Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start of experimental phase: 25 September 2017 End of experimental phase: 19 October 2017 Study completion: 18 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
adopted on 17 December 2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Version / remarks:
described in
Council Regulation (EC) No. 440/2008 and subsequent revisions
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, OPPTS Methods 870.1000 "Acute Toxicity Testing - Background"
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
Species and strain: Rat, Hsd: Sprague Dawley SD
Sex: Females (nulliparous and non-pregnant)
Age:7 weeks old
Supplier: Envigo RMS s.r.l., San Pietro al Natisone (UD), Italy
Breeder: Envigo RMS B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands
Date of arrival: 13 September 2017
Weight range at arrival: 162.6-176.3 grams
Acclimatisation period: At least 5 days
Veterinary health check: During acclimatisation period

ENVIRONMENTAL CONDITIONS
Animals per cage: 3 during the study; 5 during acclimatisation
Housing: Polisulfone solid bottomed cages measuring 59.5×38×20 cm with nesting material provided into suitable bedding bags
Cage control: Daily inspected and changed as necessary (at least 3 times/week)
Water Drinking: water supplied to each cage via a water bottle
Water supply: ad libitum
Diet: 4 RF 18 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy)
Diet supply: ad libitum throughout the study except for the dosing procedure indicated in section 4.2.2
Room lighting: Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours
Air changes: Approximately 15 to 20 air changes per hour
Temperature range: 22 °C±2 °C
Relative humidity range: 55%±15%

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 30 and 5mg/mL.
- Amount of vehicle (if gavage): 10 mL/kg

Doses:
300 mg/kg; 50 mg/kg
No. of animals per sex per dose:
3 Females at 300 mg/kg; 6 females at 50 mg/kg
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Animals were observed for clinical signs as indicated: Day of dosing: on dosing, approximately 0.5 hour after dosing, approximately 2 hours after dosing, approximately 4 hours after dosing, daily thereafter for a total of 14 days. All animals were weighed at allocation to the study (Day -1), on the day of dosing (Day 1) and
on Days 2, 8 and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Results and discussion

Effect levels
Sex:
female
Dose descriptor:
LD50
Effect level:
> 50 - < 300 mg/kg bw
Based on:
test mat. (total fraction)
Mortality:
All animals dosed at 300mg/kg bodyweight were found dead after a few minutes from the administration.
One animal dosed at 50mg/kg bodyweight was found dead in cage at 2 hours after dosing.
In the second group dosed with 50mg/kg bodyweight, one animal was found dead in cage 2
hours after dosing.
Clinical signs:
All animals dosed at 300mg/kg bodyweight appeared prone at observation performed at 30 minutes after dosing and died after few minutes.
One animal dosed at 50 mg/kg body weight, found dead at 2 hours after dosing, did not show any clinical sign. One animal, dosed at 50 mg/kg body weight,
showed decreased activity 30 minutes after dosing and was found dead at 2 hours after dosing.
Hunched posture and piloerection were observed in two animals dosed at 50 mg/kg body weight at 2 and 4 hours after dosing. These signs recovered on Day 2 of study. Two animal dosed at the same dose level did not show any clinical sign.
Body weight:
Changes in body weight observed during the study were within the expected range for this strain and age of animals.
Gross pathology:
Unscheduled deaths:
Orange mucosa and orange mucoid content in duodenum, jejunum and stomach and orange staining on the muzzle were observed in all early deceased animals
treated at 300mg/kg bodyweight. In addition, single animals showed abnormal colour and contents (orange mucosa and orange mucoid, respectively) in ileum
(animal nos. 191 and 195) and red mucosa of the caecum (animal no. 191). Abnormal colour and/or abnormal contents in duodenum, jejunum and stomach were observed in early deceased animals treated at 50mg/kg bodyweight. Moreover, orange fluid in ileum was observed in animal no. 201 and dark colour of the liver,
swollen spleen, dark red pancreas, multiple dark red pinpoint on thymus, dark colour of mesenteric lymph nodes, orange staining on muzzle and brown
staining on urogenital region in animal no. 207.

Final sacrifice:
No abnormalities were observed at necropsy examination performed at the end of the observation period on animals treated at 50mg/kg bodyweight.

Applicant's summary and conclusion

Interpretation of results:
Category 3 based on GHS criteria
Conclusions:
The acute toxicity of Basic Orange 22 was investigated following a single oral administration (10 mL/kg bodyweight in purified water) to the Sprague Dawley rat
followed by a 14-day observation period.
All animals treated at 300 mg/kg bodyweight died. At 50 mg/kg bodyweight, two animals out of 6 died. Piloerection, hunched posture and decreased activity were observed in 2 of the surviving animals following dosing at 50 mg/kg bodyweight.
These results demonstrate the expected acute oral LD50 of Basic Orange 22 to be lower than 300 mg/kg bodyweight, but higher than 50 mg/kg bodyweight.
Executive summary:

The acute toxicity of Basic Orange 22 was investigated following a single oral administration to the Sprague Dawley rat followed by a 14-day observation period. A first group of 3 female animals was initially dosed at 300 mg/kg bodyweight (Step 1). All animals died on the day of dosing. The animals appeared prone at observation performed at 30 minutes after dosing and died after few minutes.

A second group of 3 female animals was then dosed at 50 mg/kg bodyweight (Step 2). One animal showed decreased activity 30 minutes after dosing and was found dead at 2 hours after dosing.

Hunched posture and piloerection were observed in one animal at 2 and 4 hours after dosing. These signs recovered on Day 2 of study. The third animal in this group did not show any clinical sign. A third group, similarly composed, was then dosed at the same dose level (Step 3). One animal was found dead 2 hours after dosing. A single animal showed piloerection, hunched posture and decreased activity at 2 and 4 hours after dosing. These signs recovered on Day 2 of study. The third animal did not show any clinical sign. Body weight changes recorded during the study were within the expected range for this strain and age of animals. Orange mucosa and orange mucoid content in duodenum, jejunum and stomach and orange staining on the muzzle were observed in all early deceased animals treated at 300 mg/kg bodyweight. In addition, single animals showed abnormal colour and contents (orange mucosa and orange mucoid, respectively) in ileum (animal nos.191 and 195) and red mucosa of the caecum (animal no. 191). Abnormal colour and/or abnormal contents in duodenum, jejunum and stomach were observed in early deceased animals treated at 50 mg/kg bodyweight. Moreover, orange fluid in ileum was observed in animal no. 201 and dark colour of the liver, swollen spleen, dark red pancreas, multiple dark red pinpoint on thymus, dark colour of mesenteric lymph nodes, orange staining on muzzle and brown staining on urogenital region in animal no. 207. No abnormalities were observed at necropsy examination performed at the end of the observation period on animals treated at 50mg/kg bodyweight. These results demonstrate the expected acute oral LD50 to be lower than 300 mg/kg bodyweight, but higher than 50 mg/kg bodyweight.

European Directives concerning the classification, packaging and labelling of dangerous substances (Council Regulation (EC)No. 1272/2008 and subsequent revisions) would indicate the following:

Classification Category 3

Signal word Danger

Hazard statement Toxic if swallowed