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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Sep 2017 - 15 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Sep 2017 - 15 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developme ntal Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Specific gravity/density: 1.3183 at 20°C (specific gravity used for adjustment at preparation of dosing formulations)
Species:
rat
Strain:
other: Crl: WI(Han)
Details on test animals or test system and environmental conditions:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 12 weeks (males), 13 weeks (females)
- Weight at study initiation: 247 - 287 g (males), 201 - 247 g (females)
- Fasting period before study: no (except for males that were fasted overnight (water was available) with a maximum of 24 hours before blood sampling)
- Housing: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type); During the mating phase, males and females were coha
bitated on a 1:1 basis in Macrolon plastic cages (MIII type); During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type) with a maximum of 5 males/cage.
Females were individually housed in Macrolon plastic cages (MIII type); During the lactation phase, females were housed in Macrolon plastic cages (MIII type), pups were housed with the dam; During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage enrichment, bedding material, food and water
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum (except during motor activity measurements)
- Water: municipal tap water, ad libitum (except during motor activity measurements)
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants . Periodic analysis of the water is performed, and it is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 39-59
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 04 Sep 2017 - 15 Feb 2018
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate sets of samples were collected in week one to analyze concentration (all dose groups), homogeneity and stability (low and high doses). The homogeneity results obtained from the top, middle and bottom for the low and high dose group formulations were averaged and utilized as the concentration results. Stability of the test item in the vehicle was tested upon storage for 6 hours at room temperature under normal laboratory light conditions. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation.
Details on mating procedure:
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males. Detection of mating was not confirmed in first instance for female nos. 53 and 79. Evidence of mating was obtained by palpation and indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continuation of di-estrus during the mating period in these females. The mating date of these animals was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 postcoitum.
Duration of treatment / exposure:
Males: 29 days
Females: 50-56 days or 64 days (females that delivered); 52-55 days (females without offspring); 38-42 days (females with total litter loss)
Frequency of treatment:
7 days/ week
Duration of test:
Same as treatment
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a 10-day dose range finder with oral administration of Carob Bean Extract in rats. This dose range finder was conduct ed to select dose levels for the main study, and to determine the peak effect of occurrence of clinical signs after dosing. No guidelines were applicable as this study was intended for dose level selection purposes only. If not mentioned otherwise, test system, procedures and techniques were identical to those used during the main study. In short, the test item, formulated in water, was administered to the appropriate animals by daily oral gavage for 10 consecutive days. Dose levels tested were 500 and 1000 mg/kg bw/day (dose volume 5 mL/kg bw), 3 females per group were tested. The following parameters were included: Mortality: Twice daily throughout the study.
Clinical Observations: At least daily on days 1-10, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing.
Body Weights: On day 1 prior to dosing and on Days 5 and 10.
Food Consumption Over days 1-5 and 5-10.
At termination, all animals were subjected to an external, thoracic and abdominal examination.
Terminal body weight, kidney and liver weight were determined at scheduled necropsy.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily; in addition, clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment. These observations were conducted after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on post natal days 1, 4, 7, and 13. Terminal body weights were recorded on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on post natal days 1, 4, 7, and 13.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day of sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: males were fasted, females were not fasted
- How many animals: all
- All parameters according to guidelines were examined. In addition T4 was measured in F0 males and PND 14-16 pups.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4 (males) or during the last week of lactation (post natal days 6-13)
- Dose groups that were examined: all dose groups (F0 animals), 5 animals/dose
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength and locomotor activity (total movements and ambulations)

ESTROUS CYCLICITY
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment
(pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until ter
mination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for females with total litter loss


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups.
At necropsy (post natal day 14-16) sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (one male and one female pup) was preserved in 10% buffered formalin. Pups selected for blood sampling were the same pups as selected for thyroid preservation.
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy
date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the
comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: low dose vs. control, mid dose vs. low dose and high dose vs. low dose. Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set
was compared using an overall Kruskal-Wallis. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Indices:
- Post-implantation survival index: (Total number of offspring born/Total number of uterine implantation sites) x 100
- Live birth index: (Number of live offspring on Day 1 after littering/ Total number of offspring born) x 100
- Percentage live males at First Litter Check: (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check: (Number of live female pups at First Litter Check/ Number of live pups at First Litter Check) x 100
- Percentage of postnatal loss Days 0-4 of lactation: (Number of dead pups on Day 4 of lactation/ Number of live pups at First Litter Check) x 100
- Viability index: (Number of live pups on Day 4 of lactation / Number of pups born alive) x 100
- Lactation index: (Number of live offspring on Day 13 after littering/ Number live offspring on Day 4 (after culling)) x 100
Historical control data:
Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were noted during daily clinical observations or during weekly arena observations. Any clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period. Two females were euthanized on day 1 of lactation due to total litter loss (at 100 and 300 mg/kg bw/day, respectively).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights and body weight gain were not affected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption (before and after correction for body weight) was not affected by treatment. An isolated, statistically significant difference noted in females at 100 mg/kg bw/day (lower relative food consumption between post-coitum days 14-17) was regarded as unrelated to treatment due to the lack of a dose-related trend.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment up to 1000 mg/kg bw/day was not associated with changes in red blood cell parameters, white blood cell parameters or number of platelets. A statistically significantly higher mean activated partial thromboplastin time (APTT) was noted at 1000 mg/kg bw/day in females (relative difference from controls +20%). APTT values of all 1000 mg/kg bw/day females remained within the normal range of which the control females were slightly below the historical control mean and the high dose females were slightly above the historical control mean. This statistical significant change was therefore not regarded toxicologically relevant. There were no treatment-related changes in prothrombin time.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical chemistry parameters showed no differences between control and treated rats that were considered to be toxicologically relevant. The mean concentration of bile acids appeared higher in females at 1000 mg/kg bw/day. The difference from controls (74%) was not statistically significant and could largely be explained by a high value in a single female. In the absence of associated adverse anatomic pathology changes, this isolated finding was considered not to represent an adverse effect of the test item. Isolated statistically significant variations noted in clinical chemistry values were regarded as unrelated to treatment due to the lack of a dose-related response (lower inorganic phosphate at 300 mg/kg bw/day in females) or slightly high control value (lower total protein at 1000 mg/kg bw/day in males).
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. A statistically significantly lower hind limb grip strength was noted in males at 1000 mg/kg bw/day (relative difference from control: -22%). Grip strength values of all males remained in the normal range of which the control males were slightly above the historical control mean and the high dose males were slightly below the historical control mean. This statistical significant change was therefore not regarded toxicologically relevant. Hind limb grip strength in females and forelimb grip strength in both sexes were unremarkable. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related changes were noted for organ weights and organ:body weight ratios. Statistically significant changes between adrenals and thyroids weights of treated and control females were considered not to be a sign of toxicity. The increased adrenals weights noted for females of all treated groups were considered to have arisen as a result of slightly low control values, and in the absence of a treatment-related distribution and histopathological correlate, considered to be of no toxicological significance. The increased relative thyroid weight for females at 1000 mg/kg bw/day was not regarded toxicologically relevant as this change was only slight and no effect was noted for absolute thyroid weight.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic observations. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Other effects:
no effects observed
Description (incidence and severity):
Gestation index and duration of gestation were not affected by treatment. The gestation indices were 100, 80, 90 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
Three pregnant females had no live offspring: one at 100 mg/kg bw/day, one at 300 mg/kg bw/day (which had only dead pups at first litter check (total litter loss)) and one at 100 mg/kg bw/day which had no pups (she had only one implantation site). These cases of failed pregnancy occurred without related histopathology changes in reproductive organs and their incidence showed no dose-related trend. They were therefore considered to be unrelated to treatment.

No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects seen up to and including the highest dose tested (1000 mg/kg bw/day)
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were not affected by treatment.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
The viability index was not affected by treatment. The viability indices were 100% for all test item groups and 98% for the control group.
Two pups of the control group went missing (presumably cannibalized) or were found dead at post natal day 3 or 4. This incidental pup mortality was within normal limits.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean number of living pups at first litter check (live litter size) was not affected by treatment.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The lactation index was not affected by treatment. No pups died after post natal day 4, resulting in a lactation index of 100% for all groups.
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the few findings noted remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Other effects:
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment.
Treatment up to 1000 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at post natal day 13.

Serum T4 levels in male and female post natal day 14-16 pups were judged to be unaffected by treatment. At 1000 mg/kg bw/day, T4 levels were slightly lower than the concurrent control (9%). However, as this difference was not statistically significant and all values remained within normal limits, it was not regarded to be toxicologically relevant.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects seen up to and including highest dose tested.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
According to the results of a repeated dose study with screening for reproduction and developmental toxicity performed with Carob Bean Extract according to OECD guideline 422 and GLP principles, the NOAEL for development was found to exceed 1000 mg/kg bw/day.
Executive summary:

A repeated dose study with screening for reproduction and development toxicity was performed with Carob Bean Extract according to OECD guideline 422 and GLP principles. Wistar Han rats were treated with Carob Bean Extract by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, water, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and 13-15 days of lactation (mostly for 50-56 days). Females without offspring were treated for 52-55 days and females that had a total litter loss were treated for 38-42 days. Formulation analysis showed that the formulations were prepared accurately and homogeneously, and that the formulations were stable for at least 6 hours at room temperature under normal laboratory light conditions. No parental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in the developmental parameters investigated (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance (post natal day 1), areola/nipple retention (post natal day 13 males), T4 thyroid hormone levels (post natal day 14-16) and macroscopy). Based on this outcome, the NOAEL for developmental toxicity was concluded to exceed 1000 mg/kg bw/day.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Sep 2017 - 15 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Specific gravity/density: 1.3183 at 20°C (specific gravity used for adjustment at preparation of dosing formulations)
Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 12 weeks (males), 13 weeks (females)
- Weight at study initiation: 247 - 287 g (males), 201 - 247 g (females)
- Fasting period before study: no (except for males that were fasted overnight (water was available) with a maximum of 24 hours before blood sampling)
- Housing: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type); During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type); During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type); During the lactation phase, females were housed in Macrolon plastic cages (MIII type), pups were housed with the dam; During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage enrichment, bedding material, food and water
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum (except during motor activity measurements)
- Water: municipal tap water, ad libitum (except during motor activity measurements)
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water is performed, and it is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 39-59
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 04 Sep 2017 - 15 Feb 2018
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 6 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate sets of samples were collected in week one to analyze concentration (all dose groups), homogeneity and stability (low and high doses). The homogeneity results obtained from the top, middle and bottom for the low and high dose group formulations were averaged and utilized as the concentration results. Stability of the test item in the vehicle was tested upon storage for 6 hours at room temperature under normal laboratory light conditions. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation.
Duration of treatment / exposure:
Males: 29 days
Females: 50-56 days or 64 days (females that delivered); 52-55 days (females without offspring); 38-42 days (females with total litter loss)
Frequency of treatment:
7 days/ week
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 10-day dose range finder with oral administration of Carob Bean Extract in rats. This dose range finder was conducted to select dose levels for the main study, and to determine the peak effect of occurrence of clinical signs after dosing. No guidelines were applicable as this study was intended for dose level selection purposes only. If not mentioned otherwise, test system, procedures and techniques were identical to those used during the main study. In short, the test item, formulated in water, was administered to the appropriate animals by daily oral gavage for 10 consecutive days. Dose levels tested were 500 and 1000 mg/kg bw/day (dose volume 5 mL/kg bw), 3 females per group were tested.
The following parameters were included:
Mortality: Twice daily throughout the study.
Clinical Observations: At least daily on days 1-10, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing.
Body Weights: On day 1 prior to dosing and on Days 5 and 10.
Food Consumption Over days 1-5 and 5-10.
At termination, all animals were subjected to an external, thoracic and abdominal examination.
Terminal body weight, kidney and liver weight were determined at scheduled necropsy.
Positive control:
Not included in the study, but Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily; in addition, clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment. These observations were conducted after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on post natal days 1, 4, 7, and 13. Terminal body weights were recorded on the day of necropsy.
Variables:
Body Weight Gains: Calculated against the body weight on day 1 (premating, mating and lactation periods) or day 0 (postcoitum period);
Relative Food Consumption Calculated against the body weight for scheduled intervals;
Organ Weight Relative to Body Weight: Calculated against the terminal body weight.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on post natal days 1, 4, 7, and 13.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day of sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: males were fasted, females were not fasted
- How many animals: all
- All parameters according to guidelines were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
on day of sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: males were fasted, females were not fasted
- How many animals: all
- All parameters according to guidelines were examined. In addition T4 was measured in F0 males and PND 14-16 pups.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4 (males) or during the last week of lactation (post natal days 6-13)
- Dose groups that were examined: all dose groups (F0 animals), 5 animals/dose
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength and locomotor activity (total movements and ambulations)

Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for females with total litter loss
Sperm parameters (parental animals):
Parameters examined in male parental generation:
Testis weight and epididymis weight (all males); Histopathological examination: epididymis, prostate gland, seminal vesicle gland and testes.
For the testes of all selected males of the control group and the high dose group, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS:
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes, full post mortem examination, with special attention for the reproductive organs. Major organs were weighed (paired organs were weighed together)
HISTOPATHOLOGY: Yes, all tissues according to guidelnes were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. For the testes of all selected males of the low and the high dose groups, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 14-16 days of age.

GROSS NECROPSY
- Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (one male and one female pup) was preserved in 10% buffered formalin. Pups selected for blood sampling were the same pups as selected for thyroid preservation.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: low dose vs. control, mid dose vs. low dose and high dose vs. low dose. Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
For each group, the following calculations were performed:
- Mating index: (Number of females mated/Number of females paired) x 100
- Fertility index: (Number of pregnant females/Number of females paired) x 100
- Gestation index: (Number of females bearing live pups on Day 1/Number of pregnant females) x 100

- Precoital time: Number of days between initiation of cohabitation and confirmation of mating
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
- Post-implantation survival index: (Total number of offspring born/Total number of uterine implantation sites) x 100
- Live birth index: (Number of live offspring on Day 1 after littering/ Total number of offspring born) x 100
- Percentage live males at First Litter Check: (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check: (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage of postnatal loss Days 0-4 of lactation: (Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check) x 100
- Viability index: (Number of live pups on Day 4 of lactation / Number of pups born alive) x 100
- Lactation index: (Number of live offspring on Day 13 after littering/ Number live offspring on Day 4 (after culling)) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were noted during daily clinical observations or during weekly arena observations. Any clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period. Two females were euthanized on day 1 of lactation due to total litter loss (at 100 and 300 mg/kg, respectively).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights and body weight gain were considered not to be affected by treatment. A finding of note at 1000 mg/kg bw/day consisted of reduced weight gain of one male throughout the 4-week treatment period. All other 1000 mg/kg bw/day males grew normally. There were no associated signs of toxicity and mean body weights of 1000 mg/kg bw/day males remained close to control values (6% difference at the end of the treatment period, not statistically significant). Therefore, the reduced weight gain of a single high-dose male was considered not to reflect an adverse effect of the test item.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption (before and after correction for body weight) was not affected by treatment. An isolated, statistically significant difference noted in females at 100 mg/kg bw/day (lower relative food consumption between post-coitum days 14-17) was regarded as unrelated to treatment due to the lack of a dose-related trend.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment up to 1000 mg/kg bw/day was not associated with changes in red blood cell parameters, white blood cell parameters or number of platelets. An isolated, statistically significant difference noted in males (higher red blood cell distribution width at 100 mg/kg bw/day) was considered to be unrelated to treatment due to the lack of a dose-related trend.
A statistically significantly higher mean activated partial thromboplastin time (APTT) was noted at 1000 mg/kg bw/day in females (relative difference from controls +20%). APTT values of all 1000 mg/kg bw/day females remained within the normal range of which the control females were slightly below the historical control mean and the high dose females were slightly above the historical control mean. This statistical significant change was therefore not regarded toxicologically relevant. There were no treatment-related changes in prothrombin time.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical chemistry parameters showed no differences between control and treated rats that were considered to be toxicologically relevant. The mean concentration of bile acids appeared higher in females at 1000 mg/kg bw/day. The difference from controls (74%) was not statistically significant and could largely be explained by a high value in a single female. In the absence of associated adverse anatomic pathology changes, this isolated finding was considered not to represent an adverse effect of the test item. Isolated statistically significant variations noted in clinical chemistry values were regarded as unrelated to treatment due to the lack of a dose-related response (lower inorganic phosphate at 300 mg/kg bw/day in females) or slightly high control value (lower total protein at 1000 mg/kg bw/day in males). Serum levels of T4 in males were not affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. A statistically significantly lower hind limb grip strength was noted in males at 1000 mg/kg bw/day (relative difference from control: -22%). Grip strength values of all males remained in the normal range of which the control males were slightly above the historical control mean and the high dose males were slightly below the historical control mean. This statistical significant change was therefore not regarded toxicologically relevant. Hind limb grip strength in females and forelimb grip strength in both sexes were unremarkable. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic observations. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment. All females had regular cycles of four days.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and sperm stage evaluation of the testes did not show any indication for abnormal spermatogenesis.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Two couples had no offspring: one female at 100 mg/kg bw/day had implantation sites only and one female at 1000 mg/kg bw/day showed no evidence of mating. Two females had total litter loss at post natal day 1: one female at 100 mg/kg bw/day and one female at 300 mg/kg bw/day. No abnormalities were seen in the reproductive organs which could account for the reproductive failure.
Mating index was considered not to be affected by treatment. Except for one female of the 1000 mg/kg bw/day group, all females showed evidence of mating.
The mating indices were 90% for the 1000 mg/kg bw/day group and 100% for the other groups.
This incidental case of unsuccessful mating, without supportive morphological changes in reproductive organs, was considered to be unrelated to treatment.

Precoital time was considered not to be affected by treatment. Most females showed evidence of mating within four days. Longer precoital intervals were noted for three females
at 100 mg/kg bw/day (13 or 14 days) and two females at 1000 mg/kg bw/day (7 or 14 days). These longer intervals were considered not to reflect an effect of the test item due to the lack of a dose-related trend.

The number of implantation sites was not affected by treatment. One pregnant female at 100 mg/kg bw/day had only one implantation site (this female had no offspring). This incidental finding in the lowest dose group was regarded as unrelated to treatment.

Fertility index was not affected by treatment. All mated females were pregnant (fertility index 100% for all groups).
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects seen up to and including the highest dose tested (1000 mg/kg bw/day)
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment. The clinical signs noted incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The viability index was not affected by treatment. The viability indices were 100% for all test item groups and 98% for the control group.
Two pups of the control group went missing (presumably cannibalized) or were found dead at post natal day 3 or 4. This incidental pup mortality was within normal limits.

The lactation index was not affected by treatment. No pups died after post natal day 4, resulting in a lactation index of 100% for all groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were not affected by treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Serum T4 levels in male and female post natal day 14-16 pups were judged to be unaffected by treatment.
At 1000 mg/kg bw/day, T4 levels were slightly lower than the concurrent control (-9%). However, as this difference was not statistically significant and all values remained within normal limits, it was not regarded to be toxicologically relevant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of the few findings noted remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment.
Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment.
Treatment up to 1000 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at post natal day 13.
Key result
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 adverse effects seen up to and including the highest dose tested (1000 mg/kg bw/day)
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
According to the results of a repeated dose study with screening for reproduction and developmental toxicity performed with Carob Bean Extract according to OECD guideline 422 and GLP principles, the NOAEL for reproduction was found to exceed 1000 mg/kg bw/day.
Executive summary:

A repeated dose study with screening for reproduction and development toxicity was performed with Carob Bean Extract according to OECD guideline 422 and GLP principles. Wistar Han rats were treated with Carob Bean Extract by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, water, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and 13-15 days of lactation (mostly for 50-56 days). Females without offspring were treated for 52-55 days and females that had a total litter loss were treated for 38-42 days. Formulation analysis showed that the formulations were prepared accurately and homogeneously, and that the formulations were stable for at least 6 hours at room temperature under normal laboratory light conditions. No parental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted the reproductive parameters examined (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Based on this outcome, the NOAEL for reproduction toxicity was concluded to exceed 1000 mg/kg bw/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents
Version / remarks:
2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Test material form:
liquid
Details on test material:
- Physical appearance: Dark brown to black liquid
- Storage of test material: At room temperature
Specific details on test material used for the study:
Specific gravity/density: 1.3183 at 20°C (specific gravity used for adjustment at preparation of dosing formulations)

Test animals

Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 12 weeks (males), 13 weeks (females)
- Weight at study initiation: 247 - 287 g (males), 201 - 247 g (females)
- Fasting period before study: no (except for males that were fasted overnight (water was available) with a maximum of 24 hours before blood sampling)
- Housing: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type); During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type); During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type); During the lactation phase, females were housed in Macrolon plastic cages (MIII type), pups were housed with the dam; During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage enrichment, bedding material, food and water
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum (except during motor activity measurements)
- Water: municipal tap water, ad libitum (except during motor activity measurements)
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water is performed, and it is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 39-59
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 04 Sep 2017 - 15 Feb 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 6 hours after adding the vehicle to the test item.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate sets of samples were collected in week one to analyze concentration (all dose groups), homogeneity and stability (low and high doses). The homogeneity results obtained from the top, middle and bottom for the low and high dose group formulations were averaged and utilized as the concentration results. Stability of the test item in the vehicle was tested upon storage for 6 hours at room temperature under normal laboratory light conditions.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%. Stability results were considered acceptable if the sample analysis results were within or
equal to ±10% of the concentration determined by the initial analysis of each formulation.
Duration of treatment / exposure:
Males: 29 days
Females: 50-56 days or 64 days (females that delivered); 52-55 days (females without offspring); 38-42 days (females with total litter loss)
Frequency of treatment:
7 days/ week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 10-day dose range finder with oral administration of Carob Bean Extract in rats.
This dose range finder was conducted to select dose levels for the main study, and to determine the peak effect of occurrence of clinical signs after dosing. No guidelines were applicable as this study was intended for dose level selection purposes only. If not mentioned otherwise, test system, procedures and techniques were identical to those used during the main study. In short, the test item, formulated in water, was administered to the appropriate animals by daily oral gavage for 10 consecutive days. Dose levels tested were 500 and 1000 mg/kg bw/day (dose volume 5 mL/kg bw), 3 females per group were tested.
The following parameters were included:
Mortality: Twice daily throughout the study.
Clinical Observations: At least daily on days 1-10, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing.
Body Weights: On day 1 prior to dosing and on Days 5 and 10.
Food Consumption Over days 1-5 and 5-10.
At termination, all animals were subjected to an external, thoracic and abdominal examination. Terminal body weight, kidney and liver weight were determined at scheduled necropsy.
Positive control:
Not included in the study, but Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily; in addition, clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment. These observations were conducted after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on post natal days 1, 4, 7, and 13. Terminal body weights were recorded on the day of necropsy.
Variables:
Body Weight Gains: Calculated against the body weight on day 1 (premating, mating and lactation periods) or day 0 (postcoitum period);
Relative Food Consumption Calculated against the body weight for scheduled intervals;
Organ Weight Relative to Body Weight: Calculated against the terminal body weight.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on post natal days 1, 4, 7, and 13.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day of sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: males were fasted, females were not fasted
- How many animals: all
- All parameters according to guidelines were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
on day of sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: males were fasted, females were not fasted
- How many animals: all
- All parameters according to guidelines were examined. In addition T4 was measured in F0 males and PND 14-16 pups.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4 (males) or during the last week of lactation (post natal days 6-13)
- Dose groups that were examined: all dose groups, 5 animals/dose
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength and locomotor activity (total movements and ambulations)

OESTRUS CYCLE DETERMINATION:
- Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for females with total litter loss

Sacrifice and pathology:
GROSS PATHOLOGY: Yes, full post mortem examination, with special attention for the reproductive organs
Major organs were weighed (paired organs were weighed together)
HISTOPATHOLOGY: Yes, all tissues according to guidelnes were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. For the testes of all selected males of the low and the high dose groups, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made:
low dose vs. control, mid dose vs. low dose and high dose vs. low dose.
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were noted during daily clinical observations or during weekly arena observations. Any clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period. Two females were euthanized on day 1 of lactation due to total litter loss (at 100 and 300 mg/kg, respectively).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights and body weight gain were considered not to be affected by treatment. A finding of note at 1000 mg/kg bw/day consisted of reduced weight gain of one male throughout the 4-week treatment period. All other 1000 mg/kg bw/day males grew normally. There were no associated signs of toxicity and mean body weights of 1000 mg/kg bw/day males remained close to control values (6% difference at the end of the treatment period, not statistically significant). Therefore, the reduced weight gain of a single high-dose male was considered not to reflect an adverse effect of the test item.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption (before and after correction for body weight) was not affected by treatment. An isolated, statistically significant difference noted in females at 100 mg/kg bw/day (lower relative food consumption between post-coitum days 14-17) was regarded as unrelated to treatment due to the lack of a dose-related trend.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment up to 1000 mg/kg bw/day was not associated with changes in red blood cell parameters, white blood cell parameters or number of platelets. An isolated, statistically significant difference noted in males (higher red blood cell distribution width at 100 mg/kg bw/day) was considered to be unrelated to treatment due to the lack of a dose-related trend.
A statistically significantly higher mean activated partial thromboplastin time (APTT) was noted at 1000 mg/kg bw/day in females (relative difference from controls +20%). APTT values of all 1000 mg/kg bw/day females remained within the normal range of which the control females were slightly below the historical control mean and the high dose females were slightly above the historical control mean. This statistical significant change was therefore not regarded toxicologically relevant. There were no treatment-related changes in prothrombin time.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical chemistry parameters showed no differences between control and treated rats that were considered to be toxicologically relevant. The mean concentration of bile acids appeared higher in females at 1000 mg/kg bw/day. The difference from controls (74%) was not statistically significant and could largely be explained by a high value in a single female. In the absence of associated adverse anatomic pathology changes, this isolated finding was considered not to represent an adverse effect of the test item. Isolated statistically significant variations noted in clinical chemistry values were regarded as unrelated to treatment due to the lack of a dose-related response (lower inorganic phosphate at 300 mg/kg bw/day in females) or slightly high control value (lower total protein at 1000 mg/kg bw/day in males). Serum levels of T4 in males were not affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. A statistically significantly lower hind limb grip strength was noted in males at 1000 mg/kg bw/day (relative difference from control: -22%). Grip strength values of all males remained in the normal range of which the control males were slightly above the historical control mean and the high dose males were slightly below the historical control mean. This statistical significant change was therefore not regarded toxicologically relevant. Hind limb grip strength in females and forelimb grip strength in both sexes were unremarkable. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test
period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related changes were noted for organ weights and organ:body weight ratios. Statistically significant changes between adrenals and thyroids weights of treated and control females were considered not to be a sign of toxicity. The increased adrenals weights noted for females of all treated groups were considered to have arisen as a result of slightly low control values, and in the absence of a treatment-related distribution and histopathological correlate, considered to be of no toxicological significance. The increased relative thyroid weight for females at 1000 mg/kg bw/day was not regarded toxicologically relevant as this change was only slight and no effect was noted for absolute thyroid weight.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic observations. All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment. All females had regular cycles of four days.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to and including the highest dose tested (1000 mg/kg bw/day)

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Results of dose range finding study:

No dose limiting effects were noted up to 1000 mg/kg bw/day. Alopecia was seen in 1/3 animals on days 5 -10 at 500 mg/kg bw/day. Reduced body weight gain was seen over days 1-10 in 1/3 animals at 1000 mg/kg bw/day, normal weight gain was seen in 2/3 animals. No effects were seen on liver and kidney weights. Since no peak effect of occurrence of clinical signs was observed in the dose range finder, clinical observations were conducted and functional observations were started in the main study after dosing at no specific time point, but within a similar time period after dosing for the respective animals.

Results of formulation analysis:

The concentrations analyzed in the formulations of the test item groups were in agreement with target concentrations (i.e. mean accuracies between 100% and 102%).

No test item was detected in the conrol group formulation.

The formulations of the low and the high dose groups were homogeneous (i.e. coefficient of variation ≤ 1.9%).

Formulations of the low and the high dose groups were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Applicant's summary and conclusion

Conclusions:
According to the results of a repeated dose study with screening for reproduction and development toxicity performed with Carob Bean Extract according to OECD guideline 422 and GLP principles, the NOAEL was found to exceed 1000 mg/kg bw/day.
Executive summary:

A repeated dose study with screening for reproduction and development toxicity was performed with Carob Bean Extract according to OECD guideline 422 and GLP principles. Wistar Han rats were treated with Carob Bean Extract by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, water, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and 13-15 days of lactation (mostly for 50-56 days). Females without offspring were treated for 52-55 days and females that had a total litter loss were treated for 38-42 days. Formulation analysis showed that the formulations were prepared accurately and homogeneously, and that the formulations were stable for at least 6 hours at room temperature under normal laboratory light conditions. No parental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day).

No treatment-related changes were noted in any of the parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations (including male T4 thyroid hormone levels), macroscopic examination, organ weights, and microscopic examination). Based on this outcome, the NOAEL for repeated dose toxicity was concluded to exceed 1000 mg/kg bw/day.