Registration Dossier

Administrative data

acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 August - 03 September 2010
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
according to guideline
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
according to guideline
EPA OPPTS 870.1100 (Acute Oral Toxicity)
according to guideline
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent partial revisions.
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): Custom Red #2
- Substance type: Violet powder
- Physical state: Solid
- Lot/batch No.: C50APR0805D
- Stability under storage conditions: Stable
- Storage condition of test material: At room temperature in the dark
- Expiration date of the lot/batch: 08 January 2011

Test animals

other: Crl:WI (Han)
Details on test animals or test system and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 8 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- Housing: Group housing of 3 animals per cage in labeled Macrolon cages (MIV type; height 18 cm.) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.
- Health inspection: A health inspection was performed prior to treatment, to ensure that the animals were in a good state of health.

Results of analysis for diet (nutrients and contaminants), sawdust, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

- Temperature (°C): 19.5 - 21.3ºC
- Humidity (%): 46 - 81%
Temporary deviations from the maximum level of relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations.
- Air changes (per hr): approximately 15 air changes per hour- Photoperiod (hrs dark / hrs light): 12 hours artificial
fluorescent light and 12 hours darkness per day.

IN-LIFE DATES: From: 17 August - 03 September 2010

Administration / exposure

Route of administration:
oral: gavage
propylene glycol
Details on oral exposure:
GAVAGE METHOD: plastic feeding tubes.

Frequency: single dosage, on Day 1.

VEHICLE: Propylene glycol (Merck, Darmstadt, Germany) (specific gravity 1.036).
- Justification for choice of vehicle: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

MAXIMUM DOSE VOLUME APPLIED: 2000 mg/kg (10 mL/kg) body weight.

DOSAGE PREPARATION: The formulations (w/w) were prepared within 4 hours prior to dosing.
Homogeneity was accomplished to a visually acceptable level.
Adjustment was made for specific gravity of the vehicle.
2000 mg/kg body weight
No. of animals per sex per dose:
6 (2 groups of three females in a stepwise manner)
Control animals:
Details on study design:
Animals were deprived of food overnight prior to dosing and until 3-4 hours after administration of the test substance. Water was available ad libitum.

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Mortality/Viability: Twice daily.
Body weights: Days 1 (pre-administration), 8 and 15.
Clinical signs: At periodic intervals on the day of dosing (Day 1) and once daily thereafter, until Day 15. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded.
- Necropsy of survivors performed: At the end of the observation period, animal nos. 1-3 were sacrificed by oxygen/carbon dioxide procedure and animal nos. 4-6 were deeply anaesthetized using an isoflurane (Abbott B.V., Hoofddorp, The Netherlands) in nitrous oxide/oxygen (Air Products, Amsterdam, The Netherlands) combination followed by exsanguination. All animals were subjected to necropsy. Descriptions of all internal macroscopic
abnormalities were recorded.
- Other examinations performed: none.
No statistical analysis was performed (The method used is not intended to allow the calculation of a precise LD50 value).

Results and discussion

Effect levels
Dose descriptor:
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
No mortality occurred.
Clinical signs:
Lethargy, hunched posture, piloerection, uncoordinated movements and/or abnormal gait were shown
by all animals between Days 1 and 3.
Body weight:
The body weight gain shown by the animals over the study period was considered to be similar to that
expected of normal untreated animals of the same age and strain.
Gross pathology:
No abnormalities were found at macroscopic post mortem examination of the animals.

Applicant's summary and conclusion

Interpretation of results:
not classified
Migrated information Criteria used for interpretation of results: EU
The oral LD50 value of Custom Red #2 in Wistar rats was established to exceed 2000 mg/kg body weight.
According to the OECD 423 test guideline, the LD50 cut-off value was considered to be 5000 mg/kg body weight.
Based on these results, Custom Red #2 should be classified as: may be harmful if swallowed (Category 5) for acute toxicity by the oral route according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007) and does not have to be classified and has no obligatory labelling requirement for oral toxicity according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.