[No public or meaningful name is available]

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June-Nov 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was performed after an ECHA decision was received on a testing proposal.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

453-140-3

EC Name:

-

Cas Number:

667465-46-5

Molecular formula:

C20H38N6O13 (not dissociated) C20H44N6O13 (dissociated in water)

IUPAC Name:

3,6,9-tris(2-{[(2-hydroxyethyl)amino]oxy}-2-oxoethyl)-3,6,9-triazaundecan-1,11-dioic acid

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Water

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): DHX2
- Description: clear colourless to light yellow liquid
- Storage condition of test material: at room temperature in the dark in well-sealed container

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 12 weeks
- Weight at study initiation: 200-244 g
- Fasting period before study: no
- Housing:
Pre-mating: During acclimatization, females were housed in groups of maximum 5 animals/cage in Macrolon plastic cages.
Mating: Females were placed in the males’s homecage in Macrolon plastic cages. During the weekend, mating procedures were suspended and the females were housed in groups of maximum 5 animals/cage in Macrolon plastic cages.
Post-coitum: Females were individually housed in Macrolon plastic cages.
General: Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied.
- Diet: free access to pelleted rodent diet (SM R/M-Z From SSNIFF Spezialdiaten GmbH, Soest, Germany)
- Water: free access to tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 July 2014 To: 20 August 2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test substance. A correction factor of 1.2628 was applied for the purity/composition of the test substance, because in previous studies a different batch (RH020604) was used that had a purity of 61.5%. For comparison, a correction factor of 1.2628 was applied to correct the current purity (approximately 48.7%) to the purity in the previous batch.

VEHICLE
- Amount of vehicle: 5ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Details on mating procedure:

Mating procedure: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the female was separated from the male. When sufficient mated females had been obtained for each dose group, the surplus females were removed from the study. During the weekend, mating procedures were suspended.
- If cohoused:
- M/F ratio per cage: 1:1

Duration of treatment / exposure:

from day 6 to 19 post-coitum, inclusive

Frequency of treatment:

daily

Duration of test:

from day 6 to 19 post-coitum, inclusive

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results of the dose range finding study. In this study no mortality, no clinical signs, no effect on body weight, food consumption, no macroscopic abnormalities were noted for dams. No effect on litter size, viable/dead foetuses, early/late resorptions, sex ratio, foetal weight, or external abnormalities was observed.

Examinations

Maternal examinations:

CLINICAL OBSERVATIONS:
- Time schedule: daily; twice daily for mortality/viability

BODY WEIGHT:
- Time schedule for examinations: days 0, 3, 6, 9, 12, 15, 17 and 20 post-coitum

FOOD CONSUMPTION: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 20
- Organs examined: according to OECD 414 (2001): all females (including the one that aborted on day 19) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs

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:

- Number and distribution of live and dead foetuses, weight and sex of each foetus.
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, all per litter
- Skeletal examinations: Yes, all per litter for the control and highest dose group
- Head examinations: Yes, half per litter

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

Pre-implantation loss (%): (number of corpora lutea - number of implantation sites)/ number of corpora lutea x 100
Post-implantation loss (%): (number of implantation sites - number of live foetuses)/number of implantation sites x 100
Viable foetuses affected/litter (%): (number of viable foetuses affected/litter)/ (number of viable foetuses/litter) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were noted with treatment with DHX2 up to 1000 mg/kg bw/day.
Alopecia was noted for one control female on the last day of the observation period. As this female
was treated with the vehicle (water) alone, this finding was not related to treatment with DHX2.

Mortality:

no mortality observed

Description (incidence):

One control female was euthanized on day 19 post-coitum due to abortion.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicologically significant changes in body weights and body weight gain were noted. Likewise, corrected terminal maternal body weight and corrected body weight gain were unaffected by treatment up to 1000 mg/kg bw/day.
The significantly higher mean body weight recorded at 100 mg/kg bw/day on Day 20 post-coitum was considered not to be toxicologically relevant. Body weights at this low dose level were higher
compared to controls throughout the entire observation period. They remained within the normal range of biological variation and no dose-related distribution could be established.

The mean results are attached below in tabular form.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No toxicologically significant changes in food consumption before or after correction for body weight were noted up to 1000 mg/kg bw/day.
The slightly lower relative food consumption from Days 17-20 post-coitum was not considered a sign of toxicity as the difference compared to controls was only slight and values remained within the normal range of biological variation.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
The only finding in this study was a diaphragmatic hernia on the left median liver lobe of a female in the 100 mg/kg w/day group. This abnormality was considered to be a physiological defect rather than treatment-related.

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

One control female had an abortion on day 19, delivering 11 live foetuses; she had 11 implantation sites.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

At the highest dose level, there were two litters with 6 early resorptions each. These
early resorptions were seen next to 8 and 6 live fetuses, respectively. Furthermore, also in the control group there was one litter with 4 early resorptions, and mean litter proportions of early resorptions in the high dose group fell within the normal range of biological variation (9.1% versus max: 11.2% per litter). Therefore, this finding was not considered to be related to treatment.

Dead fetuses:

no effects observed

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal body weight was unaffected by treatment up to 1000 mg/kg bw/day.
For one litter each in the 100 and 1000 mg/kg bw/day groups unrealistic high fetal weights were recorded. These fetal weights were excluded from group mean values.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Litter sizes were unaffected by treatment with DHX2 up to 1000 mg/kg bw/day.
Mean litter sizes were 10.8, 11.7, 11.0 and 11.4 fetuses per group for the control, 100, 300 and
1000 mg/kg bw/day groups, respectively.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on the fetal sex ratio in any group.

External malformations:

no effects observed

Description (incidence and severity):

There were no treatment related effects on external morphology up to 1000 mg/kg bw/day.
One fetus in the 1000 mg/kg bw/day group had an omphalocele in the abdominal wall (protrusion of several loops of intestine through a defect in the abdominal wall at the umbilicus; not covered by a thin, translucent sac). At the isolated incidence, it was considered a chance finding rather than to be caused by the test substance. No external variations were observed in fetuses of any group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal examination did not reveal any toxicologically relevant findings up to 1000 mg/kg bw/day. Malformations of the skeleton were noted in 3(2) fetuses(litters) in the high dose group. Two fetuses had vertebral anomaly (with or without associated rib anomaly). In the latter
litter another fetus was found with schisis of sternebra # 2. Neither of these findings was
considered to be treatment-related as they occurred infrequently and the mean litter proportion
remained within the available historical control data range (0.7% and 0.4% versus maximum values of 0.8%).
Neither of the skeletal variations seen in DHX2 treated groups was considered to be treatment-related as they occurred infrequently, were observed at lower frequencies than in the concurrent control group, at frequencies that were within or only slightly above the range of available historical control data, or were limited to control fetuses.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no findings for visceral morphology that were considered to be toxicologically relevant. Visceral malformations included abnormal lobation of the lungs and liver together with situs inversus for one fetus at 300 mg/kg bw/day, absent liver lobe for another fetus from the same litter, and situs inversus for one fetus at 1000 mg/kg bw/day. As these were isolated observations that occurred in the absence of a dose-related incidence, they were considered to be chance findings and thus not related to treatment.
Visceral variations seen in DHX2 treated groups were not considered to be treatment related as they occurred infrequently and/or in the absence of a dose-related incidence trend, were observed either at lower frequencies as in the concurrent control group or at frequencies that were within the range of available historical control data.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter sizes were unaffected by treatment: 10.8, 11.7, 11.0 and 11.4. The number of viable or dead foetuses, and early or late resorptions were all within the normal range of biological variation. At the highest dose level, there were two litters with 6 early resorptions each. These early resorptions were seen next to 8 and 6 live fetuses, respectively. Furthermore, also in the control group there was one litter with 4 early resorptions, and mean litter proportions of early resorptions in the high dose group fell within the normal range of biological variation (9.1% versus max: 11.2% per litter). Therefore, this finding was not considered to be related to treatment.
No treatment-related effects were noted on sex ratio and foetal body weight.
No treatment-related effects were seen on external, visceral and skeletal malformations and variations. One foetus had an omphalocele in the abdominal wall, which was considered to be chance finding and not related to treatment.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were seen up to and including the highest dose level (1000 mg/kg bw/day)

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Formulation analysis: The concentrations analysed in the formulations at 100, 300 and 1000 mg/kg bw/d were in agreement with target concentrations (i.e. mean accuracies between 92.4 -94.7%). No test substance was detected in the control formulation.

The formulations at 100 and 1000 mg/kg bw/d were homogeneous (i.e. coefficient of variation 0.9 -3.8%).

Formulations at the entire range were stable when stored at room temperature under normal light conditions for at least 5 hours. The long term storage samples were stable at ≤ -70°C for at least 353 hours.

Applicant's summary and conclusion

Conclusions:

In a developmental toxicity study in rats performed with DHX2 according to OECD 414 and GLP, the NOAEL for maternal and developmental toxicity was shown to be at least 1000 mg/kg bw/d.

Executive summary:

A developmental toxicity study was performed according to OECD 414 and GLP administering 0, 100, 300 and 1000 mg/kg bw/d DHX2 to rats by gavage from days 6 to 19 post-coitum. No treatment-related effects on clinical signs, body weight, food consumption and macroscopy in dams was noted. No treatment-related effect on the litter size, number of viable and dead foetuses, early and late resorptions, sex ratio and foetal weight were seen. No treatment-related effects were observed on external, visceral and skeletal malformations and variations. The NOAEL for maternal and developmental toxicity was determined to be at least 1000 mg/kg bw/d based on the absence of adverse effects at any dose level.