Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 April 2017 - 27 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Justification for Test System and Number of Animals:

The New Zealand White rabbit was chosen as the animal model for this study as it is an accepted non-rodent species for developmental toxicity testing by regulatory agencies. Charles River Den Bosch has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants.
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
This type of study plan was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of Charles River Den Bosch as required by the Dutch Act on Animal Experimentation (February 1997).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Test material form:
liquid: viscous
Specific details on test material used for the study:
- Storage condition of test material: At room temperature protected from light
- Stability of the test substance in the solvent/vehicle 0.5% CMC + 1.25% Tween 80: Stability for at least 6 hours at room temperature, 8 days in the refrigerator and 3 weeks in freezer is confirmed over the concentration range 1 mg/g (1 mg/mL) to 469 mg/g (500 mg/mL), Test Facility Study No.517011.

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 17-19 weeks
- Weight at study initiation: between 2965 and 4307 g
- Fasting period before study: no
- Housing: individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles
- Diet: Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) was provided ad libitum throughout the study, except during designated procedures. In addition, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were provided during the study period.
- Water: tap water, ad libitum
- Acclimation period: at least 5 days prior to treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 55 to 97%
- Air changes (per hr): Ten or greater
- Photoperiod (hrs dark / hrs light):12 hour light/12 hour dark cycle

IN-LIFE DATES: From: 5 June 2017(first delivery of mated females) To: 6 July 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous carboxymethyl cellulose with 1.25% Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
When solidified, the test item was heated to a maximum of 75±5°C for around 2 hours to reach the liquid state and obtain visual homogeneity. Test item dosing formulations (w/w) were homogenized to visually acceptable levels. The dosing formulations were prepared daily and dosed within 6 hours after completion of the preparation of the test item. Formulations were heated to a maximum of 75±5°C for maximally 25±5 minutes to obtain visual homogeneity. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment were made for specific gravity of the test item. No correction was made for the purity/composition of the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed on two days during the treatment phase (12 and 26 June 2017) by using a validated analytical procedure. Samples of formulation were analyzed for homogeneity (lowest and highest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations within or equal to ± 15% for suspensions of target concentrations. Homogeneity was demonstrated if the coefficient of variation of concentrations was smaller or equal to 10%.
Details on mating procedure:
- Impregnation procedure: rabbits were purchased timed pregnant, arriving at test facility on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
Duration of treatment / exposure:
From Days 6 to 28 post-coitum, inclusive
Frequency of treatment:
Daily
Duration of test:
Necropsy on Day 29 post-coitum
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
concurrent vehicle control
Dose / conc.:
8 mg/kg bw/day
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
75 mg/kg bw/day
No. of animals per sex per dose:
22 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of the dose range finder. In the dose-range finder pregnant females (n=6/group) were dosed at 0, 40, 75 or 100 mg/kg bw/d from day 6 to 28 post-coitum, inclusive. At 100 mg/kg bw/day, 1 female was sacrificed in extremis on Day 24 post-coitum due to a reduced food consumption, no body weight gain and the persistence of piloerection and lean appearance, in addition to severely reduced feces production, pale appearance, alopecia and red fluid on manure tray. Except for alopecia, no macroscopic alterations were noted at necropsy.
A dose-dependent increased incidence and severity of reduced feces production was observed (up to a severe degree at 75 and 100 mg/kg bw/day). Piloerection was observed for two females at 75 mg/kg bw/day and for all females at 100 mg/kg bw/day. Pale appearance was noted for one female at 75 mg/kg bw/day and two females at 100 mg/kg bw/day, which had a lean appearance in addition.
A slight body weight loss and reduced body weight and body weight gain was observed at 75 and 100 mg/kg bw/day. Reduced food consumption was observed in all treatment groups. Macroscopic observations at necropsy did not reveal any alterations that were considered to be toxicologically relevant.
All females were pregnant and had litters with viable fetuses. No treatment related changes for the number of post-implantation loss were observed. Litter sizes, sex ratio and fetal body weights were within normal limits for all groups.External examination of the fetuses did not show any abnormalities.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, beginning on Day 3 post-coitum and lasting up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 3, 6, 9, 13, 16, 20, 23, 26 and 29 post-coitum

FOOD CONSUMPTION: Yes
- Food consumption was quantitatively measured for Days 3-6, 6-9, 9-13, 13-16, 16-20, 20-23, 23-26 and 26-29 post-coitum.

WATER CONSUMPTION: Yes
- Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29 or within 24 hours of early delivery

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
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and reported at the 1% and 5% levels.
Group means were calculated for continuous data and medians were calculated for discrete data (scores). Test statistics were calculated on the basis of exact values for means and pooled variances.
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test. 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.
Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant. 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 fetuses)/number of implantation sites) x 100
Viable fetuses affected/litter (%) = ((number of viable fetuses affected/litter)/(number of viable fetuses/litter)) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Piloerection and a lean appearance were observed incidentally in individual females in all dose groups, including controls. In addition, a hunched posture was noted on a few occasions in one high dose female. At the incidence observed, these signs were considered to be unrelated to treatment. Reduced feces and/or absence of feces was noted for almost all females across the groups, including controls. In the absence of a dose-relationship, this finding was not considered to represent a sign of toxicity. A slightly shallow respiration was observed for one control female on post-coitum Days 14 and 15. This animal was therefore preventively not dosed on Day 14. In addition, a necrotic wound in the throat area was observed for this animal from post-coitum Day 24 onwards, which was considered to be background finding. No other relevant signs were noted for this control animal.
All other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female at 8 mg/kg bw/day and one female at 75 mg/kg bw/day were sacrificed in extremis on post-coitum Day 20 and 16, respectively, due to their detoriating physical condition. Prior to their early sacrifice, these females had completely stopped eating for a period of 10 consecutive days, with consequent body weight losses of 10-13%, when compared to start of treatment. In addition, up to severely reduced feces production along with piloerection (low dose female only) and a lean appearance were noted for these animals prior to sacrifice. No abnormalities were noted at necropsy. In the absence of a dose-relationship these decedents were considered to have occurred by chance and not to be related to treatment.
In addition, two females at 8 mg/kg bw/day died on post-coitum Day 26 directly after dosing, due to complications of the oral gavage procedure. No abnormal signs, food intake values or body weights were noted for these animals prior to their moribund state. Except for red fluid around the mouth and nose and in the trachea noted for both females, no macroscopic abnormalities were observed at necropsy. Complications during the oral gavage procedures are incidentally observed in pregnant rabbits and in the absence of any other relevant signs, these decedents were considered to be unrelated to treatment with the test item.
One control female and one female at 8 mg/kg bw/day were sacrificed on post-coitum Day 21 and 25, respectively, as they delivered their offspring early.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly (but statistically significantly) lower body weight gain was noted for females at 75 mg/kg bw/day on post-coitum Days 9 and 13, when compared to controls. On post-coitum Day 9, almost all females at 75 mg/kg bw/day showed a minimal body weight loss, with an average weight loss of 1%, whereas in the other groups including controls, this was noted for incidental females only.
In addition, body weight gain corrected for gravid uterus was also slightly lower for these high dose females: Mean corrected body weight gain was -202 gram (-5%), compared to -156 gram (-4%) in controls. These changes were only minimal and remained within the normal range .
Body weights, body weight gain and weight gain corrected for gravid uterus of animals treated at 8 and 25 mg/kg bw/day remained in the same range as controls over the study period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before and after correction for body weight was statistically significantly lower over post-coitum Days 6-9 and 9-13 for females at 75 mg/kg bw/day, when compared to controls. Average relative food consumptions were 41% and 35% lower than controls.
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded for females at 8 and 25 mg/kg bw/day.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Incidental findings were noted among control and treated animals, including wounds, foci located on the lungs or stomach, and cysts on the oviducts. These are occasionally seen among rabbits used in these types of study and at the incidences observed in absence of a dose-relationship, they were considered to be of no toxicological significance.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
effects observed, non-treatment-related
Description (incidence and severity):
One control female and one female at 8 mg/kg bw/day delivered their offspring early on Day 21 and 25 post-coitum, respectively. Prior to early delivery, piloerection and reduced food intake with slight body weight loss (6%) were noted for the control female. For the low dose female, only slightly reduced food intake was noted prior to early delivery. No abnormalities were noted at necropsy for either female, or their fetuses. In the absence of a dose-relationship and at the incidence observed, these early deliveries were not considered to be related to treatment with the test item.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
At scheduled necropsy, there were 17, 14, 19 and 16 pregnant females which had litters with viable fetuses in respectively the control, 8, 25 and 75 mg/kg bw/day group. In total, including the premature decedents which were all pregnant, there were 18, 18, 19 and 17 pregnant females in respectively the control, 8, 25 and 75 mg/kg bw/day group.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
In total, 16 females across the dose groups were not pregnant, based on no visually observed implantation sites at necropsy: 4, 4, 3 and 5 at 0, 8, 25 and 75 mg/kg bw/day, respectively. Despite the high number of non-pregnancy, this was considered to be unrelated to treatment, as treatment started on post-coitum Day 6.
The numbers of corpora lutea and implantation sites, and pre-implantation loss in the treatment groups remained in the same range as controls.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Remarks on result:
other: no treatment-related adverse effects observed at the highest dose level of 75 mg/kg bw/day

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean combined (male and female) fetal body weights were 38.3, 42.0, 37.0 and 38.9 grams for the control, 8, 25 and 75 mg/kg bw/day groups, respectively. The relatively high value at 8 mg/kg bw/day, reaching statistical significance for male fetal body weights, was mainly caused by one litter, having an average fetal body weight of 54.2 gram. As this was observed for one single litter in the lowest dose group, this was considered to have occurred by chance and not to be related to treatment.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
One fetus at 8 mg/kg bw/day was found dead at scheduled necropsy.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 75 mg/kg bw/day. Mean sex ratios (males:females) were 49:51, 46:54, 51:49 and 54:46 for the control, 8, 25 and 75 mg/kg bw/day groups, respectively.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on litter size of any group. Mean litter sizes were 9.6, 8.9, 9.3 and 8.7 viable fetuses/litter for the control, 8, 25 and 75 mg/kg bw/day groups, respectively. The mean litter incidences of viable fetuses, early and late resorptions and post-implantation loss were slightly lower in the 75 mg/kg bw/day group, which was caused by two litters. As the changes were only minimal and not statistically significant and as the incidences of the other 20 litters of the 75 mg/kg bw/day group remained in the same range as controls, this was considered not to be related to treatment with the test item.
External malformations:
no effects observed
Description (incidence and severity):
The numbers of fetuses (litters) available for fetal morphological examination were 164 (17), 124 (14), 176 (19) and 139 (16) in the control, 8, 25 and 75 mg/kg bw/day groups, respectively.
There were no external malformations and variations seen for any fetus in any group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment up to 75 mg/kg bw/d. Skeletal malformations in fetuses were a rib anomaly in one fetus each at 25 and 75 mg/kg bw/day and a vertebral anomaly with associated rib anomaly in one fetus at 25 mg/kg bw/day. Because these malformations occurred singly and were noted previously in historical controls, they were not considered to be treatment related. One other skeletal malformation (caudal vertebral anomaly) was observed in a control fetus, and as such considered to be spontaneous in origin.
Skeletal variations occurred at an incidence of 60.5%, 79.3%, 75.5% and 77.6% per litter at 0, 8, 25 and 75 mg/kg bw/day, respectively. All the variations noted, were not considered treatment related as they occurred infrequently, in the absence of a dose-related incidence trend and/or at frequencies that were within the range of available historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to 75 mg/kg bw/day. Four different visceral malformations were observed that occurred singly 0, 8 and 25 mg/kg bw/day. No malformations were observed at 75 mg/kg bw/day. At 25 mg/kg bw/day, three fetuses were affected and these either had a malpositioned kidney, absence of the accessory lung lobe or tetralogy of Fallot. A malpositioned kidney was also found at 8 mg/kg bw/day, whereas tetralogy of Fallot was seen in one control fetus as well. The only other malformation observed was a large intestine in one control fetus. As the above visceral malformations occurred singly in one or two groups, including the control group, and not in the high dose group, all were considered to be chance findings.
All the variations noted were considered not treatment related as they occurred infrequently, occurred at frequencies that were within the range of available historical control data or were observed in control fetuses only.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no treatment-related adverse effects observed at the highest dose level of 75 mg/kg bw/d

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Dose formulation analyses:

Accuracy

The concentrations analyzed in the formulations of low and high dose group from both week 1 and 3 of treatment were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). 

For the formulation of the mid dose group prepared for use in week 1 of treatment, the mean accuracy was above the target concentration (i.e. 120% of target). Out of scope investigation was performed and no analytical issue was found. The concentrations analyzed in the formulations of the mid dose group from week 3 of treatment were in agreement with target concentrations (i.e. mean accuracy between 85% and 115%).

No test item was detected in the control group formulations.

Homogeneity

The formulations of low dose group from both week 1 and 3 of treatment were homogeneous (i.e. coefficient of variation10%).

For the formulations of high dose group prepared for use in week 1 of treatment the coefficient of variation was above the target (i.e. 20%). Out of scope investigation was performed and no analytical issue was found. For the formulations of high dose group prepared for use in week 3 of treatment, the coefficient of variation was marginally above the target homogeneity of =10% (i.e. 10.6%), which was considered to be acceptable as the accuracies were within target concentrations.

The out of target results in week 1 of treatment were related to the duration of the heating period of the formations. On the first three days of the treatment period, including the day of sampling in week 1, and on Day 9 of treatment formulations were heated for about 15 minutes. The remainder treatment period, including the day of sample analysis in week 3, formulations were heated for about 30 minutes. As the formulations were sufficiently heated for 22 out of 26 days of the treatment period resulting in acceptable accuracy and homogeneity levels, it was considered that the formulations were appropriate for the purpose of the study.

Applicant's summary and conclusion

Conclusions:
Based on the results of a prenatal developmental toxicity study performed according to OECD/EC guidelines and GLP principles, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for 1,1’-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol was established as being at least 75 mg/kg bw/day.
The high dose of 75 mg/kg bw/day was considered to be the highest tolerated dose, based on the results of a dose range finding study.
Executive summary:

A prenatal developmental toxicity study (OECD 414) was performed in rabbits by oral gavage with 1,1'-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol. The objectives of this study were todetermine the potential of the substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post-coitum, inclusive. In addition, theNo Observed Adverse Effect Levels(NOAELs) for maternal toxicity and developmental toxicity were evaluated.

The dose levels in this study were selected to be 0 (0.5% aqueous carboxymethyl cellulose with 1.25% Tween 80), 8, 25 and 75 mg/kg/day, based on the results of a dose range finder.

Chemical analyses of formulations were conducted twice during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents.

In addition, the following parameters were determined for the F1-generation: the number of fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations.

No mortality occurred during the study period that was considered to be related to treatment with the test item. Treatment at 75 mg/kg resulted in slight body weight loss (average weight loss of 1%) on post-coitum Day 9 and significantly reduced food consumption over post-coitum Days 6-13 (relative food consumption about 38% lower than controls). As during the remaining of the treatment period, body weight and food consumption values returned back to normal, these effects were transient and therefore considered not to be adverse.

No maternal toxicity was observed in the 8 and 25 mg/kg groups.

No developmental toxicity was observedin the 8, 25 and 75 mg/kg groups.

In conclusion, based on the results in this prenatal developmental toxicity study in rabbit a maternal and developmental No Observed Adverse Effect Level (NOAEL) for 1,1’-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol of at least 75 mg/kg was established.

The high dose of 75 mg/kg was considered to be the highest tolerated dose, as in the dose range finder treatment at 100 mg/kg resulted in mortality of one female, piloerection noted for all females, body weight loss with an average of -4% on post-coitum Days 9 and 13, and significantly reduced food consumption of 70% over post-coitum Days 6 to 20.