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Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted under GLP
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited
- Age at study initiation: 8 weeks
- Weight at study initiation: males, 290-335g; females 191-215g
- Housing: Initially in groups of 5 in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbant paper. During mating cages held one male and one female. Males were returned to their original cages and females were housed individually.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: 4°C

VEHICLE
- Justification for use and choice of vehicle (if other than water): arachis oil BP
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear
- After successful mating each pregnant female was caged (how): individually in wire mesh cages
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Up to 54 consecutive days.
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 15, 150, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on results o f a preliminary range-finder study.
- Rationale for animal assignment (if not random): Random based on stratified bodyweights
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily



DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before and after dosing, and one and five hours after dosing during the working week; before and after dosing, and one hour after dosing during the weekend.


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for males; weeks 1, 2 and 3 and days 1 and 4 post partum for females.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data:


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 13, females at day 5 post-partum
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals:5 males and 5 females/dose group


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:Day 13, females at day 5 post-partum
- Animals fasted: No data
- How many animals:5 males and 5 females/dose group

URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine:
- Animals fasted:


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: End of mating phase, 5 males/dose group; Day 4 post-partum, 5 females/dose group
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals Day 5 post partum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 2 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
GROSS NECROPSY
- Gross necropsy consisted of external appearance
Statistics:
ANOVA, incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis, ANOVA and Mann-Whitney 'U' test.
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY
There were no toxicologically significant deaths during the study.

Increased salivation was detected prior to dosing and up to 5 hours after dosing for animals of either sex treated with 1000 mg/kg bw/day from Day 9 onwards. One female treated with 150 mg/kg bw/day developed clinical signs consistent with inappropriate dosing on Day 5 and was subsequently terminated. One female treated with 1000 mg/kg bw/day had given birth to a number of pups of which the majority were found dead. Several clinical signs were observed in this animal, and the animal and litter were terminated.

BODY WEIGHT AND WEIGHT GAIN
A slightly reduced bodyweight gain was observed for 1000 mg/kg bw/day males during the first two weeks of the study. Reduced bodyweight gain was also observed for 1000 mg/kg bw/day females during the later stages of the gestation period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No adverse effect on dietary intake or food efficiency were detected.

FOOD EFFICIENCY
No adverse effect on dietary intake or food efficiency were detected.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
No intergroup differences were detected.

OPHTHALMOSCOPIC EXAMINATION
N/A

HAEMATOLOGY
Haematological assessments revealed elevated platelet counts in animals of either sex treated with 1000 mg/kg bw/day. Elevated haemoglobin, erythrocyte and haematocrit was also evident for males treated at 1000 mg/kg bw/day. No such effects were detected at 150 and 15 mg/kg bw/day.

CLINICAL CHEMISTRY
An increase in aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase levels were observed for 1000 mg/kg bw/day animals of either sex, together with elevated inorganic phosphorus, bilirubin and urea, and reduced cholesterol levels.

URINALYSIS
N/A

NEUROBEHAVIOUR
Open field arena observations revealed increased salivation for individual animals of either sex treated with 1000 mg/kg bw/day from Week 3.

ORGAN WEIGHTS
Females treated with 1000 mg/kg bw/day showed elevated liver weights.

GROSS PATHOLOGY
None

HISTOPATHOLOGY: NON-NEOPLASTIC
Groups of alveolar macrophages were seen with a higher incidence for females treated with 1000 mg/kg bw/day.
A higher incidence of sinus histiocytosis and/or foamy histiocytes was observed in relation to treatment with 1000 mg/kg bw/day.
Hyperkeratosis, frequently associated with acanthosis was seen in the forestomachs of animals of either sex treated with 1000 mg/kg bw/day. Focal ulceration of the forestomach epithelium was also seen in one high dose female.
Mucosal hypertrophy was seen in three males at the top dose group. A low incidence of mucosal hypertrophy was observed in females in all dose groups.

OTHER FINDINGS
MATING
No adverse effects on mating or fertility were observed.

OFFSPRING LITTER SIZE AND VIABILITY
No effects detected.

OFFSPRING GROWTH AND DEVELOPMENT
No effects detected.

LITTER OBSERVATIONS
No effects detected.

UTERINE EXAMINATION
No effects detected.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: BAsed on systemic changes in the lungs, mesenteric lymph node, stomach and duodenum.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
OFFSPRING LITTER SIZE AND VIABILITY
No effects detected.

OFFSPRING GROWTH AND DEVELOPMENT
No effects detected.

LITTER OBSERVATIONS
No effects detected.

UTERINE EXAMINATION
No effects detected.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment effects occurred on reproduction or offspring development.
Reproductive effects observed:
not specified
Conclusions:
A NOAEL of 150 mg/kg bw/day has been identified for systemic toxicity. A NOAEL of 1000 mg/kg bw/day has been identified for reproductive parameters.
Executive summary:

In a subchronic toxicity study Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) was administered to 5 Sprague-Dawley rats/sex/dose via gavage at dose levels of 0, 15, 150, or 1000 mg/kg bw/day).

 

Systemic changes in the lungs, mesenteric lymph node, stomach and duodenum occurred at the highest dose.The NOAEL is 150 mg/kg bw/day.

 

This repeat dose/reproductive toxicity screening study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study OECD 422 in rats.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Short description of key information:
In a subacute toxicity study Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) was administered to 5 Sprague-Dawley rats/sex/dose via gavage at dose levels of 0, 15, 150, or 1000 mg/kg bw/day).
Systemic changes in the lungs, mesenteric lymph node, stomach and duodenum occurred at the highest dose. The NOAEL is 150 mg/kg bw/day. A NOAEL of 1000 mg/kg bw/day has been identified for reproductive parameters.
This repeat dose/reproductive toxicity screening study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study OECD 422 in rats.

In an OECD 414 study, conducted under GLP, in the absence of adverse findings in any of the other parameters at the lowest dose level, the systemic maternal No Observed Adverse Effect Level (NOAEL) for Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) was determined to be 100 mg/kg bw/day. A NOAEL of 300 mg/kg bw/day has been derived for fertility effects since no effects on the relevant reproductive endpoints were observed at the highest dose where females survived to necropsy.

Effects on developmental toxicity

Description of key information

In a subacute toxicity study Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) was administered to 5 Sprague-Dawley rats/sex/dose via gavage at dose levels of 0, 15, 150, or 1000 mg/kg bw/day).
Systemic changes in the lungs, mesenteric lymph node, stomach and duodenum occurred at the highest dose. The NOAEL is 150 mg/kg bw/day. A NOAEL of 1000 mg/kg bw/day has been identified for reproductive parameters as no adverse effects on the development of offspring were observed at the highest test dose. This repeat dose/reproductive toxicity screening study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study OECD 422 in rats.


In an OECD 414 study, conducted under GLP, in the absence of adverse findings in any of the other parameters at the lowest dose level, the systemic maternal No Observed Adverse Effect Level (NOAEL) for Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) was determined to be 100 mg/kg bw/day. A NOAEL of 300 mg/kg bw/day has been derived for foetal development since no effects on the relevant developmental endpoints were observed at the highest dose where females survived to necropsy.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted under GLP
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
other: OECD 422
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited
- Age at study initiation: 8 weeks
- Weight at study initiation: males, 290-335g; females 191-215g
- Housing: Initially in groups of 5 in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbant paper. During mating cages held one male and one female. Males were returned to their original cages and females were housed individually.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: 4°C

VEHICLE
- Justification for use and choice of vehicle (if other than water): arachis oil BP

Details on mating procedure
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear
- After successful mating each pregnant female was caged (how): individually in wire mesh cages

Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear
- After successful mating each pregnant female was caged (how): individually in wire mesh cages

Duration of treatment / exposure:
Up to 54 consecutive days.
Frequency of treatment:
Daily
Duration of test:
28 days up to 54 day.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on results o f a preliminary range-finder study.
- Rationale for animal assignment (if not random): Random based on stratified bodyweights

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily



DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before and after dosing, and one and five hours after dosing during the working week; before and after dosing, and one hour after dosing during the weekend.


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for males; weeks 1, 2 and 3 and days 1 and 4 post partum for females.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data:


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 13, females at day 5 post-partum
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals:5 males and 5 females/dose group


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:Day 13, females at day 5 post-partum
- Animals fasted: No data
- How many animals:5 males and 5 females/dose group

URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine:
- Animals fasted:


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: End of mating phase, 5 males/dose group; Day 4 post-partum, 5 females/dose group
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: No

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead. GROSS NECROPSY
- Gross necropsy consisted of external appearance


Statistics:
ANOVA, incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis, ANOVA and Mann-Whitney 'U' test.
Indices:
Mating index, pregnancy index, parturition index, live birth index, viability index, sex ratio, pre-implantation loss, post-implntation loss
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
There were no toxicologically significant deaths during the study.

Increased salivation was detected prior to dosing and up to 5 hours after dosing for animals of either sex treated with 1000 mg/kg bw/day from Day 9 onwards. One female treated with 150 mg/kg bw/day developed clinical signs consistent with inappropriate dosing on Day 5 and was subsequently terminated. One female treated with 1000 mg/kg bw/day had given birth to a number of pups of which the majority were found dead. Several clinical signs were observed in this animal, and the animal and litter were terminated.

BODY WEIGHT AND WEIGHT GAIN
A slightly reduced bodyweight gain was observed for 1000 mg/kg bw/day males during the first two weeks of the study. Reduced bodyweight gain was also observed for 1000 mg/kg bw/day females during the later stages of the gestation period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No adverse effect on dietary intake or food efficiency were detected.

FOOD EFFICIENCY
No adverse effect on dietary intake or food efficiency were detected.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
No intergroup differences were detected.

OPHTHALMOSCOPIC EXAMINATION
N/A

HAEMATOLOGY
Haematological assessments revealed elevated platelet counts in animals of either sex treated with 1000 mg/kg bw/day. Elevated haemoglobin, erythrocyte and haematocrit was also evident for males treated at 1000 mg/kg bw/day. No such effects were detected at 150 and 15 mg/kg bw/day.

CLINICAL CHEMISTRY
An increase in aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase levels were observed for 1000 mg/kg bw/day animals of either sex, together with elevated inorganic phosphorus, bilirubin and urea, and reduced cholesterol levels.

URINALYSIS
N/A

NEUROBEHAVIOUR
Open field arena observations revealed increased salivation for individual animals of either sex treated with 1000 mg/kg bw/day from Week 3.

ORGAN WEIGHTS
Females treated with 1000 mg/kg bw/day showed elevated liver weights.

GROSS PATHOLOGY
None

HISTOPATHOLOGY: NON-NEOPLASTIC
Groups of alveolar macrophages were seen with a higher incidence for females treated with 1000 mg/kg bw/day.
A higher incidence of sinus histiocytosis and/or foamy histiocytes was observed in relation to treatment with 1000 mg/kg bw/day.
Hyperkeratosis, frequently associated with acanthosis was seen in the forestomachs of animals of either sex treated with 1000 mg/kg bw/day. Focal ulceration of the forestomach epithelium was also seen in one high dose female.
Mucosal hypertrophy was seen in three males at the top dose group. A low incidence of mucosal hypertrophy was observed in females in all dose groups.

OTHER FINDINGS
MATING
No adverse effects on mating or fertility were observed.

OFFSPRING LITTER SIZE AND VIABILITY
No effects detected.

OFFSPRING GROWTH AND DEVELOPMENT
No effects detected.

LITTER OBSERVATIONS
No effects detected.

UTERINE EXAMINATION
No effects detected.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
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
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No effects occurred.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No effects observed on offspring development
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
A NOAEL of 150 mg/kg bw/day has been identified for systemic toxicity. A NOAEL of 1000 mg/kg bw/day has been identified for reproductive parameters.

Executive summary:

In a subchronic toxicity study Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) as administered to 5 Sprague-Dawley rats/sex/dose via gavage at dose levels of 0, 15, 150, or 1000 mg/kg bw/day).

 

Systemic changes in the lungs, mesenteric lymph node, stomach and duodenum occurred at the highest dose.The NOAEL is 150 mg/kg bw/day.

 

This repeat dose/reproductive toxicity screening study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study OECD 422 in rats.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31st October to 22nd November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
OECD TG414 dated June 2018
Deviations:
yes
Remarks:
Due to severe toxicity of the test substance that was observed in females treated at 1000 mg/kg bw/day, this complete high dose group was euthanized in extremis on Days 12-18 post-coitum.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
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 171 and 266 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 to 22°C.
- Humidity (%): Relative target humidity of 40 to 70% with the actual daily mean relative humidity being 40 to 53%.
- 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:
Route of administration:
oral: gavage
Vehicle:
propylene glycol
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 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 90% and 110%).
For the formulation of 100 mg/kg bw/day (low dose), the mean accuracy was 89% of target, which was only slightly below the criterion of 90-110% and, therefore, the results were accepted.
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
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, propylene glycol, 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: These doses were selected based on the results of a 14 day range finding test for the OECD TG408 study
- Rationale for animal assignment (if not random):
- Other:
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 at least once daily from Day 2 post-coitum up to and including Days 6-8 post-coitum. During this period, animals were observed 1.5 hours after dosing on dosing days (i.e. Days 6-8 post-coitum). From Days 6-9 post-coitum onwards, clinical observations were performed at least twice daily: immediately after dosing and 1.5 hours post-dose

BODY WEIGHT: Yes
- Time schedule for examinations: Time schedule: Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum. In order to monitor their health status, animal numbers. 73 and 84-88 from the 1000 mg/kg bw/day group were also weighed on Day 11-13 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.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Due to the severe toxicity of the test substance that was observed in females treated at the highest dose of 1000 mg/kg bw/day, this complete high-dose group was euthanized in extremis on Days 12-18 post-coitum. In these animals, severe clinical signs (e.g. hunched posture, piloerection, respiratory difficulties, uncoordinated movements, diarrhoea) were observed, along with other physiological and histopathological responses.

Salivation was observed directly after dosing amongst animals at all dose levels, with the incidence increasing with increasing dose. This was possibly related to the severe forestomach morphological effects seen during necropsy in all dose groups.
Mortality:
no mortality observed
Description (incidence):
No mortalities were observed in the controls and the 100 and 300 mg/kg bw/day treatment groups
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight gain, before and after correction for the weight of the uterus of the pregnant female (up to 0.83x and 0.5x of the controls, respectively) was observed from the initiation of treatment onwards, together with lower food consumption (up to 0.77x of the controls).
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
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No changes in behaviour were noted in the 100 and 300 mg/kg bw/day treatments compared to the controls.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no treatment-related organ weight or organ/body weight changes in the thyroid glands of females treated at 100 and 300 mg/kg bw/day. No organ weights were determined at the dose level of 1000 mg/kg bw/day as all dams were sacrificed in extremis.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related macroscopic findings were noted in the gastro-intestinal tract from 100 mg/kg bw/day.

An irregular surface of the forestomach was noted in 7/22 females treated at 100 mg/kg bw/day, in 19/22 females treated at 300 mg/kg bw/day and in all animals treated at 1000 mg/kg bw/day. In 5/22 animals treated at 1000 mg/kg bw/day, the stomach had grown together with other structures like the diaphragm, spleen and/or liver. Additionally, the duodenum was thickened in 1/22 and 7/22 females at 300 and 1000 mg/kg bw/day, respectively, and the jejunum was thickened in 4/22 females treated at 1000 mg/kg bw/day. For one animal treated at 1000 mg/kg bw/day, gelatinous content in the duodenum, jejunum and ileum was observed.

All other macroscopic findings were within the range of background gross observations encountered in rats of this age and strain used in these types of studies.

Watery fluid in the uterus, found in one high dose female was related to the stage in the estrous cycle and is a normal finding.

Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At the lowest test dose of 100 mg/kg bw/day, test substance-related findings were limited to an irregular surface of the forestomach and/or thickening of the duodenum, which was observed in 7/22 (32%) animals, but was not considered to be adverse based on a histopathological assessment of the severity of these local effects.

At the 300 mg/kg bw/day dose level, the test substance caused adverse local effects on the surface of the forestomach in 19/22 (86%) of females.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No neoplastic histopathological effects were evident in the controls and treatment groups of 100 and 300 mg/kg bw/day.
Other effects:
no effects observed
Description (incidence and severity):
THYROID HORMONE ANALYSIS
Analysis of the blood samples taken from adult females at necropsy for thyroid (T3, T4 and TSH) hormones showed a minimal decrease in mean total T3 and mean total T4 levels in animals treated at 300 mg/kg bw/day (0.9x levels in controls and 0.85x levels in controls, respectively). Slightly increased serum levels of the thyroid stimulating hormone (TSH) were evident at 300 mg/kg bw/day (1.4x levels in controls). However, these increases were not statistically significant and were within the available historical control data.
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 100 and 300 mg/kg bw/day 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 100 and 300 mg/kg bw/day 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 100 and 300 mg/kg bw/day compared to the controls.

A relatively high incidence (5.0%, compared with 0.4% in controls) of late resorptions in dams treated at 100 mg/kg bw/day was observed, exceeding the historical control data, which resulted in a slightly lower percentage of viable fetuses in the low dose group. As this increased incidence of late resorptions was only observed in dams treated at 100 mg/kg bw/day, which was mainly caused by one female that had a single resorption only, this was considered not to be test substance-related.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the number of dead foetuses at 100 and 300 mg/kg bw/day 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 100 and 300 mg/kg bw/day compared to the controls.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
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 300 mg/kg/day.

Due to the premature sacrifice of the dams treated at 1000 mg/kg/day, no data on fetal parameters were determined at this dose level.

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: embrytoxicity
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Key result
Developmental effects observed:
no
Conclusions:
In the absence of adverse findings in any of the other parameters at the lowest dose level, the systemic maternal No Observed Adverse Effect Level (NOAEL) for Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) was determined to be 100 mg/kg bw/day.

The developmental NOAEL for Distilled Grade was established as being at least 300 mg/kg bw/day, as no litters were available at 1000 mg/kg bw/day as these dams were sacrificed in extremis.
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 propylene glycol 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 14 day range finding test for the OECD TG408 study. The study included a vehicle (propylene glycol) treated control group. Each study group contained 22 mated females.

 

Initially the protocol designated a high dose group of 1000 mg/kg bw/day. However, this group was euthanizedin extremison Days 12-18post-coitumdue to severe adverse clinical signs.

 

The test substance administered at dose levels of 100 mg/kg bw/day was not considered to cause maternal toxicity. No systemic toxic effects and non-adverse local effects on the surface of the forestomach were observed. At the 300 mg/kg bw/day dose level, the test substance was maternally toxic causing both adverse systemic toxic effects (on body weight gain) and adverse local effects on the surface of the forestomach. However, there was no evidence of embryotoxic, fetotoxic or teratogenic effects at the exposure doses of 100 and 300 mg/kg bw/day.

 

No effects on endocrine-disruption related apical morphological and histological endpoints in females (thyroid weight and histopathology and indicators of thyroid hormonal activity) and foetuses (i.e. anogenital distance and sex ratio) were observed in the study at either 100 or 300 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Additional information

.

Justification for classification or non-classification

No adverse effects on reproduction or development were observed in a 28 day reproductive screening study, therefore classification is not considered appropriate.

In an OECD 414 study, in the absence of adverse findings in any of the other parameters at the lowest dose level, the systemic maternal No Observed Adverse Effect Level (NOAEL) for Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) was determined to be 100 mg/kg bw/day. No adverse effects on reproduction or development were observed in the study.

Additional information