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Repeated dose toxicity: oral

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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:
Feb 13 to Oct 8, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
2008
Deviations:
yes
Remarks:
One deviation reported; considered uncritical
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Batch No. 6F11027000
- Expiration date of the lot/batch: 17.11.2016
- Purity: 96%

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Han (IGS)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Germany)
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 7-8 weeks
- Weight at study initiation: males between 180 g and 208 g, females between 131 g and 149 g
- Housing: Groups of 5 animals per gender in open macrolon cages type 2000P, Tecniplast (size slightly larger than GV-SOLAS Type IV )
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70%
- Air changes (per hr): Air conditioning with approx. 10 air changes per hour
- Photoperiod (hrs dark / hrs light): artificial lighting, 12 h light/12 h

IN-LIFE DATES: First application (Day 1) February 23, 2015 to end of in-life phase March 24, 2015

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily by oral gavage to 5 male and 5 female rats in three groups at dosages of 1000 mg/kg bw (high dose), 333 mg/kgbw (medium dose) and 111 mg/kg bw (low dose) over a period of 28 days (males) or 29 days (females). Further 10 animals (5 males and 5 females) received the vehicle solution (corn oil) as control.
Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: The test item was diluted in corn oil. The preparation was intended for an application volume of 4 ml per kg body weight. The test item was administered daily by oral gavage.

- VEHICLE: Corn oil
- Justification for use and choice of vehicle: As the test item’s solubility in water is poor, corn oil was used as an organic solvent. Pre- tests showed that the solubility of the test item in corn oil was sufficient.
- Amount of vehicle: 4 mL per kg body weight
- Lot/batch no.: Sigma, Batch No. MKBQ9948V
- Purity: 100%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Due to the stability data presented, the test item preparation was performed once at the beginning of the 28-day period of the in-life phase. For application on day 29 of the female animals, the solutions were prepared freshly 3 days before application.
Each formulated test item solution was analyzed after preparation to reassure correct consistency and concentration.
From each application solution of the first test item preparation, a sample was sent to Principal Investigator 1 (PI1) at the day of the first application, after the first application week and after the last application on day 28. From the second application solution one sample of each concentration was sent to PI1 on day 29 of the in-life phase. Thus, a total of 12 samples, 4x 250 mg/mL, 4x 83.3 mg/mL and 4x 27.8 mg/mL in corn oil, were sent to PI1.
The sample transfers to PI1 followed vivo Science´s SOP MAT-3-0003 “Versand von Prüfsubstanzen, Blutproben Serumproben und Materialien, in-und extern” (“Shipment of test items, blood samples, serum samples and materials, internal and external”). Samples were shipped cooled, with minimal headspace and closed properly.

Accuracy was shown for the test item in corn oil at 250 mg/mL, 83.3 mg/mL and 27.8 mg/mL. The recovery rate lay in the range 100 ± 4 % for all concentrations.
Duration of treatment / exposure:
The test item was administered daily by oral gavage over a period of 28 days (males) or 29 days (females).
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
333 mg/kg bw/day (nominal)
Dose / conc.:
111 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males and 5 females/dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In preparation for this study a dose range finding study with dose escalation was performed. The test item was administered orally in escalating doses every 3rd day up to 1000 mg/kg body weight over a period of 18 days. This resulted in slight detectable effects (reddened thyroid (5/6) and slightly reddened fundus of the stomach (1/6). A concrete sign of toxicity or morbidity was not detected. Consequently, the highest dose for the main study was set to 1000 mg/kg bodyweight. Two graduated (1 in 3) serial dilutions thereof (333 mg/kg, 111 mg/kg) will be assigned as medium and low dose.
- Rationale for selecting satellite groups: Not included. Considering the multitude and severity of the effects of the test substance observed in the dose range finding study performed prior to the main study, potential delayed toxic effects are not likely to be more severe or to increase the toxicity rating of the test item. The potential reversibility of the toxic effects induced by the test item has no influence on the assessment of the toxicity of a test substance. In accordance with the Sponsor a satellite group was therefore not included in the study.

Examinations

Observations and examinations performed and frequency:
General clinical signs, viability, reactions to treatment and conspicuous behavioural traits of all experimental animals were monitored daily during the in-life phase. Group food consumption and individual body weight were recorded once weekly. Group water consumption was recorded twice weekly.

At the end of the treatment period, blood samples from all animals were drawn to provide data on haematology, clinical biochemistry and blood clotting time. All animals were sacrificed after bleeding and examined by gross necropsy.

According to the study plan, weights of selected organs were recorded, and tissues and organs were preserved. Samples from the high dose groups and the vehicle groups were processed for histopathological examination.
Sacrifice and pathology:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Performed daily throughout the study up to the day of necropsy

DETAILED CLINICAL OBSERVATIONS: Yes, IRWIN Test
- Time schedule: Once weekly, including once before the application start


BODY WEIGHT: Yes
- Time schedule for examinations: Once weekly, including once before the application start

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Once weekly, including once before the application start

WATER CONSUMPTION: Yes
- Time schedule for examinations: Twice weekly, including once before the application start

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the in-life phase
- Anaesthetic used for blood collection: Yes
- How many animals: All animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the in-life phase
- How many animals: All animals

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during the last exposure week
- Dose groups that were examined: All animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Grip-strength and beam-walking tests

OTHER: Necropsy
- The in-life phase will be terminated on day 29. All animals will be sacrificed by asphyxiation in a CO2 enriched atmosphere. A full macroscopic examination will be performed. All occurring lesions will be noted. Data will be recorded on checklists for each individual animal.
Statistics:
- The arithmetic mean, standard deviation and median calculated for all grouped numerical data originating from the monitoring of grip strength, gross pathology (organ weights) and parts of the hemogram. Where appropriate, detailed column statistics applied (minimum / maximum data, 25% quartiles, standard error, upper and lower confidence interval 95%).

- For basic analysis the respective test item groups compared to the vehicle group by assessing of statistical significance using a two-tailed unpaired Student´s t-test. For all calculations, the significance level set to 0.05. The adequate analysis methods were one-way ANOVA (analysis of variance) followed by a post-hoc Dunnett´s t-test (for hemograms and organ weights), or two- way ANOVA followed by a post-hoc Bonferroni test (for body weight analysis).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals treated with the high and medium dose of the test item showed increased saliva production and wiped their mouth in the cage bedding after application. A mild test item effect was noted.
Mortality:
no mortality observed
Description (incidence):
No animal died or had to be euthanized during the in-life phase of this study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No indication for toxicological effects on the body weight and weight gain could be observed during in-life phase. All parameters were within the normal range of rats of this strain, sex and age. Statistically, no significant changes were noted in any of the treated animals.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption of the male animals of the high, medium and low dose groups showed a slight increase in consumption with rising test item concentration. This effect was less prominent in the female dose groups. A minor test item related effect was therefore noted, but not considered adverse.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
The water consumption showed a similar increase in uptake, with minor differences in the female animals but marked changes in the male rats of the high dose group. A test item related effect could therefore not be excluded.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No relevant test item-induced effects on any haematological parameter could be detected.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Analysis of the clinical biochemistry parameters revealed a significant increase in the cholesterol concentration (males and females), for the urea concentration (males) and for the aspartate aminotransferase (females). In males a non-significant aspartate aminotransferase increase was noted.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No differences in grip strength were observed in relation to the vehicle control group, and none of the animals showed significant differences to the vehicle group during the beamwalking test.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The most prominent finding during gross necropsy was a statistically significant increase of the liver weights in both the male and female high dosed animals. Some minor changes were observed in the weights of spleens and kidneys in some dose groups, but with no clear treatment related tendency.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
During gross pathology of the female animals, no abnormal finding was recorded. Some individual findings were made but without test item related tendency. During gross pathology of the male animals reddened thyroid glands were found in all high dose animals (5/5), two medium dose animals (2/5), four low dose animals (4/5) and two of the vehicle animals (2/5).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Based upon the evaluation of control and high dose animals, treatment with 1,3,5-Triisopropylbenzene under the conditions of this study caused histopathological changes in the liver, thyroid gland and thymus at 1000 mg/kg/day, out of which the liver effects were considered adverse.
Histopathological findings: neoplastic:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 333 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
yes

Any other information on results incl. tables

Deviation reported in the recovery of the last test item preparation (23.03.2015): a recovery of only 80.8 % in the high dose group was measured (202.3 mg/ml instead of 250.0 mg/ml). This would be outside of the acceptable range of 94.5 – 99.7 % of the nominal value. The animals received the intended concentration for 28 days and only once the insufficiently concentrated test item preparation. Also, the application period of these animals was extended by one day. The deviation was thus rated as uncritical for the outcome of the study. During the histopathological examination, additional organs were analyzed which were not included in the original study plan. As well, peyer's patch was not present on slides for a proportion of control and high dose animals, and no slides were presented for macroscopic lesions in the low and mid dose group. The noted deviations were not considered to have a negative impact on the evaluation or interpretation of the study results.

Applicant's summary and conclusion

Conclusions:
The several liver related findings point to an adverse effect of the test item at a concentration of 1000 mg/kg bw/day. Thus, we would recommend the NOAEL at a concentration of 333 mg/kg bw/day.
Executive summary:

The aim of the study was to assess data on the oral toxicity of the substance 1,3,5-Triisopropylbenzene, dissolved in corn oil, after daily administration to Wistar rats. The test item was administered daily by oral gavage at dosages of 1000 mg/kg, 333 mg/kg or 111 mg/kg over a period of 28 days (males) and 29 days (females).

General clinical signs and behavior

Animals treated with the high and medium dose of the test item showed increased saliva production and wiped their mouth in the cage bedding after application. A mild test item effect was noted.

No differences in grip strength were observed in relation to the vehicle control group, and none of the animals showed significant differences to the vehicle group during the beam-walking test.

Body weight, food and water consumption

No indication for toxicological effects on the body weight and weight gain could be observed during in-life phase. All parameters were within the normal range of rats of this strain, sex and age. Statistically, no significant changes were noted in any of the treated animals.

The food consumption of male and female animals of the high, medium and low dose groups showed a slight increase in consumption with rising test item concentration. A minor test item related effect was therefore noted, but not considered adverse.

The water consumption showed a similar increase in uptake, with minor differences in the female animals but marked changes in the male rats of the high dose group. A test item related effect could therefore not be excluded.

Haematology and clinical biochemistry

No relevant test item induced effects on any haematological parameter could be detected.

Analysis of the clinical biochemistry parameters revealed a significant increase in the cholesterol concentration (males and females), for the urea concentration (males) and for the aspartate aminotransferase (females). In males a non-significant aspartate aminotransferase increase was noted.

Necropsy

The gross necropsy revealed a significant increase of the liver weights in both the male and female high dose animals as most prominent effect. Some minor changes were observed in the weights of spleens and kidneys in some dose groups, but with no clear treatment related tendency.

Histology

Based upon the evaluation of control and high dose animals, treatment with 1,3,5-Triisopropylbenzene under the conditions of this study caused histopathological changes in the liver, thyroid gland and thymus at 1000 mg/kg bw/day, out of which the liver effects were considered adverse.

Conclusion

In the present study, administration of the test item 1,3,5-Triisopropylbenzene once daily to Wistar rats over a time period of 28 days (male) or 29 days (females) resulted in morphological findings in the liver of animals treated with 1000 mg/kg bw/day. The histopathological findings in the liver correlate with an increase of the liver weight (males and females), an increase in the blood cholesterol concentration (males and females) as well as an increase in the aspartate aminotransferase concentration (females).

Combined, the several liver related findings point to an adverse effect of the test item at a concentration of 1000 mg/kg bw/day. Thus, we would recommend the NOAEL at a concentration of 333 mg/kg bw/day.