Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled, oligomerisation products with 1-chloro-2,3-epoxypropane

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL >= 1000 mg/kg bw (no findings regarding reproduction success were observed) - OECD 422, GLP

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

some minor deviations

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sandhofer Weg 7 97633 Sulzfeld, Germany
- Age at study initiation: 11-12 weeks
- Weight at study initiation: 259 g – 296 g (males); 173 g – 196 g (females) (at acclimatisation)
- Fasting period before study: no information
- Housing: Clean conventional housing
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6-9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 10 cycles/hour
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not soluble in water in concentrations necessitated for this study. Formulations prepared with the vehicle are suitable for oral administration as it is the anticipated route of human exposure to the test item.
- Concentration in vehicle:
- Amount of vehicle (if gavage): Treatment volume was 5 mL/kg body weight in all groups
- Lot/batch no. (if required): N83746634

Details on mating procedure:

- Proof of pregnancy: vaginal plug and sperm in vaginal smear
- M/F ratio per cage: 1/1
- After successful mating each pregnant female was caged (how): single

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

males: 48 -61 days
females: >= 46 days

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: preliminary study

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day
- Cage side observations: Viability and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: at least once weekly

OTHER: see study record under 'Repeated dose toxicity'

Sperm parameters (parental animals):

Parameters examined in male parental generation:
testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, weight gain

GROSS EXAMINATION OF DEAD PUPS: no

Postmortem examinations (parental animals):

Sacrifice: Animals were euthanised when found to be moribund or when the adequate number of litters according to guideline OECD 422 was achieved. All animals were sacrificed humanely by asphyxiation in a CO2 atmosphere.

Organ weights: The body weight of all animals killed at the end of the treatment period was recorded before sacrifice, and the organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

Macroscopic post-mortem examination: A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mild discomfort throughout the whole application period was observed for the animals treated with the high and the medium dose of the test item (wiping of nose and mouth through the cage bedding, salivation after application, bleeding of mucous membranes at nose and mouth, respirators sounds). Moreover, some male animals of the high dose group became lethargic after application of the test item on individual days. A biological and particularly toxicological relevance of these observations could not be excluded.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Regarding the body weight and the body weight gain, no significant differences were observed between all test item treated animals (male and female) and their respective vehicle control animals. Occasional differences observed for the females were assumed to be of natural origin based on the pregnancy status of the animals.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Regarding the body weight and the body weight gain, no significant differences were observed between all test item treated animals (male and female) and their respective vehicle control animals. Occasional differences observed for the females were assumed to be of natural origin based on the pregnancy status of the animals.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Regarding the body weight and the body weight gain, no significant differences were observed between all test item treated animals (male and female) and their respective vehicle control animals. Occasional differences observed for the females were assumed to be of natural origin based on the pregnancy status of the animals.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No relevant test item induced effects on any haematology or clinical biochemistry parameter
could be detected

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No relevant test item induced effects on any haematology or clinical biochemistry parameter
could be detected

Endocrine findings:

not examined

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No alterations regarding general behaviour of the rats were observed during in-life phase

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The type, incidence and severity of all microscopic findings observed did not indicate a relationship to the treatment with the test item. The repeated oral administration of the test item did not produce any evidence of pathomorphological findings that are considered to be due to a toxic effect of the test item.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

ORGAN WEIGHTS (PARENTAL ANIMALS)
For females of the high dose group (1000 mg/kg bw) ovary weight was increased.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: No adverse effects observed

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

A statistical significant decrease in the amount of pups born alive was detected for the animals of the high and medium dose groups

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:

not examined

Histopathological findings:

not examined

The total amount of live pups born is statistically significant reduced in the test item treated groups when compared to the vehicle control animals. A total of 11 animals of the high dose group gave birth to 69 pups (mean: 6,3). Ten animals of the medium dose group gave birth to 82 pups (mean: 8,2) and 9 animals of the low dose group gave birth to 77 pups (mean: 8,6). In contrast, 12 animals of the vehicle control group gave birth to 124 pups (mean: 10,3). The data were analysed additionally by Dunnett’s post-hoc t-test after One-Way ANOVA. Analysis revealed a statistically significant reduced amount of live born pups in the animals treated with the high and the medium dose of the test item. Regarding the loss of offspring it is striking, that six animals of the high dose group, eight animals of the medium dose group and seven animals of the low dose group had three or more pre-implantation losses (difference between corpora lutea and implantation sites), whereas only three animals of the vehicle control group had more than three pre-implantation losses (see table 10). Moreover, the mean amount of pre-implantation losses is about 30 to 50 % higher in the test item treated animals (High dose: 4,5; Medium dose: 3,9; Low dose: 4,1) when compared to the vehicle control animals (3,0).
No further abnormalities were detected during gestation and lactation phase. In summary, an increase in pre- implantation losses and a corresponding decrease in the amount of pups born alive was detected in all dose groups compared to the vehicle control group. However, pup growth and development, as far as monitored, was normal for all test item treated animals when compared to the vehicle control animals. No further differences were observed regarding reproduction success (achievement and duration of pregnancy, post- implantation losses, survival rate of pups, development of pups) for all test item treated animals when compared to the vehicle control animals. In the absence of another test item related effect the reduced amount of live born could not be rated as clear evidence for a toxic effect. Nonetheless, a test item related effect could not be excluded.

Dose descriptor:

NOAEL

Remarks:

Development

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Pup growth and development (day 0 and day 4 post-partum) were normal for all test item treated animals when compared to the vehicle control animals

Dose descriptor:

NOAEL

Remarks:

Embryo-/fetotoxicity

Generation:

F1

Effect level:

62.5 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: number of pups born alive

Reproductive effects observed:

not specified

Conclusions:

Under the conditions of the present study, CARDOLITE NC-513 did not cause toxic changes and did not influence male and female reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Wistar rats after repeated dose oral administration at 62.5, 250 or 1000 mg/kg bw/day.
The number of pups born alive was lower with respect to control at 250 and 1000 mg/kg bw/day.
Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL (F0 systemic) females/males: 250 mg/kg bw/day
NOAEL (F0 reproduction) females/males: >=1000 mg/kg bw/day
NOAEL (F1 embryo-/fetotoxicity) 62.5 mg/kg bw/day
NOAEL (F1 development): >=1000 mg/kg bw/day (pup growth and development (day 0 and day 4 post-partum) were normal for all test item treated animals when compared to the vehicle control animals)

Executive summary:

 

In a GLP-study according to OECD Tes Guideline 422 the toxic effects of the test item CARDOLITE NC-513 at doses of 62.5, 250 and 1000 mg/kg body weight on the subacute toxicity and the development and reproduction of Wistar rats after oral administration were under examination.The substance was at least applied for 46 days daily to rats of both sexes. The following observations were made.

 

General and detailed clinical signs:

Mild discomfort throughout the whole application period was observed for the animals treated with the high and the medium dose of the test item (wiping of nose and mouth through the cage bedding, salivation after application, bleeding of mucous membranes at nose and mouth, respirators sounds). Moreover, some male animals of the high dose group became lethargic after application of the test item on individual days. A biological and particularly toxicological relevance of these observations could not be excluded.

 

Body weight, food and water consumption: Regarding the body weight and the body weight gain, no significant differences were observed between all test item treated animals (male and female) and their respective vehicle control animals. Occasional differences observed for the females were assumed to be of natural origin based on the pregnancy status of the animals.

No test item related tendencies regarding food and water consumption of all test item treated animals (male and female) could be observed throughout the whole study phase when compared to their respective vehicle control animals. All fluctuations observed were most likely of natural origin.

 

Haematology and clinical biochemistry: no effects

 

Reproduction and Development:

A statistical significant decrease in the amount of pups born alive was detected for the animals of the high and medium dose groups. This was not statistically significant for the animals of the low dose group. The increase in pre-implantation losses was not significant for any dose group. Pup growth and development (day 0 and day 4 post-partum) were normal for all test item treated animals when compared to the vehicle control animals. No further findings regarding reproduction success (achievement and duration of pregnancy, post- implantation losses, survival rate of pups, development of pups) were observed in animals of any test item treated group when compared to the vehicle control group.

 

Necropsy:

The determination of organ weights, as part of the necropsy, showed a statistically significant increase of the liver weight (male; absolute and relative), a statistically significant decrease of the prostate weight (absolute and relative), a statistically significant increase of the brain weight (female; absolute), and a statistically significant weight increase of the right ovary (absolute and relative) within the animals treated with the high dose of the test item. However, the changed organ weights were not accompanied by structural changes (see below 'Histology'). Moreover, with increasing dose levels the amount of male animals having slightly developed mammary glands declined. Besides some further individual findings, heterogeneously distributed over all dose groups, no further apparent observations were made that could be related to the administration of the test item.

 

Histology: no effects

 

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL (F0 systemic) females/males: 250 mg/kg bw/day
NOAEL (F0 reproduction) females/males: >=1000 mg/kg bw/day
NOAEL (F1 embryo-/fetotoxicity) 62.5 mg/kg bw/day
NOAEL (F1 development): >=1000 mg/kg bw/day (pup growth and development (day 0 and day 4 post-partum) were normal for all test item treated animals when compared to the vehicle control animals)

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Quality of whole database:

GLP-compliant OECD Guideline study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Subacute NOAEL (F1 embryo-/fetaltoxicity): 62.5 mg/kg bw/day (reduced number of pubs born alive) - OECD 422, GLP

Subacute NOAEL (F1 developmental): >= 1000 mg/kg bw/day (no effects were observed on pup growth and development (day 0 and day 4 post-partum)) - OECD 422, GLP

Subacute NOAEL (F1 development): No effect level could be derived - OECD 414, GLP

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

some minor deviations

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sandhofer Weg 7 97633 Sulzfeld, Germany
- Age at study initiation: 11-12 weeks
- Weight at study initiation: 259 g – 296 g (males); 173 g – 196 g (females) (at acclimatisation)
- Fasting period before study: no information
- Housing: Clean conventional housing
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6-9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 10 cycles/hour
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not soluble in water in concentrations necessitated for this study. Formulations prepared with the vehicle are suitable for oral administration as it is the anticipated route of human exposure to the test item.
- Concentration in vehicle:
- Amount of vehicle (if gavage): Treatment volume was 5 mL/kg body weight in all groups
- Lot/batch no. (if required): N83746634

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Proof of pregnancy: vaginal plug and sperm in vaginal smear
- M/F ratio per cage: 1/1
- After successful mating each pregnant female was caged (how): single

Duration of treatment / exposure:

males: 48 -61 days
females: >= 46 days

Frequency of treatment:

daily

Duration of test:

males: 48 -61 days
females: >= 46 days

Dose / conc.:

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

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: preliminary study

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day
- Cage side observations: Viability and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: at least once weekly

OTHER: see study record under 'Repeated dose toxicity'

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: No

Fetal examinations:

- External examinations: No
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
For females of the high dose group (1000 mg/kg bw) findings in ovary weight.

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The total amount of live pups born is statistically significant reduced in the test item treated groups when compared to the vehicle control animals. A total of 11 animals of the high dose group gave birth to 69 pups (mean: 6,3). Ten animals of the medium dose group gave birth to 82 pups (mean: 8,2) and 9 animals of the low dose group gave birth to 77 pups (mean: 8,6). In contrast, 12 animals of the vehicle control group gave birth to 124 pups (mean: 10,3). The data were analysed additionally by Dunnett’s post-hoc t-test after One-Way ANOVA. Analysis revealed a statistically significant reduced amount of live born pups in the animals treated with the high and the medium dose of the test item.
Regarding the loss of offspring it is striking, that six animals of the high dose group, eight animals of the medium dose group and seven animals of the low dose group had three or more pre-implantation losses (difference between corpora lutea and implantation sites), whereas only three animals of the vehicle control group had more than three pre-implantation losses (see table 10). Moreover, the mean amount of pre-implantation losses is about 30 to 50 % higher in the test item treated animals (High dose: 4,5; Medium dose: 3,9; Low dose: 4,1) when compared to the vehicle control animals (3,0).

Dose descriptor:

NOAEL

Remarks:

Development

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Pup growth and development (day 0 and day 4 post-partum) were normal for all test item treated animals when compared to the vehicle control animals

Dose descriptor:

NOAEL

Remarks:

Embryo-/fetotoxicity

Effect level:

62.5 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Number of pups born alive

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Under the conditions of the present study, CARDOLITE NC-513 did not cause toxic changes and did not influence male and female reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Wistar rats after repeated dose oral administration at 62.5, 250 or 1000 mg/kg bw/day.
The number of pups born alive was lower with respect to control at 250 and 1000 mg/kg bw/day. Pup growth and development (day 0 and day 4 post-partum) were normal for all test item treated animals when compared to the vehicle control animals.
Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL (F0 systemic) females: 250 mg/kg bw/day
NOAEL (F0 reproduction) females: >=1000 mg/kg bw/day
NOAEL (F1 embryo-/fetotoxicity) 62.5 mg/kg bw/day
NOAEL (F1 development): >=1000 mg/kg bw/day

Executive summary:

In a GLP-study according to OECD Tes Guideline 422 the toxic effects of the test item CARDOLITE NC-513 at doses of 62.5, 250 and 1000 mg/kg body weight on the subacute toxicity and the development and reproduction of Wistar rats after oral administration were under examination.The substance was at least applied for 46 days daily to rats of both sexes. The following observations were made.

 

General and detailed clinical signs:

Mild discomfort throughout the whole application period was observed for the animals treated with the high and the medium dose of the test item (wiping of nose and mouth through the cage bedding, salivation after application, bleeding of mucous membranes at nose and mouth, respirators sounds). Moreover, some male animals of the high dose group became lethargic after application of the test item on individual days. A biological and particularly toxicological relevance of these observations could not be excluded.

 

Body weight, food and water consumption: Regarding the body weight and the body weight gain, no significant differences were

observed between all test item treated animals (male and female) and their respective vehicle control animals. Occasional differences observed for the females were assumed to be of natural origin based on the pregnancy status of the animals.

No test item related tendencies regarding food and water consumption of all test item treated animals (male and female) could be observed throughout the whole study phase when compared to their respective vehicle control animals. All fluctuations observed were most likely of natural origin.

 

Haematology and clinical biochemistry: no effects

 

Reproduction and Development:

A statistical significant decrease in the amount of pups born alive was detected for the animals of the high and medium dose groups. This was not statistically significant for the animals of the low dose group. The increase in pre-implantation losses was not significant for any dose group. Pup growth and development (day 0 and day 4 post-partum) were normal for all test item treated animals when compared to the vehicle control animals. No further findings regarding reproduction success (achievement and duration of pregnancy, post- implantation losses, survival rate of pups, development of pups) were observed in animals of any test item treated group when compared to the vehicle control group.

 

Necropsy:

The determination of organ weights, as part of the necropsy, showed a statistically significant increase of the liver weight (male; absolute and relative), a statistically significant decrease of the prostate weight (absolute and relative), a statistically significant increase of the brain weight (female; absolute), and a statistically significant weight increase of the right ovary (absolute and relative) within the animals treated with the high dose of the test item. However, the changed organ weights were not accompanied by structural changes (see below 'Histology'). Moreover, with increasing dose levels the amount of male animals having slightly developed mammary glands declined. Besides some further individual findings, heterogeneously distributed over all dose groups, no further apparent observations were made that could be related to the administration of the test item.

 

Histology: no effects

 

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL (F0 systemic) females: 250 mg/kg bw/day
NOAEL (F0 reproduction) females: >=1000 mg/kg bw/day
NOAEL (F1 embryo-/fetotoxicity) 62.5 mg/kg bw/day
NOAEL (F1 development): >=1000 mg/kg bw/day (pup growth and development (day 0 and day 4 post-partum) were normal for all test item treated animals when compared to the vehicle control animals)

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 31.05.2016 to 08.05.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material:KK-1883
- Expiration date of the batch:October 2017
- Purity test date:Feb 2015


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature


OTHER SPECIFICS:
Purity: 100%

Species:

rat

Strain:

other: Wistar (Cmdb: WI)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Wistar female rats (Cmdb: WI; outbred) coming from the husbandry of laboratory animals of the Experimental Medicine Centre at the Medical University in Białystok kept behind the breeding barrier (Register No: 0043). Males used for mating with nulliparous female rats came from the same husbandry
- Age at study initiation:
- Weight at study initiation: 252.6 g. (sighting); 242.3 g. (main)
- Housing: The females were kept in plastic cages with metal wire lid and dimensions: 37 x 22 x 15 cm (length x width x height). UV-sterilized wood shavings were used as bedding [SOP/T/16, SOP/T/48]. At mating one female was placed in one cage with one male. At pregnancy the females were housed individually.
- Diet: Sighting range-finding study, during the acclimatization and the experiment the animals were given ad libitum access to “Murigran” standard laboratory fodder. Main study, :ad libitum access to “Laboffed H Standard” standard laboratory fodder
- Water: ad libitum tap water
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 28°C
- Humidity (%): 45 – 90%
- Air changes (per hr): 12 hours light / 12 hours dark
- Photoperiod (hrs dark / hrs light): 1bout 16 times/h

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Sighitng study: The test item was diluted in corn oil directly before administration, in the appropriate concentration for each dose. A constant volume of solution i.e. 0.52 mL of solution /100 g b.w. was used at every level of dosage.
Main study: The test item was diluted in the corn oil once a week, in the appropriate concentration for each dose and in volume sufficient for the weekly demand. Prepared concentrations were stored at room temperature. A constant volume of solution i.e. 0.52 mL of solution /100 g b.w. was used at every level of dosage.


VEHICLE
- Justification for use and choice of vehicle: Use of the corn oil is recommended by the OECD Guidelines in case water cannot be used as a vehicle.
- Lot/batch no. (if required): L5294; expiry date: October 2017 - Sighting range-finding study and Lot No.: 6037; expiry date: February 2018 – main study

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples of the test item solutions were chemically analyzed three times (every two weeks). The Phase Report for the formulation analysis can be found in the Appendix No. 1 to the Report (GLP Certificate of Test Facility is in the Appendix No. 7). The % nominal concentration for the samples measured during the 3 sessons was 76-108.6% (Appendix 1).

Details on mating procedure:

- Impregnation procedure: Pregnant females in the sighting study and the main study were obtained by mating with not related males.

- M/F ratio per cage: One male was used to mate with two females. For each mating one female was placed in a cage with one male

- Length of cohabitation: Length of cohabitation was from 1 to 2 days.

- Proof of pregnancy: Every day in the morning vaginal smears were taken from females. The smears collected with the use of inoculating loop were transferred to 2-3 drops of physiological fluid on a slide, fixed over a flame, stained with Giemza and May-Grunwald stains and then evaluated with the use of microscope [6]. The sperm-positive females were accepted as mated and the day of observation of sperm in the smear was accepted as day 0 of gestation.

Duration of treatment / exposure:

Day 5 to 19 of gestation (sighting)
Day 0 to 19 of gestation (main)

Frequency of treatment:

Daily

Duration of test:

On day 20 of gestation, the females were euthanised

Dose / conc.:

6.25 mg/kg bw/day (nominal)

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Dose / conc.:

625 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The sighting study was conducted on 28 females divided into four experimental groups: three treated groups and one control group. Each group consisted of 7 females. From day 5 to 19 of gestation the females of treated group were given by gavage the test item dissolved in corn oil. The following doses of the test item were used: in group 1 – 12.0 mg/kg b.w., in group 2 – 62.5 mg/kg b.w. and in group 3 – 325.0 mg/kg b.w. Females of the control group (group 0) were given the corn oil.

All 28 mated females were pregnant No pathological changes were noticed in females at the necropsy.
One early resorption was noticed in control group. One early resorption was noticed in group 1. Two early resorptions were noticed in group 2 and one early resorption was noticed in group 3.
Cranial vault fissure (cranioschisis) in one fetus and hemorrhage in five fetuses were noticed in the control group. Hemorrhage in three fetuses was found in group 1. Hemorrhage in six fetuses was found in group 2. Hemorrhage
in two fetuses and conjoined placenta of two fetuses were found in group 3.
In group 1 statistically significantly higher weight of the fetuses without placenta and higher weight of placenta were noticed in comparison with control group.
In group 2 statistically significantly higher weight of the fetuses with placenta and without placenta were noticed in comparison with control group.
In group 3 statistically significantly higher weight of the fetuses without placenta was noticed in comparison with control group.
In group 1, group 2 and group 3 the average length of the fetuses with tail and the fetuses without tail was statistically significantly greater in comparison with the control group.
In group 1, group 2 and group 3 the average number of ossification points of metacarpus were statistically significantly greater in comparison with the control group.

Based on the data of the sighting study the doses for the main study were determined (6.25, 62.5 adn 625 mg/kg bw/day) and the treatment period was increased up to the entire period of gestation (DG 0-19).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
General evaluation of animals, i.e. observation for morbidity and mortality was performed twice a day during labour week and once a day on days off.

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule: Day of beginning of experiment (day 0), and then on 2nd, 5th, 8th, 11th, 14th, 17th and 20th day of gestation.(main study)

BODY WEIGHT: Yes
0, 2nd, 5th, 8th, 11th, 14th, 17th and 20th day of gestation (main study)


POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day # 20
- Organs examined: At necropsy the females were examined macroscopically for any abnormalities in body structure or pathological changes which could have influenced the gestation. After caesarean section the uterus from each female with fetuses was dissected.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
After removing of the fetuses left and right uteri horn were examined. Number of viable and dead fetuses as well as number of resorptions was determined in each horn. Each pregnant uterus after removing of fetuses was weighed. The uteri of non-pregnant females and non-pregnant horns of the uteri were fixed between two glass plates and overexposed by electric lamp to confirm the non-pregnant status. Then these uteri were stained using ammonium sulphide and then again overexpose with light by electric lamp in order to confirm the non-pregnant status. at the necropsy ovaries from each pregnant female were collected and fixed in 10% solution of formalin in order to determine number of corpora lutea.

Fetal examinations:

- External examinations: Yes:
Sex of each fetus, body weight, length with tail and without tail was determined.
Weight of the fetuses with placenta and fetal membranes was determined first and then weight of the fetuses without placenta and fetal membranes as well as weight of placenta were measured. After removal of the fetal membranes each fetus was measured with tail and without tail, and detailed gross evaluation was performed.half of fetuses from each litter was subjected to evaluation of skeleton and in the other half, formation of particular cavities and presence of internal organs was evaluated

Statistics:

The obtained results (body weight of pregnant females, food consumption of pregnant females, number of corpora lutea, number of fetuses in litter, number of males and females in litter, weight of uteri, weight of fetuses, length of fetuses, number of ossification points in sternum and limbs) were elaborated statistically with the use of t-Student test with p ≤ 0.05. The elaborated results are presented in Tables in form of average values and standard deviation

Indices:

Mating, viability

Historical control data:

No speicifc historical control data provided.

Clinical signs:

no effects observed

Description (incidence and severity):

No changes in behavior were observed in females.

Mortality:

no mortality observed

Description (incidence):

Among 100 mated females, six of them were not pregnant: two in control group, one in group 1, three in group 2.
94 pregnant females were used for evaluation: twenty three females in control group, twenty four females in group 1, twenty two females in group 2 and twenty five females in group 3.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Average body weights of the females in particular groups, recorded on days of weighing, are presented in Graph 1 and Table 8.
During the entire period of the experiment average body weight of the females of the treated groups
did not differ statistically significantly from average body weight of the females of the control group.
Transient body weight losses observed in some pregnant females were slight and did not cause
statistically significant differences in average body weights of the females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The average food consumption of the females of the treated groups and control group is presented in
Graph 2 and Table 10.
During the experiment, food consumption of the females in group 1 was in the same level with
average food consumption of the females of the control group and was not statistically significantly
different.
Food consumption of the females in group 2 was statistically significantly greater in comparison with
the control group from 17th to 20th gestation day.
Food consumption of the females group 3 was statistically significantly greater in comparison with the
control group from 8th to 20th gestation day.

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):

The average weight of uterus of pregnant females is presented in Graph 3 and Table 12.
The average weight of the uterus of pregnant females of the treated groups did not differ statistically
significantly from average weight of the uterus of pregnant females of the control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No pathological changes were stated at gross examination of the females performed at caesarean
section.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

not examined

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Mean pre implantation losses / group (%0 and Mean post implantation losses / group (%): No abnormalities detected. (Table 16)

Total litter losses by resorption:

not examined

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

10 early resorptions (Control); 12.48 fetuses and 0.44 resorptions fell on average to one female
8 early resorptions were noticed (Group 1); 2.00 fetuses and 0.34 resorptions fell on average to one female.
12 early resorptions (Group 2); 12.05 fetuses and 0.55 resorptions fell on average to one female
23 early resorptions ( Group 3); 12.48 fetuses and 0.92 resorptions fell on average to one female.
(Table 16)

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

Four fetuses were dead: one in the control group, two in group 1 and one fetus in group 3 (Table 15)

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The average number of corpora lutea are presented in Table 16.

In control group 287 fetuses were obtained from 23 pregnant females, the litters numbered from 1 to 17 fetuses. Average number of corpora lutea amounted to 14.13.
In group 1, 288 fetuses were obtained from 24 pregnant females, the litters numbered from 1 to 16 fetuses. Average number of corpora lutea amounted to 13.33.
In group 2, 265 fetuses were obtained from 22 pregnant females, the litters numbered from 5 to 16 fetuses. Average number of corpora lutea amounted to 14.05.
In group 3, 312 fetuses were obtained from 25 pregnant females, the litters numbered from 6 to 17 fetuses. Average number of corpora lutea amounted to 14.44.

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

6.25 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

food consumption and compound intake

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The average weights of the fetuses and average weights of placenta in particular groups are
presented in Graph 7, Graph 8, Graph 9 and Table 18.
The average weight of the fetuses with placenta and weight of fetuses without placenta were
statistically significantly greater in all treated groups compared with the control group.
The average weight of placenta was statistically significantly greater in group 1 and group 3 compared
with the control group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

not examined

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The detailed pathological changes observed in the fetuses are presented in Table 22.
At gross necropsy blood hemorrhages in different parts of body were noticed in 61 fetuses.
In the control group blood hemorrhages were noticed in 15 fetuses: on a head and a metatarsus, on a mandible, on a neck, on a thorax, two on a back, on an abdomen, on a forelimb , on a axilla, two on a shank, on a carpus and metacarpus, on a metatarsus, two on a foot.
In group 1 blood hemorrhages were noticed in 20 fetuses: three on an ear, on a mandible, four on a back, two on an abdomen, four on a scapula, two on a groin, on a thigh, on a shank, two on a foot.
In group 2 blood hemorrhages were noticed in 15 fetuses: two on a thorax, two on a back, four on an abdomen, on an anus, on a tail, on a scapula, on an elbow, two on a shank, on a metatarsus.
In group 3 blood hemorrhages were noticed in 11 fetuses: on an eye, on an ear, three on a neck, two on a back, on an abdomen, two on a shank, on a foot.
Schistocelia (congenital fissure of the abdominal wall with protrusion of the viscera), micrognathia, lack of skull fornix were noticed in one fetus of female No 11 in group 1.
Two properly formed fetuses (male and male) had conjoined placenta in female No 25 in group 2.
All fetuses of control group and groups treated with test item (except one fetus in group 1 described above) had normally developed body coverings, limbs, fingers and toes, auricular conchas, nasal apertures, eyelids. Normally developed heads and oral cavities as well as presence of an anus and a tail were noticed in all fetuses.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Changes in the skeletal system noticed in the fetuses are presented in Table 23.
In control group a misalignment of the two halves of ossification center in 2nd, 3rd and 4th ossification point of sternum in one fetus of female No 18 and a misalignment of the two halves of ossification center in 4th ossification point of sternum in one fetus of female No 4 were observed.
In group 1 no ossification points in sternum and unossified center of pubis bone were noticed in one fetus of female No. 2. Wavy ribs were noticed in three fetuses: in female No 1, No 9 and No 10.
In group 2 a misalignment of the two halves of ossification center in 2nd, 3rd and 4th ossification point of sternum in one fetus of female No 22 and a misalignment of the two halves of ossification center in 3rd and 4th ossification point of sternum in one fetus of female No 4 were noticed. Three ossification points in sternum were stated in six fetuses: two in female No 2 and one in female No 6, No 7, No 10 and No 25.
In group 3 a misalignment of the two halves of ossification center in 2nd and 3rd ossification point of sternum in one fetus of female No 20, a misalignment of the two halves of ossification center in 4th ossification point of sternum in one fetus of female No 15, and a misalignment of the two halves of ossification center in 4th and 5th ossification point of sternum in one fetus of female No 19. Three ossification points in sternum were stated in two fetuses: one in female No 2 and one in female No 20.

Percentage of fetuses with number of ossification points in sternum is presented in Table 24.
In control group, in group 1 and in group 3 the greatest percentage of fetuses had 6 points of ossification in sternum, however this value was the greatest in group 1. In group 2 the greatest percentage of fetuses had 5 ossification points of sternum. Some similar percentage of fetuses in control group and in groups 2 and 3 had 4 ossification points of sternum. The percentage of fetuses with 4 points of ossification in the sternum in group 1 is two times lower than the control group.
Furthermore, 1 fetus in group 1 (0.67% of all fetuses in the group) had no points of ossification in sternum, 6 fetuses in group 2 (4.41 % of all fetuses in the group) and 2 fetuses in group 3 (1.24% of all fetuses in the group) had 3 ossification points in sternum.

Percentage of fetuses with number of ossification points in limbs is presented in Table 25.
In the control group and in the treated group the greatest percentage of fetuses had 4 ossification points of metacarpus. In group 2 the percentage of fetuses with 4 and 3 ossification points of metacarpus is similar to the same values in the control group. In group 1 and group 3 percentage of fetuses with 4 ossification points is greater and percentage of fetuses with 3 ossification points is lower compared with the same values in the control group.
In the control group and in group 3 all fetuses had 4 ossification points of metatarsus.
In group 1 one fetus (0.67%) and in group 2 one fetus (0.74%) had 3 points of ossification in metatarsus.

The average number of ossification points in sternum and limbs – sighting study is presented in Table 26. Statistically significantly greater number of ossification points in metacarpus was observed in all treated groups compared with the control group.

The average number of ossification points in sternum and limbs – main study is presented in Graph 12, Graph 13, Graph 14 and Table 27. Statistically significantly greater number of ossification points in sternum and statistically significant greater number of ossification points in metacarpus were observed in group 1 compared with the control group.
Statistically significantly lower number of ossification points in sternum was observed in group 2
compared with the control group. Statistically significantly greater number of ossification points in metatarsus was observed in group 3 compared with the control group.

Visceral malformations:

no effects observed

Description (incidence and severity):

Evaluation of sagittal sections indicated that fetuses from the females of control group and the treated groups in the main study had normally developed skull bones, back-bone, ribs, acromial and pelvic girdles as well as internal organs and body cavities. No differences in formation and location of internal organs in body cavities were stated in fetuses of treated groups compared with fetuses of control group. The sagittal section of the fetus of female No 11 in group 1 in the main study confirmed changes which had been observed in this fetus during the gross necropsy: schistocelia, micrognathia and the lack of the skull fornix.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The average weights of the fetuses and average weights of placenta in particular groups are presented in Graph 7, Graph 8, Graph 9 and Table 18. The average weight of the fetuses with placenta and weight of fetuses without placenta were statistically significantly greater in all treated groups compared with the control group. The average weight of placenta was statistically significantly greater in group 1 and group 3 compared with the control group.
The average lengths of the fetuses in particular groups are presented in Graph 10, Graph 11 and
Table 20. The average lengths of the fetuses with tail and lengths of the fetuses without tail were statistically significantly greater in all treated groups compared with the control group.

Details on embryotoxic / teratogenic effects:

Greater average body weights and lengths of the fetuses in all treated groups and some changes in the average number of ossification points of the sternum and the metacarpus in the treated groups seem to be caused by the test item.
However, the authors themselves pointed out that for some of the changes in number of ossification points, no dose-response relationship was observed. Also, the information was given that it is known that a decrease in number of ossification points does not influence the postnatal development is later compensated, and an increase, on the other hand, is a result of the intensive growth of fetuses.
Changes in the fetus in group 1, which included malformation of the head and the trunk, were extensive and can be defined as polydysplasia. Since the test item at all doses used in the sighting and the main studies affected the growth of the fetuses, this malformation could be its teratogenic effect. However it should be pointed out that only one fetus in the low doses group was affected, and no such malformations occurred in animals of the higher dose groups.

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable because of methodological limitations

Abnormalities:

not specified

Developmental effects observed:

not specified

Results of Sighting Study

No differences in appearance and behavior between females of treated groups and females of control group were stated. Coat thinning on the forelimbs was observed in one female in group 2. No clinical signs were observed in the remaining females during the entire experiment. All females survived the period of experiment.

Slight body weight losses (from 1 to 7 g) were observed in some females during the initial period of experiment (0 – 8th day): in control group – in one female, in group 1 – in two females and in group 2 – in three females. Average body weight of pregnant females of treated groups was comparable with average body weight of pregnant females of control group during the entire experiment.

Average food consumption of pregnant females of treated groups was comparable with average food consumption of pregnant females of control group during the entire experiment.

All 28 mated females were pregnant. No pathological changes were noticed in females at the necropsy. One early resorption was noticed in control group. One early resorption was noticed in group 1. Two early resorptions were noticed in group 2 and one early resorption was noticed in group 3.Cranial vault fissure (cranioschisis) in one fetus and hemorrhage in five fetuses were noticed in the control group. Hemorrhage in three fetuses was found in group 1. Hemorrhage in six fetuses was found in group 2. Hemorrhage in two fetuses and conjoined placenta of two fetuses were found in group 3. In group 1 statistically significantly higher weight of the fetuses without placenta and higher weight of placenta were noticed in comparison with control group. In group 2 statistically significantly higher weight of the fetuses with placenta and without placenta were noticed in comparison with control group. In group 3 statistically significantly higher weight of the fetuses without placenta was noticed in comparison with control group. In group 1, group 2 and group 3 the average length of the fetuses with tail and the fetuses without tail was statistically significantly greater in comparison with the control group. In group 1, group 2 and group 3 the average number of ossification points of metacarpus were statistically significantly greater in comparison with the control group.

Conclusions:

Based on the resutls of a pre-natal developmental study, CARDOLITE ® NC-513 Cashew Nutshell Liquid, polymer with epichlorohydrin did not cause clinical changes and changes in behavior of pregnant females in the tested dose groups. At the doses of 62.5 mg/kg b.w. and 625.0 mg/kg b.w. the test item led to greater food consumption at the last period of pregnancy. The test item caused faster growth of fetuses in all tested dose groups. There is a possibility of embryotoxic influence at highest used dose - 625.0 mg/kg bw. Teratogenic effect and malformation induction cannot be excluded which is why no NOAEL was derived (with regard to a possible teratogenic effect, it should be pointed out that the only one fetus was affected, and no such malformations occurred in animals of the higher dose groups.)

Executive summary:

In a developmental toxicity study (T-24/16), CARDOLITE ® NC-513 was administered to 25 female Wistar (Cmdb: WI) rats/dose by gavage in corn oil at dose levels of 0, 6.25, 62.5 and 625 mg/kg bw/day from days 0 through 19 of gestation.

 

There were no mortalities or clinical signs. Statistically significantly greater food consumption at 62.5  mg/kg bw/day between day 17 and day 20 of gestation and at 625 mg/kg bw/day between day 8 and day 20 was noted. However, greater food consumption did not lead to statistically significantly greater weights of the females from these groups during the gestation period. No pathological changes were noted at gross examination of the females performed at caesarean section. The average weight of the uterus of pregnant females of the treated groups did not differ statistically significantly from average weight of the uterus of pregnant females of the control group. There was no difference in mean pre- or post-implantation losses between control and treated groups. After caesarean section, there were four fetuses dead which was not related to the test item. The numbers of resorptions at 6.25 and 62.5 mg/kg bw/day did not differ from the control group. The number of resorptions at 625 mg/kg bw/day was more than twice the control group. The number of resorptions at  625 mg/kg bw/day indicated significant intrauterine mortality (23/312; 7.37%) and could be an embryotoxic effect of the test item. The average number of corpora lutea in all treated groups are comparable with the number of corpora lutea in the control group. The maternal NOAEL is 6.25 mg/kg bw/day.

 

The average weight of the fetuses with placenta and weight of fetuses without placenta were statistically significantly greater in all treated groups compared with the control group.The average weight of placenta was statistically significantly greater at 6.25 mg/kg bw/day and 625 mg/kg bw/day compared with the control group. Subcutaneous blood hemorrhages in different parts of the fetal bodies, which occurred in the control and the treated groups, were not considered a treatment-related effect.

 

Schistocelia (congenital fissure of the abdominal wall with protrusion of the viscera), micrognathia, and the lack of skull fornix were noticed in one fetus at 6.25 mg/kg bw/day. This malformation of the head and the trunk were extensive and was defined as polydysplasia. Although single cases of malformation could be considered as incidental spontaneous defects referred to as congenital defects, the laboratory considered that the test item at all doses used in the sighting and the main studies affected the growth of the fetuses, this malformation could be its teratogenic effect. The registrant of the substance points out that the observations were only made in one fetus in the low dose group, which is why a toxicological relevance is highly unlikely.

 

Wavy ribs were observed in three fetuses at 6.25 mg/kg bw/day.  Misalignment of the two halves of ossified center in sternum was observed in some fetuses in all groups: in two fetuses in the control group, in two fetuses at 62.5 mg/kg bw/day and in three fetuses at 625 mg/kg bw/day.  Three ossification points of sternum were observed in six fetuses at 62.5 mg/kg bw/day and in two fetuses at 625 mg/kg bw/day. No ossification points of sternum and unossified center of pubis bone were observed in one fetus at 6.25 mg/kg bw/day.  Statistically significantly higher weight of the fetuses with and without placenta was noticed in all treated groups in comparison with the control group.  Statistically significantly higher weight of placenta was noticed at 6.25 mg/kg bw/day and 625 mg/kg bw/day in comparison with the control group.  Statistically significantly greater length of fetuses with tail and without tail was noticed in all treated groups in comparison with the control group. Statistically significantly greater number of ossification points in sternum and metacarpus were noticed at 6.25 mg/kg bw/day compared with the control group.  Statistically significantly lower number of ossification points in metatarsus was noticed at 62.5 mg/kg bw/daycompared with the control group. Statistically significantly greater number of ossification points in metacarpus was noticed at 625 mg/kg bw/day compared with the control group.

 

Based on the greater average body weights and lengths of the fetuses in all treated groups and some changes in the average number of ossification points of the sternum and the metacarpus in the treated groups appear to be related to the test item. Additionally, the changes observed in one fetus at 6.25 mg/kg bw/day could be a teratogenic effect. Therefore, based on the doses used in the study, the NOAEL for fetal toxicity cannot be determined. However, it should be noted that the authors themselves pointed out that for some of the changes in number of ossification points, no dose-response relationship was observed. Also, the information was given that it is known that a decrease in number of ossification points does not influence the postnatal development is later compensated, and an increase, on the other hand, is a result of the intensive growth of fetuses. Further malformations were observed in only one fetus in the low dose group.

 

This pre-natal developmental toxicity study is acceptable and satisfies the guideline requirement for an oral pre-natal developmental study (OECD 414) in rats. It should be noted that no specific historical control data for any parameters were included in the report, though the laboratory does make reference to experience in previous internal studies and literature sources.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

6.25 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

There are 2 key studies that have a Klimisch socre of 1 so the quality of the database is high.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

There were 2 studies for the Toxicity to Reproduction endpoint

(i) Combined Oral Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in rats

(ii) Oral pre-natal developmental toxicity study in rats

 

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

 

In a GLP-study according to OECD Test Guideline 422 the toxic effects of the test item CARDOLITE NC-513 at doses of 62.5, 250 and 1000 mg/kg body weight on the subacute toxicity and the development and reproduction of Wistar rats after oral administration were under examination. The substance was at least applied for 46 days daily to rats of both sexes. The following observations were made.

 

General and detailed clinical signs:

Mild discomfort throughout the whole application period was observed for the animals treated with the high and the medium dose of the test item (wiping of nose and mouth through the cage bedding, salivation after application, bleeding of mucous membranes at nose and mouth, respirators sounds). Moreover, some male animals of the high dose group became lethargic after application of the test item on individual days. A biological and particularly toxicological relevance of these observations could not be excluded.

 

Body weight, food and water consumption: Regarding the body weight and the body weight gain, no significant differences were

observed between all test item treated animals (male and female) and their respective vehicle control animals. Occasional differences observed for the females were assumed to be of natural origin based on the pregnancy status of the animals.

No test item related tendencies regarding food and water consumption of all test item treated animals (male and female) could be observed throughout the whole study phase when compared to their respective vehicle control animals. All fluctuations observed were most likely of natural origin.

 

Haematology and clinical biochemistry: no effects

 

Reproduction and Development:

A statistically significant decrease in the amount of pups born alive was detected for the animals of the high and medium dose groups. This was not statistically significant for the animals of the low dose group. The increase in pre-implantation losses was not significant for any dose group. Pup growth and development (day 0 and day 4 post-partum) were normal for all test item treated animals when compared to the vehicle control animals. No further findings regarding reproduction success (achievement and duration of pregnancy, post- implantation losses, survival rate of pups, development of pups) were observed in animals of any test item treated group when compared to the vehicle control group.

 

Necropsy:

The determination of organ weights, as part of the necropsy, showed a statistically significant increase of the liver weight (male; absolute and relative), a statistically significant decrease of the prostate weight (absolute and relative), a statistically significant increase of the brain weight (female; absolute), and a statistically significant weight increase of the right ovary (absolute and relative) within the animals treated with the high dose of the test item. However, the changed organ weights were not accompanied by structural changes (see below 'Histology').

Moreover, with increasing dose levels the amount of male animals having slightly developed mammary glands declined. Besides some further individual findings, heterogeneously distributed over all dose groups, no further apparent observations were made that could be related to the administration of the test item. 

 

Histology: no effects

 

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL (F0 systemic) females/males: 250 mg/kg bw/day
NOAEL (F0 reproduction) females/males: >=1000 mg/kg bw/day
NOAEL (F1 embryo-/fetaltoxicity): 62.5 mg/kg bw/day
NOAEL (F1 development): >=1000 mg/kg bw/day (no effect level was derived, as pup growth and development (day 0 and day 4 post-partum) were normal for all test item treated animals when compared to the vehicle control)

 

 

Oral pre-natal developmental toxicity study in rats

 

In a developmental toxicity study (T-24/16), CARDOLITE NC-513 was administered to 25 female Wistar (Cmdb: WI) rats/dose by gavage in corn oil at dose levels of 0, 6.25, 62.5 and 625 mg/kg bw/day from days 0 through 19 of gestation. This pre-natal developmental toxicity study is acceptable and satisfies the guideline requirement for an oral pre-natal developmental study (OECD 414) in rats. It should be noted that no specific historical control data for any parameters were included in the report, though the laboratory does make reference to experience in previous internal studies and literature sources.

 

There were no mortalities or clinical signs. Statistically significantly greater food consumption at 62.5  mg/kg bw/day between day 17 and day 20 of gestation and at 625 mg/kg bw/day between day 8 and day 20 was noted. However, greater food consumption did not lead to statistically significantly greater weights of the females from these groups during the gestation period. No pathological changes were noted at gross examination of the females performed at caesarean section. The average weight of the uterus of pregnant females of the treated groups did not differ statistically significantly from average weight of the uterus of pregnant females of the control group. There was no difference in mean pre- or post-implantation losses between control and treated groups. After caesarean section, there were four fetuses dead which was not related to the test item. The numbers of resorptions at 6.25 and 62.5 mg/kg bw/day did not differ from the control group.The number of resorptions at 625 mg/kg bw/day was more than twice the control group. The number of resorptions at  625 mg/kg bw/day indicated significant intrauterine mortality (23/312; 7.37%) and could be an embryotoxic effect of the test item. The average number of corpora lutea in all treated groups are comparable with the number of corpora lutea in the control group.

Since the statistically significant greater food consumption in groups 2 and 3 seems to be caused by the test item, the NOAEL for maternal toxicity can be defined as 6.25 mg/kg bw.

 

The average weight of the fetuses with placenta and weight of fetuses without placenta were statistically significantly greater in all treated groups compared with the control group. The average weight of placenta was statistically significantly greater at 6.25 mg/kg bw/day and 625 mg/kg bw/day compared with the control group. Subcutaneous blood hemorrhages in different parts of the fetal bodies, which occurred in the control and the treated groups, were not considered a treatment-related effect.

 

Schistocelia (congenital fissure of the abdominal wall with protrusion of the viscera), micrognathia, and the lack of skull fornix were noticed in one fetus at 6.25 mg/kg bw/day. This malformation of the head and the trunk were extensive and was defined as polydysplasia. Although single cases of malformation could be considered as incidental spontaneous defects referred to as congenital defects, the laboratory considered that the test item at all doses used in the sighting and the main studies affected the growth of the fetuses, this malformation could be its teratogenic effect. The registrant of the substance points out that the observations were only made in one fetus in the low dose group, which is why a toxicological relevance is highly unlikely.

 

Wavy ribs were observed in three fetuses at 6.25 mg/kg bw/day.  Misalignment of the two halves of ossified center in sternum was observed in some fetuses in all groups: in two fetuses in the control group, in two fetuses at 62.5 mg/kg bw/day and in three fetuses at 625 mg/kg bw/day.  Three ossification points of sternum were observed in six fetuses at 62.5 mg/kg bw/day and in two fetuses at 625 mg/kg bw/day. No ossification points of sternum and unossified center of pubis bone were observed in one fetus at 6.25 mg/kg bw/day.  Statistically significantly higher weight of the fetuses with and without placenta was noticed in all treated groups in comparison with the control group.  Statistically significantly higher weight of placenta was noticed at 6.25 mg/kg bw/day and 625 mg/kg bw/day in comparison with the control group.  Statistically significantly greater length of fetuses with tail and without tail was noticed in all treated groups in comparison with the control group. Statistically significantly greater number of ossification points in sternum and metacarpus were noticed at 6.25 mg/kg bw/day compared with the control group.  Statistically significantly lower number of ossification points in metatarsus was noticed at 62.5 mg/kg bw/day compared with the control group. Statistically significantly greater number of ossification points in metacarpus was noticed at 625 mg/kg bw/day compared with the control group.

 

The authors suggested that based on the greater average body weights and lengths of the fetuses in all treated groups and some changes in the average number of ossification points of the sternum and the metacarpus in the treated groups could be related to the test item. Additionally, they speculated that the changes observed in one fetus at 6.25 mg/kg bw/day might be teratogenic. Therefore, based on the doses used in the study, a NOAEL for fetuses was not determined. 

 

However, the authors themselves pointed out that for some of the changes in number of ossification points, no dose-response relationship was observed. Also, the information was given that it is known that a decrease in number of ossification points does not influence the postnatal development is later compensated, and an increase, on the other hand, is a result of the intensive growth of fetuses. Increased growth was seen in all treated fetuses, but this was attributed by the registrant to the increased food intake of the treated dams. Further malformations were observed in one fetus in the low dose group. However, the registrant of the substance concluded that they should not be evaluated as toxicologically relevant, because only one fetus was affected, and no such malformations occurred in animals of the higher dose groups.

 

In summary, the registrants of the substance concluded that the above-mentioned observations in the fetuses do not represent relevant toxicological effects in relation to prenatal development in rats. This assumption is supported by the results of the OECD 422, which show that growth and development of pups (day 0 and day 4 post-partum) were normal in all treated animals compared with the control.

Justification for classification or non-classification

Based on the available information in the dossier, the substance CARDOLITE NC-513 (EC/List no. 701-477-4) does not need to be classified for reproductive toxicity when the criteria outlined in Annex I of 1227/2008/EC are applied.