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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
Male experimental data from the OECD 422
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
not applicable
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl 2-[(7-hydroxy-3,7-dimethyloctylidene)amino]benzoate
EC Number:
201-908-1
EC Name:
Methyl 2-[(7-hydroxy-3,7-dimethyloctylidene)amino]benzoate
Cas Number:
89-43-0
Molecular formula:
C18H27NO3
IUPAC Name:
methyl 2-[(7-hydroxy-3,7-dimethyloctylidene)amino]benzoate
Constituent 2
Reference substance name:
Reaction products of 7-hydroxy-3,7-dimethyloctanal and methyl 2-aminobenzoate
IUPAC Name:
Reaction products of 7-hydroxy-3,7-dimethyloctanal and methyl 2-aminobenzoate
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Aurantiol Pure (Product Code 2365901)
- Molecular formula (if other than submission substance): UVCB
- Molecular weight (if other than submission substance): 151 to 160 (range for identified components)
- Substance type: UVCB
- Physical state: Yellow viscous liquid
- Lot/batch No.:SC00010629
- Expiration date of the lot/batch: 26 November 2014
- Stability under test conditions: stable until the expiry date
- Storage condition of test material: Refrigerated and protected from light
- Volatility : Vapour pressure 0.4 Pa at 293K
Specific details on test material used for the study:
Appearance: Yellow viscous liquid
Batch: SC00020302
Purity/Composition: UVCB substance
Test item storage: In refrigerator (2-8°C) protected from light
Stable under storage conditions until: 22 February 2018 (expiry date)
Additional information
Test Facility test item number: 205387/D
Purity/Composition correction factor: No correction factor required
Test item handling: Use amber glassware or wrap container in aluminum-foil
Specific gravity/density 1.057,97 kg/m3 at 20°C
Chemical name (IUPAC), synonym or trade name Reaction products of 7-hydroxy-3,7-dimethyloctanal and methyl 2-aminobenzoate

Test animals

Species:
rat
Strain:
Wistar
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.
Sex:
male
Details on test animals or test system and environmental conditions:
male Crl:WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. At initiation of administration, males were 10 weeks old and weighed between 261 and 287 g
The animals were allowed to acclimate to the Test Facility toxicology accommodation for 5 days prior to start of the pretest period (females) or 5 days before the commencement of dosing administration (males).
Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately.

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
Prepared diets were provided ad libitum throughout the study, except during designated procedures. During the acclimatization period, animals had free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals had no access to food for a maximum of 2 hours.
The test item and control item were administered to the appropriate animals by inclusion in the diet ad libitum from Day 1 onwards for a minimum of 28 days. Males were exposed for 28 days, up to and including the day before scheduled necropsy. This included a minimum of two weeks prior to mating and during the mating period.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
The oral route of administration via dietary inclusion was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
The amount of test item incorporated in the diet was kept at a constant level in terms of ppm, throughout the study period. After termination, the actual test item intake was estimated based on the body weight and food consumption values.
The diet was provided in stainless steel containers, covered by a stainless steel grid to prevent spillage.
The same diets remained in the food hopper for a maximum of 1 day. During the daily weighing occasion, the remaining diet in the food hopper was replaced with new diet retained from the freezer acclimated to room temperature conditions for at least 1 hour prior to opening the diet bag.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item was mixed without the use of a vehicle, directly with the required amount of powder feed. Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used. Diets were prepared freshly for use at room temperature for a maximum of 1 day. Diets were kept in the freezer (≤-15°C) until use, if not used on the day of preparation for a maximum of 17 days.
All samples to be analyzed were transferred (at room temperature under normal laboratory light conditions) to the analytical laboratory at the Test Facility.
Residual samples were discarded after completion of the sample analysis.
Analyses were performed by by UPLC-UV using a validated analytical procedure (Test Facility Study No. 505073).
Duplicate sets of samples (approximately 5 g) for each sampling time point were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were discarded.
Duration of treatment / exposure:
Males - 28 days exposure.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 ppm
Dose / conc.:
3 000 ppm
Dose / conc.:
10 000 ppm
No. of animals per sex per dose:
10 males
Control animals:
yes, concurrent vehicle
Details on study design:
Prior to start of the treatment period (males), each animal was identified using earmark and tattoo.
The animals were allowed to acclimate to the Test Facility toxicology accommodation for 5 days prior to start of the commencement of dosing administration (males).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4).
Sacrifice and pathology:
Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
All animals surviving to scheduled necropsy were fasted (overnight with a maximum of a 24 hours) before their scheduled necropsy. Water was available.
Other examinations:
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs as part of the OECD 422 protocol.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.
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 \or 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 matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

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:
no effects observed
Mortality:
no mortality observed
Description (incidence):
All males survived, mated and were culled on day 29.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes were observed up to 10000 ppm.
Slightly lower body weights (not statistically significant) and body weight gain were noted for the males at 10000 ppm from Day 11 of treatment onwards. At the end of treatment, average body weight gain was 96% of the concurrent controls. Based on the slight change, lack of statistical significance and in the absence of correlating findings, this was considered to be non-adverse.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded for males up to 10000 ppm.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
Reticulocyte levels were unaffected in males up to 10000 ppm.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
• Decreased total bilirubin in males at 10000 ppm (0.83x of controls).
• Slightly decreased chloride levels in males at 3000 and 10000 ppm (0.97x and 0.96x of controls, respectively).
• Slightly decreased total protein levels in females at 10000 ppm (0.94x of controls).
• Slightly increased creatinine levels in females at 10000 ppm (1.09x of controls). A similar trend was noted for males at 10000 ppm (1.07x of controls), but this difference was not statistically significant.
• Slight dose related increase in glucose levels in females (1.08x, 1.17x and 1.18x of controls for 1000, 3000 and 10000 ppm, respectively), but these differences were not statistically significant.

All values remained within the normal range for rats of this strain and age.
No toxicologically relevant changes were noted in clinical biochemistry parameters at dose levels at 1000 ppm in males and up to 3000 ppm in females.
Any other 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.
For males of all treatment groups, a slight decrease in T4 levels was observed; 0.89x, 0.90x and 0.83x of controls for 1000, 3000 and 10000 ppm treated males, respectively. All values remained within the historical control data .

Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation parameters were not considered to be affected by treatment.
Motor activity was similar between treated and control groups.
All groups showed a similar motor activity habituation profile with very high activity in the first interval that decreased over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
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.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
10 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: See remarks

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
10 000 ppm
System:
other: WHole animal
Organ:
other: Whole animal

Applicant's summary and conclusion

Conclusions:
The NOAEL for male rats after 28 days is greater than 10000 ppm (710 mg/kg bw/day).
Executive summary:

Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test an parental, reproduction and developmental no-observed-adverse-effect level (NOAEL) of at least 10000 ppm for Aurantiol Pure was established, corresponding to an overall mean test item intake of 710 mg/kg bw/day for males.

For the exposure scenarios assessment and DNELs, the male NOAEL has been chosen.