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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:
31 July to 05 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study undertaken at GLP accredited laboratory to internationally accepted guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
The deviations were considered to have not affected the integrity or validity of the study.
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3050, Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.
Deviations:
yes
Remarks:
The deviations were considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
S-205
IUPAC Name:
S-205
Constituent 2
Chemical structure
Reference substance name:
2'-anilino-6'-[ethyl(3-methylbutyl)amino]-3'-methylspiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one
EC Number:
274-641-1
EC Name:
2'-anilino-6'-[ethyl(3-methylbutyl)amino]-3'-methylspiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3-one
Cas Number:
70516-41-5
Molecular formula:
C34 H34 N2 O3
IUPAC Name:
6'-[ethyl(3-methylbutyl)amino]-3'-methyl-2'-(phenylamino)-3H-spiro[2-benzofuran-1,9'-xanthen]-3-one
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): S-205
- Substance type: Industrial chemical with colourant properties
- Physical state: Pale coloured powder
- Analytical purity: 99.7%
- Lot/batch No.: 1301L
- Expiration date of the lot/batch: 1 April 2014
- Storage condition of test material: Room temperature, protected from light

Test animals

Species:
rat
Strain:
other: Crl: CD® (SD)]
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Commercial laboratory animal supplier.
- Age at study initiation: 42 to 48 days
- Weight at study initiation: 222 to 268 g for males and 177 to 217 g for females.
- Fasting period before study: Fasting overnight before routine blood sampling
- Housing: The animals were housed five of one sex per cage made of a polycarbonate body with a stainless steel mesh lid.
- Diet: The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance Diet)
- Water: Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C
- Humidity (%): 40 to 70%
- Air changes: Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod: Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: aqueous 1% methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): All formulations were prepared freshly each week and prepared in advance of the first day of dosing. Formulations were stored in a fridge (nominally 4°C) pending use or for up to 24 hours at room temperature (nominally 21°C).

- Justification for use and choice of vehicle: The vehicle used was aqueous 1% methylcellulose and is an accepted medium.
- Concentration in vehicle: 3, 30 and 100 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg
:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity and stability was confirmed for S-205 in aqueous 1% methylcellulose formulations at nominal concentrations of 1 mg/mL and 100 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 1 day and refrigerated storage for up to 15 days. The storage times represented the expected maximum time from preparation to completion of administration.
The mean concentrations of S-205 in test formulations analysed for Groups 3 and 4 in the study were within +10%/-15% of nominal concentrations, confirming accurate formulation. The mean concentrations of S-205 in test formulations analysed for Group 2 were outside the applied limits; this was considered to be due to a problem with homogeneity rather than an incorrectly prepared formulation.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses of 250, 500 and 1000 mg/kg/day were selected for a preliminary toxicity study in the rat by oral gavage administration for seven days (Huntingdon Life Sciences Study Number JAR0004) based on an acute oral toxicity study in the rat (HLS Study Number 82203D/YMD26/AC) in which no death was apparent following a single dose of 16 g/kg and signs of reaction to treatment were restricted to piloerection, hunched posture and abnormal gait and lower than anticipated bodyweight gain during the first week after administration. On study JAR0004 doses of 250, 500 and 1000 mg/kg/day were well tolerated with no death or treatment-related clinical sign. Possible treatment-related findings were slightly increased water consumption (by visual assessment) in animals treated at 500 mg/kg/day or above and slightly higher than expected liver weights in males treated at 1000 mg/kg/day. Accordingly, doses of 0, 30, 300 and 1000 mg/kg/day were selected for this
study.
Positive control:
None

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29
- Anaesthetic used for blood collection: Yes; Animals were held under light general anaesthesia induced by isoflurane and blood samples were withdrawn from the sublingual vein.
- Animals fasted: Yes
- How many animals: All

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There was no unscheduled death during the treatment period. There was no post dosing sign and no toxicologically significant clinical sign observed during the treatment period.
Mortality:
no mortality observed
Description (incidence):
There was no unscheduled death during the treatment period. There was no post dosing sign and no toxicologically significant clinical sign observed during the treatment period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Bodyweight gains over the treatment period were considered to be unaffected by treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The haematological investigation on Day 29 revealed elongated prothrombin times for females receiving 300 or 1000 mg/kg/day (1.08 and 1.11x Control, respectively). Males were considered to be unaffected with regards to all parameters.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Motor activity scores for males and females showed some inter-group variation but there was no difference considered to be associated with S-205.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Analysis of the organ weight data from animals killed after 4 weeks of treatment did not reveal any toxicologically significant changes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination performed after 4 weeks of treatment revealed no intergroup difference.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with S-205 were seen in the kidneys
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There was no unscheduled death during the treatment period. There was no post dosing sign and no toxicologically significant clinical sign observed during the treatment period.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gains over the treatment period were considered to be unaffected by treatment.

HAEMATOLOGY
The haematological investigation on Day 29 revealed elongated prothrombin times for females receiving 300 or 1000 mg/kg/day (1.08 and 1.11x Control, respectively). Monocyte counts were considered to be low for females receiving 1000 mg/kg/day (0.57x Control) and total white cell counts were slightly low (0.76x Control). Individual monocyte and total white blood cell numbers for animals treated at 1000 mg/kg/day were
within the Control individual ranges. Accordingly, the variations from Control of the mean values were considered to be normal biological variation. Males were considered to be unaffected with regards to all parameters.

CLINICAL CHEMISTRY
Biochemical analysis of the blood plasma on Day 29 revealed slightly low aspartate amino transferase activity for males receiving 300 or 1000 mg/kg/day (0.81 and 0.91x Control respectively), although this showed no dose related trend and is, therefore, considered to be fortuitous and due to high inter animal variability.

NEUROBEHAVIOUR
Motor activity scores for males and females showed some inter-group variation but there was no difference considered to be associated with S-205.

ORGAN WEIGHTS
Analysis of the organ weight data from animals killed after 4 weeks of treatment did not reveal any toxicologically significant changes.
Thymus weights were considered to be slightly low for males receiving 300 or 1000 mg/kg/day (0.89x Control), however, statistical significance was not attained and, in the absence of a microscopic correlate, this variance from Control was considered to be normal biological variation.

GROSS PATHOLOGY
The macroscopic examination performed after 4 weeks of treatment revealed no intergroup difference.
The nature and incidence of all findings were consistent with the commonly seen background of macroscopic changes in Sprague Dawley rats at these laboratories.

HISTOPATHOLOGY: NON-NEOPLASTIC
Changes related to treatment with S-205 were seen in the kidneys.
An increased incidence and severity of cortical tubular basophilia was seen in the kidneys of female animals given 1000 mg/kg/day.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Oral administration of S-205 (an industrial chemical with colourant properties) to Crl: CD® (SD) rats at doses up to 1000 mg/kg/day daily for a period of four weeks was well tolerated without any evidence of overt toxicity; following histopathological examination, the kidney was identified as a target organ.
Cortical tubular basophilia was evident in the kidneys of females given S-205 at 1000 mg/kg/day. Under the conditions of this study, this was not considered to be adverse as the changes were predominantly minimal, occurred unilaterally in the majority of animals and were without a correlate.
It was concluded that, under the conditions of this study, the no-observed-adverse-effect-level (NOAEL) was 1000 mg/kg/day.