and severity):
There were no intergroup differences in the delivery index, implantation index, parturition index of neonates on day 4 of lactation.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no abnormality in the implantation sites, delivered offspring and live delivered offspring showed almost the same values.
Changes in pregnancy duration:
effects observed, treatment-related
Description (incidence and severity):
The gestation period was significantly shortened in the 100 and 1000 mg/kg groups compared with the control group. There was no abnormality in the conditions of parturition.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: n.a.
Abnormalities:
effects observed, treatment-related
Localisation:
other: gestation period
Description (incidence and severity):
low incidence and severity
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Body weight during lactation period was significantly low on days 0 and 4 of lactation in males and day 4 in females in the 100 mg/kg group and on day 4 in males in the 300 mg/kg group, which was the change not associated with the dose.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no abnormality in the delivered offspring and live delivered offspring showed almost the same values.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no intergroup differences in the sex ratio and viability index of neonates on day 4 of lactation.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In the external examination in neonates, anophthalmia and polydactyly were observed in 1 animal each in the 300 mg/kg group. These anomalies are considered to be spontaneous, because the incidences of these anomalies were extremely low and these are of types seen in historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No abnormal findings considered to be attributable to administration of this compound were observed in dead pups during lactation and pups at scheduled sacrifice.

In the necropsy on day 4 of lactation, red patches on the plantar were observed in 15 males and 13 females in the 100 mg/kg group, and the number of occurrences significantly increased in both males and females compared with the control group. However, this finding occurred in litters in 2 broods in both males and females. In addition, dilation of the ureter was observed in 3, 4 and 3 males and 5, 1 and 2 females in the 100, 300 and 1000 mg/kg groups, respectively, and the number of occurrences significantly increased in the male 100 mg/kg group. However, dilation of the ureter in the female 100 mg/kg group was observed in litters in 4 of 5 animals.
Other findings included thymic remnant in the neck in 3, 1, 2 and 3 male animals in the control, 100, 300 and 1000 mg/kg groups and in 1, 2 and 2 female animals in the control, 100 and 300 mg/kg groups, nodes in the liver in 1 animal each in the male and female 1000 mg/kg groups, white patches in the liver in 1 animal in the male 100 mg/kg group, pyelectasia in 3 animals each in the male 100, 300 and 1000 mg/kg groups and in 2 and 1 animal in the female 300 and 1000 mg/kg groups, anophthalmia in 1 animal in the female 300 mg/kg group, cysts in the hindlimbs in 1 animal in the female 100 mg/kg group and polydactyly in 1 animal in the female 300 mg/kg group.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: n.a.
Abnormalities:
effects observed, non-treatment-related
Localisation:
other: visceral and external malformation, body weight
Description (incidence and severity):
low incidence and severity
Developmental effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
not specified
Relevant for humans:
not specified
Conclusions:
The NOAEL for reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day.
Executive summary:

Method

The study was conducted according to an OECD combined repeated dose toxicity study with the reproduction/developmental toxicity screening test guideline [TG 422] [MHLW, Japan: 2002] under GLP.

During the test Crj: CD (SD) IGS rats (12 animals/sex/dose) were administered with the test article by gavage at doses of 0 (vehicle: distilled water), 100, 300, or 1000 mg/kg bw/day. Males were dosed for 52 days from day 14 before mating and females were dosed from day 14 before mating to day 4 of lactation throughout the mating and pregnancy period.

Observations

No compound-related effects on the estrous cycle, copulation index, fertility index, gestation length, gestation index, number of corpora lutea, or number of implantation sites were found in dams. No compoundrelated effects on the number, sex ratio, or viability were observed in pups on days 0 and 4 of lactation. Anophthalmia and polydactyly were observed in one pup at 300 mg/kg bw/day. These anomalies are considered to be spontaneous, because the incidences of these anomalies were extremely low and these are of types seen in historical control data. There were no compound-related changes in body weights of pups. No abnormal findings considered to be attributable to administration of this compound were observed in dead pups during lactation and pups at scheduled sacrifice.

Results

Based on these findings, the NOAEL of test item for reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day in rats.

Data source

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Test material

1
Chemical structure
Reference substance name:
trisodium (2S)-2,6-bis(3-carboxylatopropanamido)hexanoate
EC Number:
948-778-9
Molecular formula:
C14O8H19N2Na3
IUPAC Name:
trisodium (2S)-2,6-bis(3-carboxylatopropanamido)hexanoate
Test material form:
liquid
Details on test material:
Active ingredient (%): 48.9% dry substance
Purity (%): 95.50 %
Stability: 5 years
Sterilization: None
Solubility: Soluble in water
Storage: Room Temperature

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
15 600 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
food efficiency

Results (fetuses)

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
15 600 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
fetal/pup body weight changes

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

The NOAEL for maternal and foetal toxicity is equal to 15.6 g/kg diet.

The NOAEL for reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day.

Applicant's summary and conclusion

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