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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18th August to 5th September 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
OECD TG414 dated June 2018
Deviations:
no
Remarks:
No deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
No IUPAC name is currently defined for Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled (“Distilled Residue Grade”).
EC Number:
941-212-1
Molecular formula:
Cardanol (saturated side chain): Formula: C21 H36 O Cardanol (monoene): Formula: C21 H34 O Cardanol (diene): Formula: C21 H32 O Cardanol (triene): Formula: C21 H30 O
IUPAC Name:
No IUPAC name is currently defined for Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled (“Distilled Residue Grade”).
Details on test material:
- Name of test material (as cited in test report): Cashew Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade)
- Physical state: liquid
- Other: Straw coloured to dark brown liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Hannover
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: From Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: At the initiation of dosing females were 10 to 14 weeks old
- Weight at study initiation: At the initiation of dosing females were between 186 and 274 g
- Fasting period before study:
- Housing: Individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany)
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least 5 days before the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target temperatures of 18 to 24°C with the actual daily mean temperature during the study period being 21°C.
- Humidity (%): Relative target humidity of 40 to 70% with the actual daily mean relative humidity being 49 to 73%.
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms
- Photoperiod (hrs dark / hrs light): A 12 hour light/12 hour dark cycle was maintained

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Dried and Deacidified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test substance dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.


DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity/stability analyses of dosing solutions were performed at each dose level prior to the initiation of the study.
The concentrations analyzed in the formulations of the 100 mg/kg/day (low dose), 300 mg/kg bw/day (mid dose) and 1000 mg/kg bw/day (high dose) were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).
Details on mating procedure:
The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
Duration of treatment / exposure:
Days 6 - 20 post-coitum inclusive
Frequency of treatment:
Females only, Once/day. Treated from Day 6 through 20 post-coitum inclusive
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Actual ingested
Dose / conc.:
100 mg/kg bw/day
Remarks:
Actual ingested
Dose / conc.:
300 mg/kg bw/day
Remarks:
Actual ingested
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Actual ingested
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
A total of 88 pregnant Wistar Han female rats were allocated to four groups (22 per group) Three groups were administered with the test substance Distilled Grade, by oral gavage daily from day 6 to day 20 post-coitum inclusive, at the dose level of 100, 300 and 1000 mg/kg bw/day. One group of females was only administered with the vehicle, dried and deacidified corn oil, at the same constant dose volume of 5 mL/kg.
Individual volumes were adjusted according to the most recently recorded body weight.


- Dose selection rationale: The dose levels were selected based on the results of a limited range finder in the pregnant rat (Test Facility Study No. 20205156), on the results on the dose range finder in the non-pregnant rat (Test Facility Study No. 20164929), and in an attempt to produce graded responses to the test substance.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals showing pain, distress or discomfort which was considered not to be transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed once daily from Day 2 post-coitum and lasting up to the day prior to necropsy. Females were only observed immediately after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum. In order to monitor its health status, animal number 28 from the 100 mg/kg bw/day group were also weighed on Day 7, 8 and 14 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Time scheule: Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 20 post-coitum
- Organs examined: All animals (including animals sacrificed before planned necropsy) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Each ovary and uterine horn of all surviving animals was dissected and examined as quickly as possible to determine:

- Gravid uterus weight: Yes (not for animals sacrificed before planned necropsy)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes / No / No data
- Number of late resorptions: Yes / No / No data
- Other:
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The sex of each fetus based on the ano-genital distance.

In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
Fetal examinations:
- External examinations: Yes: all viable fetuses
- Soft tissue examinations: Yes: half of all viable fetuses
- Skeletal examinations: Yes: half of all viable fetuses
- Head examinations: Yes: half of all viable fetuses
Statistics:
Data were analysed between the control and treatment groups (low, mid and high doses) as appropriate depending on data availability
Indices:
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 postimplantation loss.
Historical control data:
The laboratory has historical control data in regard to developmental/teratological parameters for this strain of rat.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicological relevance (such as hunched posture, flat gait, rales, piloerection, ptosis, uncoordinated movements and/or pale appearance) were noted at any exposure dose during the observation period.
Mortality:
no mortality observed
Description (incidence):
No mortalities were observed in the controls and all the treatment groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights, body weight gain and weight gain corrected for gravid uterus of treated females were unaffected by treatment with the test substance up to 1000 mg/kg bw/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
A subjective appraisal of water consumption was maintained during the study, but no quantitative investigation was conducted as no effect was suspected.
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Endocrine findings:
no effects observed
Description (incidence and severity):
Analysis of the samples showed that mean total T3 and total T4 levels were unaffected in animals at all the exposure doses. Mean levels of thyroid stimulating hormone (TSH) were statistically significantly increased at 1000 mg/kg/day (1.74x of controls), but there was variability in the observed responses with a coefficient of variation of 57%. Individual values remained within the historical control range of 0.129 to 0.724 (µIU/ml) for 17 of the 22 females (77%), though for the other 5/22 (23%) animals the values were above the 95%ile control limit.
Urinalysis findings:
not specified
Behaviour (functional findings):
not examined
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test substance-related alterations in thyroid gland weights up to 1000 mg/kg/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant abnormalities were observed at necropsy in exposed animals up to 1000 mg/kg bw/day
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test substance-related microscopic observations in the thyroid glands up to 1000 mg/kg/day.

Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No neoplastic histopathological effects were evident in the tissues of animals from the control and treatment groups.
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on pre- and post-implantation losses at any of the exposure doses, compared to the controls.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on total losses by resorption at any of the exposure doses, compared to the controls
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on early or late resorptions at any of the exposure doses, compared to the controls.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the number of dead foetuses at any of the exposure doses, compared to the controls.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the number of pregnant females at any of the exposure doses, compared to the controls.

Effect levels (maternal animals)

Key result
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

Results (fetuses)

Details on embryotoxic / teratogenic effects:
No
No toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, post-implantation loss, fetal sex ratio, fetal body weights, fetal ano-genital distance and external, visceral and skeletal malformations and developmental variations in fetuses) after treatment up to 1000 mg/kg/day.


Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: embrytoxicity
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Key result
Abnormalities:
no effects observed
Description (incidence and severity):
There were no test substance-related effects on fetal external malformations and variations, visceral morphology and skeletal morphology following treatment up to 1000 mg/kg bw/day.

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In the absence of adverse findings at the highest dose level, the systemic maternal No Observed Adverse Effect Level (NOAEL) for Cashew Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade) was determined to be 1000 mg/kg bw/day.

The developmental NOAEL for Distillation Residue Grade was also established as being 1000 mg/kg bw/day.
Note: In the study the mean thyroid stimulating hormone (TSH) level was increased in adult rats exposed to the test-substance at 1000 mg/kg/day/day. However, the mean TSH value of females at 1000 mg/kg/day remained within the historical control range for 77% of females. The test report concluded that “under the conditions of this study no adverse effect was observed that could be linked to the increase in TSH and therefore it was not taken into account when determining the maternal NOAEL
Executive summary:

This study was conducted to evaluate the embryotoxicity, fetotoxicity and teratogenicity of the test material in the pregnant Wistar Han rat. Test material was dissolved in dried and deacidified corn oil and administered daily by gastric intubation during the Day 6 to 20post-coitum, with the dosing solutions being prepared fresh weekly. Dose levels were 100, 300 and 1000 mg/kg bw/day which were selected based on the results of a limited range finder in the pregnant rat (Test Facility Study No. 20205156) and on the results on the dose range finder in the non-pregnant rat (Test Facility Study No. 20164929). The study included a vehicle (dried and deacidified corn oil) treated control group. Each study group contained 22 mated females.


 


Analysis of the samples showed that mean total T3 and T4 levels, as well as thyroid weight and histopathology, were unaffected in animals at all the exposure doses. Mean levels of thyroid stimulating hormone (TSH) were statistically significantly increased at 1000 mg/kg/day (1.74x of controls), but there was variability in the observed responses with a coefficient of variation of 57%. Individual values remained within the historical control range of 0.129 to 0.724 (µIU/ml) for 17 of the 22 females (77%), though for the other 5/22 (23%) animals the values were above the 95%ile control limit. The test report concluded that “under the conditions of this study no adverse effect was observed that could be linked to the increase in TSH and therefore it was not taken into account when determining the maternal NOAEL”.


 


No toxicologically significant changes at exposure levels up to 1000 mg/kg bw/day were noted in any of the remaining maternal parameters investigated in this study (i.e. mortality/moribundity, clinical appearance, body weight, food consumption, gross pathology, thyroid weights, uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and pos-timplantation loss).


 


No test substance-related changes at exposure levels up to 1000 mg/kg bw/day were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, fetal anogenital distance and external, visceral and skeletal malformations and variations in fetuses).


 


No effects on endocrine-disruption related apical morphological and histological endpoints (i.e. anogenital distance and sex ratio in foetuses, thyroid weight and histopathology in females) and indicators of thyroid hormonal activity in females (i.e. T3, T4 and TSH levels) were observed in the OECD TG414 study at exposure doses up to 1000 mg/kg bw/day.