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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 June 2016 - 08 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
September 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2011 by MHLW (0331 No.7), METI (H23.03.29 SeiKyoku No. 5) and MOE (No. 110331009).
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- State of aggregation: brown liquid
- Density: 1.29 at 20 °C
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a freezer (≤ -15°C) under nitrogen
- Solubility and stability of the test substance in the solvent/vehicle: stability for at least 3 hours at room temperature is confirmed over the concentration range 1 to 200 mg/mL in water

OTHER SPECIFICS: A correction was made for the purity/composition of the test item. A correction factor of 0.2495 was used.

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: 139-166 g (males), 108-135 g (females) (main study), g (dose range finding study)
- Fasting period before study: Animals were only deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm), except during locomotor activity monitoring, when animals were housed individually in a Hi-temp polycarbonate cage; sterilized sawdust was provided as bedding material and paper as cage-enrichment.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (were set to maintain)
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 27 June 2016 - 08 November 2016

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
This study should provide a rational basis for toxicological risk assessment in man. The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 3 hours prior to
dosing, and were homogenized to visually acceptable levels. Correction was made for purity/composition and specific gravity of the test item.

Dose volume: 5 mL/kg body bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted during the treatment phase in Weeks 1, 4, and 13, according to a validated method . Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). In Week 4, stability in vehicle over 4 hours at room temperature was also determined (highest and lowest concentration). Additionally, homogeneity and accuracy of the test item was determined in Week 4, to confirm homogeneity within the same container and to confirm that the test item concentration was similar between different containers.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
at least 90 days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
88 mg/kg bw/day (actual dose received)
Dose / conc.:
264 mg/kg bw/day (actual dose received)
Dose / conc.:
880 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
3 (dose range finding study);
10 (main study)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on a 14-day range finding study (for details see below)
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena.

FUNCTIONAL OBSERVATIONS:
During week 12-13 of treatment, the following tests were performed on the first 5 surviving animals/sex/group after dosing at no specific time point, but within a similar time period after dosing for the respective animals:
- hearing ability, pupillary reflex (L/R), static righting reflex;
- fore- and hind-limb grip strength (recorded as the mean of three measurements);
- locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system, total movements and ambulations are reported).

BODY WEIGHT: Yes
- Weekly

FOOD CONSUMPTION :
- Weekly

FOOD EFFICIENCY: no

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at pretest and week 13
- Dose groups that were examined: Since no treatment-related ophthalmologic findings were noted in Week 13, the eyes of the rats of Groups 88 and 264 mg/kg bw.day dose levels were not examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, animals were deprived of food overnight (for a maximum of 24 hours), but water was available.
- How many animals: all animals
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, animals were deprived of food overnight (for a maximum of 24 hours), but water was available.
- How many animals: all animals
- Parameters checked were: According to test guidelines

URINALYSIS: Yes
- Time schedule for collection of urine: overnight
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked were: according to test guidelines
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were necropsied and descriptions of all macroscopic abnormalities recorded.

HISTOPATHOLOGY: Yes
Organ weights and tissues were collected and slides were examined by a pathologist according to the OECD 408 guideline.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores). Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 880 mg/kg bw/day, salivation and breathing related ailments such as rales and labored respiration were most commonly observed among all animals, while deep respiration and gasping were less frequent. Very occasional hunched posture, piloerection, lean appearance, squeaking were also observed in 880 mg/kg rats. One 880 mg/kg female displayed abdominal swelling.
At 264 mg/kg bw/day salivation was noted among all animals and rales were seen very occasionally. Although this was seen in all animals at 264 and 880 mg/kg, it is considered to result from the test item properties instead of a sign of systemic toxicity.
Mortality:
mortality observed, treatment-related
Description (incidence):
At 880 mg/kg bw/day, 5/10 males and 4/10 females were killed in extremis or found dead during treatment, 2 of which were treated on the day of sacrifice. Seven animals (4 males, 2 females) were killed in extremis between Days 7 to 77, and two females were found dead on days 36 and 73, respectively. Prior to sacrifice/death, most of these animals (apart from one female) displayed breathing related ailments such as labored respiration, rales or gasping. Macroscopic and/or microscopic findings were noted in the following organs: duodenum, jejunum, ileum, caecum and colon, stomach, rectum, thymus and/or spleen. These are described in the respective paragraphs.
At 88 mg/kg bw/day two females were killed in extremis. The premature sacrifice of one female on day 24 was preceded by abnormal posture of its right foreleg. The cause of moribundity for this animal was considered to be a malignant oligodendroglioma in the cervical spinal cord. The other 88 mg/kg female sacrificed prematurely on day 81 had an exophthalamic left eye. The cause of moribundity for this animal was considered to be marked inflammatory changes in one eye. Based on the very low incidence of these findings and in absence of other toxicologically relevant clinical signs or neoplastic or preneoplastic findings in any other treated animal in the study and the absence of dose response distribution, these findings were considered to be incidental with no toxicological significance ascribed to these findings.
No mortality occurred at 264 mg/kg.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 264 and 880 mg/kg bw/day, body weight and body weight gain of males was lower than vehicle controls, from week 5 of treatment onwards, achieving a level of statistical significance on most occasions.
Body weight of females at all dose levels and for males at 88 mg/kg bw/day was considered unaffected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption appeared marginally lower in 880 mg/kg bw/day than controls throughout the treatment period.
Food efficiency:
not examined
Description (incidence and severity):
to be completed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The nature and incidence of ophthalmology findings noted during pretest and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In 880 mg/kg bw/day females red blood cell counts and hemoglobin and hematocrit levels were statistically significanlty lower than in control animals.
Any other statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Any statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes in urinary parameters distinguished treated animals from control animals:
- Lower pH in males at 880 mg/kg bw/day
- Lower potassium concentration (mmol/L) in males and females at 264 and 880 mg/kg bw/day (only statistically significant for males at 880 mg/kg bw/day).
- Lower potassium excretion (mmol/TPV) in males at 264 and 880 mg/kg bw/day and in females at 880 mg/kg bw/day.
Any other statistically significant changes in urinalysis parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend or occurred within the range considered normal for rats of this age and strain.
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. Grip strength was considered to be unaffected by treatment.
Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. The statistically significant higher hindlimb grip strength of 300 mg/kg males occurred in the absence of a dose-related trend and was therefore considered to be unrelated to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no direct test item related alterations in organ weights.
In males, the higher group mean relative weights of the brain, heart, kidneys and adrenals at 880 mg/kg bw/day, and sometimes also 264 mg/kg bw/day, were considered to be due to the lower body weight at these doses. The group mean relative weight of the testes at 264 mg/kg bw/day was statistically significantly higher than control, but the individual animal values in this dose group were within the range in controls except for 1 animal, and there was no histological correlate, so this was not considered adverse.
In females, where body weights at the terminal sacrifice were not affected by treatment, group mean absolute adrenal weight was statistically significantly higher than control at the lowest dose (88 mg/kg bw/day). Three animals in this group had adrenal weights higher than the maximum seen in the controls. This was not, however, attributed to the test item, because higher doses were not affected by test item-related organ weight or histological changes in the adrenal. The higher group mean ovary weight at 264 mg/kg bw/day was probably due to cyclical variation. Group mean liver weight at 880 mg/kg bw/day was higher than controls, but the difference was only 7 % for absolute weight, and there was no histological correlate, so this was not considered adverse.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
From the 9 out of 20 animals administered 880 mg/kg bw/day that were euthanized, all of these animals had, at necropsy, distension by gas of the duodenum, jejunum, ileum, caecum and colon, sometimes also involving the stomach and/or rectum. In addition, 4 of these animals had decreased thymus and/or spleen size at necropsy. One of these animals, which had gastric distension by gas, was severely autolysed and extensively cannibalised, so that many of the abdominal organs could not be examined histologically. One surviving male at 880 mg/kg had distension by gas of the duodenum, jejunum, ileum and cecum. This animal also had decreased size of the thymus and spleen.

Other gross observations were not considered test item-related. One 300 mg/kg bw/day animal had slight erosion of the glandular mucosa of the stomach, that was apparent at necropsy as dark foci, but this was not considered test item-related because it can occur in rats due to gavage trauma, and because only one animal was affected, and this was not observed at the highest dose. Dark foci on the glandular mucosa of the stomach in other animals in this study correlated only with minimal congestion or haemorrhage (or had no correlate) and were also not considered test item-related.
The right seminal vesicle was small in two males (880 mg/kg bw/day and 88 mg/kg/day), correlating histologically with slight decreased secretory content. In view of the low incidence and severity, and the absence of dose-relationship, these changes were not considered test item-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
No microscopic correlate could be found for the gastro-intestinal distension as shown among the premature decedents and one surviving male at 880 mg/kg bw/day. The pathogenesis of the distension was not further examined by additional pathological evaluation performed in this study. Some of the histological findings at 880 mg/kg bw/day were not considered to be a direct effect of the test item but rather a secondary occurrence to stress as a result of the gastro-intestinal distension. These findings included:
- Reduced lymphoid tissue: Minimal to moderate decreased thymic cortex in 7 premature decendents; slight to moderate decreased splenic white pulp development in 2 premature decendents; minimal decreaed lymphoid follicle development in the mandibular lymph nodes in 2 premature decendents and in the mesenteric lymph node in 1 premature decendent.
- Reductions in secretion in some secretory organs: Minimal or slight decreased secretory content in the mandibular salivary glands in 1 male and 2 females euthanized in extremis at 880 mg/kg bw/day; Minimal mucification of the vaginal epithelium was noted in 2 females; minimal tubular atrophy in the kidney was noted in 1 male.
- Slight decrease in mature spermatozoa in the testes, with a marked decrease in spermatozoa in the epididymides was noted in a premature decedent. This animal also had marked decreased secretory content in the seminal vesicles, with the same change at the slight degree in the prostate and coagulating glands.
Other histologic changes were not considered to be test item-related as these occurred at low incidence and low severity, in absence of dose-relationship and/or they are frequent spontaneous findings. The findings included: minimal ulceration of the non-glandular part of the stomach and slight necrosis in the liver, slight decreased secretory content of seminal vesicle, minimal to marked ductal dilatation of the clitoral gland, tubular basophilia in the kidneys, minimal vacuolation of the spermatogenic epithelium in the testes they are frequent spontaneous findings.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
264 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
888 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
colon
duodenum
ileum
intestine
jejunum
rectum
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Analysis of formulations: During method validation, it was decided to follow ascorbic acid with the validated method, however the results have to be critically analyzed and interpreted as indicative due to stability issues of the ascorbic acid.

KGA:

Accuracy: a small response at the retention time of the test item was observed in one of the chromatograms of the control group formulation prepared for use in Week 13 (0.11 mg/g). It was considered not to derive from the formulation since the response was not observed in the duplicate study sample. In all other formulations of Group 1, no test item was detected. The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 prepared for use in Week 1 (mean 71 -74%) were not in agreement with target concentrations, possibly due to higher concentration of KGA in the test item used for calibration. The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 prepared for use in Week 4 (mean 103 -108%) were in agreement with target concentrations. The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 prepared for use in Week 13 (114 -129%), were above target concentrations.

Homogeneity: the formulations of low (group 2) and high (group 4) dose were homogeneous (1.4 -7.1%)

Stability: formulations at the entire range were stable (6.4 -8.6%) when stored at room under normal laboratory light conditions for at least 4 hours.

Homogeneity KGA in KGA Greens containers: The KGA in the KGA Greens containers was homogeneous

Ascorbic acid

Accuracy: No ascorbic acid was detected in the Group 1 formulations. The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 prepared for use in Week 1 were in agreement with target concentrations (mean 103 -105%). The concentrations analysed in the formulations of Group 2 and Group 4 prepared for use in Week 4 were in agreement with target concentrations (mean 98 -100%), whereas of Group 3 was slightly above the target concentration (mean 116%).

For the formulation of Group 2, Group 3 and Group 4 prepared for use in Week 13, the mean accuracy was below the target concentration (mean 26 -45%).

Homogeneity: The formulations of Group 2 and Group 4 prepared for use in Week 1 and 4 were homogeneous (mean 2.7 -5.7%). For the formulation of Group 2 prepared for use in Week 13, the homogeneity was above the criteria (i.e. coefficient of variation was 13 %).

Stability: Analysis of Group 2 formulations after storage yielded a relative difference of 2.7%. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 4 hours.

Analysis of Group 4 formulations after storage yielded a relative difference of 20%. However the increase of concentration occurred and therefore the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 4 hours.

Applicant's summary and conclusion

Conclusions:
In a 90-day oral repeated dose toxicity study with rats, conducted according to OECD/EC guidelines and GLP principles, toxicity was observed at the highest dose level (880 mg/kg bw.day), manifested as mortality and clinical signs in males and females due to distension by gas of parts of the GI tract and reduced body weight and body weight gain in males only. The NOAEL was set at 264 mg/kg bw/day.
Executive summary:

In a 90 -day study performed according to OECD 408 rats (n=10) were given 0, 88, 264 and 880 mg/kg bw/day of KGA Greens by gavage daily.

At 1000 mg/kg bw/day, 5/10 males and 4/10 females were killed in extremis or found dead during treatment. Seven animals (nos 31, 32, 33, 36, 40, 73, and 78) were killed in extremis between Days 7 to 77, and two animals (nos 75 and 80) were found dead on Days 36 and 73, respectively. Prior to sacrifice/death, most of these animals displayed breathing related ailments such as labored respiration, rales, gasping and salivation. At macroscopic examination all showed distension by gas of parts of the gastro-intestinal tract, which was considered to have been the cause of death and to be adverse. It was associated in some animals by secondary changes including reduced lymphoid tissue and reductions in secretion in some secretory organs.

At 100 mg/kg bw/day two females were killed in extremis due to incidental findings unrelated to test item. The cause of moribundity for this animal was considered to be a malignant oligodendroglioma in the cervical spinal cord and for the other animal was considered to be marked inflammatory changes in one eye. Based on the very low incidence of these findings and in absence of other toxicologically relevant clinical signs or neoplastic or preneoplastic findings in any other treated animal in the study and the absence of dose response distribution, these findings were considered to be incidental with no toxicological significance ascribed to these findings.

No mortality occurred at 300 mg/kg bw/day.

During the study period reduced body weight were noted at 300 and 1000 mg/kg bw/day, albeit only in males, which was accompanied with slightly reduced food intake at 1000 mg/kg bw/day. In absence of histopathological findings at 300 mg/kg no adversity was suggested at 300 mg/kg bw/day.

Histopathological examination showed several effects at 1000 mg/kg bw/day which were considered to have occurred secondary to the gastro-intestinal distension and stress, and included decreased thymic cortex and splenic white pulp, decreased lymphoid follicle development decreased (mature) spermatozoa in the testes and in the epididymides and decreased secretory content in the seminal vesicles / prostate / coagulating glands.

Haematological changes were also observed in 1000 mg/kg bw/day animals, such as reductions in red blood cell count, hemoglobin, haematocrit levels in females. Urinalysis also revealed reduced potassium levels in 1000 mg/kg males and females, reduced pH in 1000 mg/kg bw/day males, and reduced sodium levels in females, however there were no correlating kidney findings.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. functional observations, ophthalmoscopy, clinical biochemistry, and organ weights).

Based on these results a NOAEL of 264 mg/kg bw/day was established for local and systemic effects.