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 Dec 2016 to 12 Jan 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 22nd January 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Triskelion B.V., Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test material used is consistent with the substance identity as described in section 1.2 of IUCLID - company composition for the quality described under the common name Verdox.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han IGS (Crl:WI(Han))
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: The female rats were about 10 weeks old at arrival. Males for mating from the same strain and supplier were available at the test facility. At the time of mating these males were approximately 16 weeks old.
- Housing: Animals were housed in macrolon cages with a bedding of wood shavings (Lignocel) and strips of paper (Enviro-dri) and a wooden block as environmental enrichment. During the acclimatization period, the animals were housed in groups of 4/sex. For mating, one male and two females were housed together. Mated females were housed individually in macrolon cages, which were placed in another cage rack.
- Diet: From their arrival, the animals received a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England), ab libitum. The feed was provided in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage.
- Water: Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC), ab libitum. The water was given in polypropylene bottles, which were cleaned weekly and filled as needed.
- Acclimation period: At least 5 days prior to the start of the exposure.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45 - 65
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
The test item was incorporated in the basal diet by mixing in a mechanical blender. The experimental diets were prepared on once. After preparation, the experimental diets were divided into daily amounts of diets that were stored in plastic bags in a freezer (-18°C). Each day, one bag per test concentration was removed from the freezer to feed the animals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The concentrations of the test substance in the experimental diets were measured according to a validated UPLC-UV method (Sub-chronic (13-week) oral (dietary) toxicity study with Verdox in rats, D. van Berlo, 2017).
- It was shown that the test item was stable when the diets were stored in a freezer (<-18°C) but the test item was not stable in the diets stored at room temperature in the animal room. For that reason, the feed was replaced every day.
- The homogeneous distribution and achieved concentration of the test item in the VRF1 rat feed was analysed in the batch of diets prepared for this study. Directly after mixing of each diet, samples for the homogeneity experiments were taken from the mixer. Firstly, five homogeneity samples (about 50 g each) of each group were taken at left top, right top, middle, left bottom and right bottom of the container. Samples were taken in the order: top centre, middle centre, bottom centre, left centre, right centre. The samples taken for homogeneity experiments were also used for dose confirmation.
Details on mating procedure:
At the end of the acclimatisation period, males and females were placed together for mating (two females will be caged with one male). Animals were caged together until mating occurred and the required number of mated animals was achieved. Every consecutive morning during the mating period, vaginal smears were made for determination of the presence of sperm. The day on which sperm is detected in the vaginal smear was considered as gestation day 0 (GD 0). Upon evidence of copulation the females were caged individually. Females mated by the same male were placed in different groups.
Duration of treatment / exposure:
From gestation day (GD) 0 up to and including GD 21 (necropsy)
Frequency of treatment:
Continuously
Duration of test:
22 days
Doses / concentrationsopen allclose all
Dose / conc.:
800 mg/kg diet
Remarks:
Corresponding to an anticipated dose of 55 mg/kg bw/day
Dose / conc.:
2 500 mg/kg diet
Remarks:
Corresponding to an anticipated dose of 166 mg/kg bw/day
Dose / conc.:
7 700 mg/kg diet
Remarks:
Corresponding to an anticipated dose of 444 mg/kg bw/day
No. of animals per sex per dose:
Ninety-six female rats were mated and allocated to a control group and three treatment groups (24 mated females per group). Male rats were used for mating only.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a 14-day DRF for a previously performed 70-90-days extended OECD TG 422 study (Triskelion report V20063; A. Wolterbeek, 2012). In the DRF the dose of ca 15400 mg/kg diet (nominal 1000 mg/kg bw) was used. In this DRF at this high dose decrease in food consumption and body weight was seen and these effects were related to the palatability of the test item Verdox. Verdox is a fragrance which can smell awkwardly and is therefore not tasty either. At this 15400 mg/kg diet in the DRF the relative liver weight increased >=17% in males and females, which we considered to be due to metabolic saturation. This liver weight increase is also expected in combination with Verdox’s MW (196) and physico-characteristics, which indicate full and fast oral absorption.
The dietary 80-90-day Repeated dose / Reproscreen study with the highest dose of 7500 mg/kg diet (converted to nominal 500 mg/kg bw) showed decrease in body weight gain (ca. 15%), which was considered test item related but not adverse. Based on this the 7500 mg/kg diet was sufficiently high to present (absence of adverse) effects for the OECD TG 414 study and not muddle the outcome of the study due to potential palatability issues. In this Repeated dose / Reproscreen study the relative liver weights in males were increased around 14%, which we related to increased metabolic capacity and considered this sufficiently high to set the maximum dose levels at 7500 mg/kg diet in the follow up OECD TG 414 study.

Summary of key effects in DRF and extended OECD TG 422
DRF
Mg/kg diet Body weight Body weight gain Food consumption / per animal Relative liver weight
Males
1155 Not applicable Not applicable Not applicable Not applicable
2310 Not applicable Not applicable Not applicable Not applicable
7700 No effect -18% -17% +11%
15400 No effect -25% -17% +20%
Females
1155 Not applicable Not applicable Not applicable Not applicable
2310 Not applicable Not applicable Not applicable Not applicable
7700 No effects -25% -13% +5%
15400 -6% -25% -15% +17%

Extended OECD TG 422
Mg/kg diet Body weight Body weight gain Food consumption / per animal Relative liver weight
Males
800 Not applicable Not applicable Not applicable Not applicable
2500 Not applicable Not applicable Not applicable Not applicable
7500 -7% -15% Not affected +14%
Females
800 Not applicable Not applicable Not applicable Not applicable
2500 Not applicable Not applicable Not applicable Not applicable
7500 No real effects -16% only during premat Not affected No effects

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. All cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study.

BODY WEIGHT:
Body weights of the parental female animals were recorded on gestation days (GD) 0, 6, 9, 12, 15, 18 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The food consumed for each mated female was measured daily during gestation. The results were expressed in gram per animal per day.

POST-MORTEM EXAMINATIONS:
- The females were sacrificed by decapitation after CO2/O2 anaesthesia on GD 21 and examined for gross abnormalities.
- In addition to the following organs and tissues, all gross lesions were preserved.
- The following organs were weighted: uterus, containing placentas and fetuses, uterus (empty), ovaries, live fetuses (individually) and corresponding placentas.
- Abnormalities of tissues or organs in dams
Ovaries and uterine content:
All females sacrificed on GD 21 were examined for the following parameters: number of corpora lutea, number of implantation sites, number of early and late resorptions, number of live and dead fetuses, sex of the fetuses, number of grossly visible malformed fetuses and fetuses with external abnormalities, gross evaluation of placentas.
Fetal examinations:
Fetuses were examined for external alterations and sacrificed by hypothermia. Subsequently, approximately half of the fetuses of each litter was fixed in Bouin's fixative, examined for soft tissue anomalies according to a method modified after Barrow and Taylor (1969) and then discarded. Abnormal tissues were preserved. The other half of the fetuses were fixed in 70% alcohol, subsequently partly eviscerated, and then cleared in potassium hydroxide and stained with Alizarin Red S modified after Dawson (1926). They were examined for skeletal abnormalities and then retained. During the fetopathological examination, the observer was unaware of the dose group of the fetuses.
Statistics:
The resulting data were analysed using the methods mentioned below. Tests were generally performed as two-sided tests with results taken as significant where the probability of the results is p<0.05 (*) or p<0.01 (**).
Continuous data were tested for normality b the Shapiro-Wilks test. If the data was not normally distributed, log-transformation was done and the log-transformed data were tested for normality. The homogeneity of normally distributed data was tested with the Levene's median adjusted test. Homogeneous data were analysed using Anova and, for significant differences, the Dunnett or Turkey-Kramer test. Not normally distributed data or data with a lack of homogeneity were tested with the Kruskal Wallis test and, in case of significant differences, the Dunn's or Wilcoxon's test.
Dichotomous data were analysed for difference between proportions with the Chi-square test, where significant findings in the Chi-square test were followed by the Fisher's exact test. The Cochran-Armitage test was used for trends in proportions.
Indices:
The female fertility index ((number of pregnant females / number of inseminated females) x 100) and gestation index ((number of females with live fetuses / number of pregnant females) x 100) were calculated.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical signs observed (wounds, encrustations and sparsely haired areas) are considered not related to the treatment. One animal in the high dose group showed hunched posture and piloerection from gestation day 6 to 8. In addition the animal showed minimal food consumption. In order to stimulate food intake, the animal received a single oral gavage administration of approximately 1.5 gram test diet mixed with drinking water. Thereafter the animal recovered.
An overview the clinical signs can be found in tabular form in the attached study report on page 25.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No animals died unscheduled.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight and mean body weight gain were statistically significantly decreased in the high dose group throughout a major part of gestation. This effect is considered to be related to the effect on food intake in the high dose group. In the low and mid dose groups mean body weight and mean body weight gain was comparable to the control group.
An overview of body weight and body weight gain can be found in tabular form in the attached study report on page 26 and 27, respectively.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean daily food consumption was statistically significantly lower in the high dose group on gestation days 0-6, 10-12, gestation day 14-15 and 16-21. This was considered to be related to the palatability of the test substance. In the mid dose group mean food consumption was decreased at the onset of exposure (from day 0-1 of gestation). In the low dose group mean daily food consumption was comparable to the control group. The mean test substance intake was calculated from the daily food consumption. Mean test substance intake over the complete gestation period (day 0-21 of gestation was 0, 55, 166 and 444 mg/kg body weight for the control group, low dose, mid dose and high dose group, respectively.
An overview of food consumption and test substance intake can be found in tabular form in the attached study report on page 28 to 30 and 31, respectively.
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):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No statistically significant effects were observed on the mean uterine weight (including fetuses) and on the mean empty uterus weight. Mean ovary weight was comparable in all groups.
An overview of uterine weight can be found in tabular form in the attached study report on page 35. Placental weight can be found on page 36.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One animal of the control group showed a yellow stomach deposition. One animal of the low dose and mid dose were sparsely haired. One animal of the mid dose group showed a wound on the skin and another animal from this dose group showed encrustations on the skin. These observations are common in this strain of rats and not considered to be related to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No statistical significant differences were observed in the mean pre-implantation loss between the control group (6.8%) and the animals in the low, mid and high dose groups (11.3%, 11.1% and 10.2%, respectively). Mean post-implantation loss varied from 1.7% (control group) to 6.8% (mid dose group).
An overview of pre- and post-implantation loss can be found in tabular form in the attached study report on page 32 to 34.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
No increase was observed in the mean number of early and late resorptions.
An overview of early and late resoptions can be found in tabular form in the attached study report on page 32 to 34.
Dead fetuses:
no effects observed
Description (incidence and severity):
The mean number of live fetuses was comparable in all groups( 11.9, 11.5, 11.8 and 11.3 for the control group, low dose, mid dose and high dose group, respectively).
An overview of the number of dead fetuses can be found in tabular form in the attached study report on page 32 to 34.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Two animals in the mid dose group and one animal in the high dose group were not pregnant, resulting in 24 litters in the control and low dose group, 22 litters in the mid dose group and 23 litters in the high dose group. The female fertility index was therefore 100% in the control and low dose group, 92% for the mid dose group and 96% for the high dose group, respectively).
Other effects:
no effects observed
Description (incidence and severity):
All pregnant females had live fetuses, resulting in a gestation index of 100% for all groups. The mean number of implantation sites was comparable in all groups (12.1, 12.2, 12.4 and 11.8 for the control group, low dose, mid dose and high dose, respectively).

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 444 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed at highest tested dose
Remarks on result:
other: Corresponding with 7700 mg/kg diet

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean fetus weight (both sexes together) was statistically significantly lower in the high dose group. When split to males and females, the mean male fetus weight in the high dose group was statistically significantly lower, whereas the mean female fetus weight did not reach statistical significance. The decrease in fetus weight in the high dose group is considered to be related to the decrease in maternal body weight and is only minor (approximately 6% as compared to mean fetus weight in the control group, whereas mean maternal body weight in the high dose group was approximately 20% lower as compared to the control group). In addition no effects were observed on the ossification of the fetuses and therefore this slight effect on fetus weight is not considered adverse. No effect on mean fetus weight were observed in the low dose and mid dose group as compared to the control group.
An overview of fetal body weight changes can be found in tabular form in the attached study report on page 36.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
An overview of the number of live offspring can be found in tabular form in the attached study report on page 32.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The mean percentage male fetuses per litter (sex ratio) was comparable in all groups (52.4%, 46.8%, 44.4% and 52.0% for the control group, low dose, mid dose and high dose group, respectively).
An overview of changes in sex ratio can be found in tabular form in the attached study report on page 34.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No fetal external malformations were observed. One fetus in the low dose group was small and another one showed subcutaneous hemorrhages.
An overview of external malformations and variations can be found in tabular form in the attached study report on page 38 and 39, respectively.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The following malformations were observed: one fetus in the control group showed a small pubis and another fetus in the control group showed branched ribs and a fused centrum of the thoracic vertebra. One fetus in the mid dose group also showed a fused centrum of the thoracic vertebra. These observations were considered malformations. Variations observed were mainly related to the partly incomplete ossification status of the fetal bones. In addition, wavy ribs, irregular shapes, fusions or supernumerary sternebrae were observed.
An overview of skeletal malformations and variations can be found in tabular form in the attached study report on page 43 and 44 to 56, respectively.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No fetal visceral malformations were observed. Visceral variations included observations in the liver (discolored lobe), eyes (unilateral or bilateral retina fold), variations in the ureters (unilateral or bilateral bent or kinked), kidneys (dilated renal pelvis), urinary bladder (distended) and mouth (dark content). A distended urinary bladder was observed in fetuses of the treatment groups only. However, this is a common variation in this strain of rats. Historical background data show that the incidences observed in this study are considered common for this observation. Therefore the observation “distended urinary bladder” is not considered to be treatment-related.
An overview of visceral malformations and variations can be found in tabular form in the attached study report on page 40 and 41 to 42, respectively.
Other effects:
no effects observed
Description (incidence and severity):
No effects were observed on mean placenta weight in the exposed groups as compared to the control group.
An overview of placental weight can be found in tabular form in the attached study report on page 36.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 444 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at highest tested dose
Remarks on result:
other: Corresponding with 7700 mg/kg diet

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
For the substance a NOAEL of > 444 mg/kg bw/day (corresponding to > 7700 mg/kg in diet) is derived for maternal and developmental toxicity. The basis for this effect level is the absence of adverse effects at the highest dose tested. The only treatment effects observed on maternal toxicity are decreased food intake and body weight in the high dose group, which both result from reduced palatability of the high-dose diet and thus not adverse. The reduced fetal body weights are linked to the decrease in maternal food intake and body weight, and are not considered adverse.
Executive summary:

A prenatal developmental toxicity study was performed according to OECD TG 414 and GLP. The substance was administered to female Wistar Han IGS rats (Crl:WI(Han)) at dose levels of 800, 2500 and 7700 mg/kg in diet (corresponding to 55, 166 and 444 mg/kg bw total dose).

The dose levels were selected based on the results of a 14-day DRF for a previously performed 70-90-days extended OECD TG 422 study (Triskelion report V20063; A. Wolterbeek, 2012). In the DRF the dose of ca 15400 mg/kg diet (nominal 1000 mg/kg bw) was used. In this DRF at this high dose decrease in food consumption and body weight was seen and these effects were related to the palatability of the test item Verdox. Verdox is a fragrance which can smell awkwardly and is therefore not tasty either. At this 15400 mg/kg diet in the DRF the relative liver weight increased >=17% in males and females, which we considered to be due to metabolic saturation. This liver weight increase is also expected in combination with Verdox’s MW (196) and physico-characteristics, which indicate full and fast oral absorption.

The dietary 80-90-day Repeated dose / Reproscreen study with the highest dose of 7500 mg/kg diet (converted to nominal 500 mg/kg bw) showed decrease in body weight gain (ca. 15%), which was considered test item related but not adverse. Based on this the 7500 mg/kg diet was sufficiently high to present (absence of adverse) effects for the OECD TG 414 study and not muddle the outcome of the study due to potential palatability issues. In this Repeated dose / Reproscreen study the relative liver weights in males were increased around 14%, which we related to increased metabolic capacity and considered this sufficiently high to set the maximum dose levels at 7500 mg/kg diet in the follow up OECD TG 414 study.

 

The 24 females/ dose level were exposed from gestation day 0 (defined by a sperm-positive vaginal smear) to necropsy at gestation day 21. A control group on plain diet was included. Analysis of the test diets showed that the test substance was homogeneously distributed in the test diets and that the concentrations were close to intended. Based on the limited stability in the test feeds, the feed was replaced daily with fresh portions from the freezer.

Clinical observations and feed consumption were measured daily. Body weight was determined on gestation days 0, 6, 9, 12, 15, 18 and 21. At gestation day 21 the animals were sacrificed and cesarean section was performed. On the maternal animals gross pathology was performed and weights of reproductive organs were recorded. In addition, the number and distribution of implantations, fetuses and resorptions was recorded. Fetuses were weighed and examined for external observations and then sacrificed and processed for visceral or skeletal fetal evaluations.

During the study no mortalities or morbidity was observed. Statistically significant decreased mean body weight and body weight gain in the 7700 mg/kg diet group which were related to decreased food intake in the in this dose-group. The test substance is a fragrance and probably reduced palatability of the high-dose diet resulted in a lower food intake. Hence the reduced food intake and the accompanying lower body weights in the high-dose group are considered to be due to reduced palatability rather than to the test substance per se, and are not considered to be adverse. No effects on maternal reproductive organ weights (full and empty uterus, ovary weight) fertility and litter parameters (pre- and post-implantation loss, number of live fetuses and resorptions), placenta weight and fetus sex ratio were observed. No treatment related effects on external, visceral and skeletal observations were observed. In the high dose group, a decrease in mean fetus weight in the 7700 mg/kg diet group was observed. This effect is considered to be related to the decrease of maternal food intake and maternal body weight. In view of the minor effect on fetal body weight as compared to the maternal effects and in absence of a delay in ossification this fetal body weight decrease is not considered to be adverse. As the maternal effects on body weight and food consumption observed in the high dose group are considered related to the palatability of the test substance and are not considered adverse, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity is ≥ 7700 mg/kg diet (corresponding to a dose level of 444 mg/kg body weight/day). In absence of effects on ossification the slight effect on fetus weight in the high dose group was not considered adverse and therefore the NOAEL for developmental toxicity is ≥ 7700 mg/kg diet (corresponding to a dose level of 444 mg/kg body weight/day). The substance is therefore not considered to be a developmental toxicant.