Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 March - 30 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline valiity criteria were met.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Version / remarks:
European Community (EC). Council Regulation (EC) No 440/2008 (31 May 2008)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
17 December 2001
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Version / remarks:
December 2002
Deviations:
no
Qualifier:
according to
Guideline:
other: Appendix to Director General Notification, No. 12-Nousan-8147, Japanese Ministry of Agriculture, Forestry and Fisheries of Japan
Version / remarks:
November 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
Clear, light yellow liquid
Storage: 2 to 8 °C, in the dark
Expiry date: 01-Jan-18
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8 C, in the dark
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: The test article was dispersed in corn oil for the 50 mg/kg dose level. Corn oil was chosen as it was capable of forming a homogeneous solution as described in Covance GLP Study 8359421.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was used as supplied for dose levels of 300 and 2000 mg/kg. For the 50 mg/kg/day group, the test item was formulated in corn oil.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: Not reported
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Applied as supplied, or in a corn oil formulation (50 mg/kg/day group only)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) N/A

OTHER SPECIFICS: N/A

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 7-10 weeks
- Weight at study initiation: The weight variation did not exceed ±20% of the mean weight.
- Fasting period before study: Night before dosing
- Housing: The animals were housed in groups of up to five during the acclimatisation period in suspended, solid floor cages with wire lids. From the day prior to dosing (Day-1), the rats were housed in groups of three in similar cages. Bedding was provided on a weekly basis to each cage by use of clean European soft wood bedding (Datesand Ltd., Manchester, UK). Each batch of bedding was analysed for specific constituents and contaminants. No contaminants were present in bedding at levels which might have interfered with achieving the objective of the study.
- Diet (e.g. ad libitum): Throughout the study the animals had access to 5LF2 EU Rodent Diet 14%, which was freely available to the animals at all times, except for a period of fasting from the evening of the day prior to dosing (Day-1) until approximately 3 hours after dosing. Each batch of diet had been analysed for specific constituents and contaminants by the manufacturer. No contaminants were present in diet at levels which might have interfered with achieving the objective of the study.
- Water (e.g. ad libitum): Mains water was provided ad libitum via cage mounted water bottles. The water was periodically analysed for specific contaminants. No contaminants were present in water at levels which might have interfered with achieving the objective of the study.
- Acclimation period: 6-15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: 14 March 2017 To: 25 May 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
50 mg/kg/day group only
Details on oral exposure:
VEHICLE
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): 5 mL/kg bw
- Justification for choice of vehicle: Was found to form a homogenous formulation in Covance GLP Study 8359421. Corn oil is also an guideline recommended vehicle.
- Lot/batch no. (if required): Not reported
- Purity: Not reported

MAXIMUM DOSE VOLUME APPLIED: 5 mL/kg

DOSAGE PREPARATION (if unusual): N/A

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: N/A
Doses:
2000, 300 and 50 mg/kg
No. of animals per sex per dose:
6 females (2 x groups of 3)
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Treated rats were observed closely for clinical signs of reaction to treatment. Clinical signs were recorded immediately post-dose, at approximately 15 and 30 minutes post- dose, hourly between 1 and 4 hours post-dose (inclusive), twice daily on Days 2, 3 and 4 and once daily from the fifth to last day of the observation period.
Individual body weights were recorded on Day-1 (day before dosing) and on Days 1, 4, 8 and 15. Decedent carcass weights were also recorded prior to necropsy
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, free-hand histopathology (sectioning) of liver and kidneys, examination of representative sections of mucosal surfaces of the stomach, small and large intestines
Statistics:
No

Results and discussion

Effect levelsopen allclose all
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 300 - <= 2 000 mg/kg bw
Based on:
test mat.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
500 mg/kg bw
Based on:
test mat.
Remarks on result:
other: result is based on the cut-off value as determined in Annex 2 of the OECD TG 423
Mortality:
50 mg/kg = 0/6 mortality
300 mg/kg = 0/6 mortality
2000 mg/kg = 3/6 mortality
Clinical signs:
Decreased activity and hunched posture were noted 3 and 4 hours after dosing, in one animal treated at 2000 mg/kg. Hypothermia, ptosis, piloerection, laboured breathing and prone posture were noted in the animal that was killed in extremis. No clinical signs were seen prior to death in the two animals that were found dead. No clinical signs were seen in animals treated at 50 or 300 mg/kg.
Body weight:
All surviving rats achieved body weight gains during the first and second weeks of the study.
Gross pathology:
No abnormalities were noted at necropsy of animals that died or were killed in extremis during the study or at necropsy of those that were killed at the end of the study.

Any other information on results incl. tables

Table 2      Number of animals dead (and with evident toxicity)

 

Dose

(mg/kg bw)

Mortality

(# dead / total)

Time range of deaths

(hours)

Number with evident toxicity

(# / total)

Male

Female

Combined

Male

Female

Combined

2000

-

3 / 6

3/ 6

24 -48

-

2 / 6

2 / 6

 300

-

0 / 6

0 / 6

n/a

-

0 / 6

0 / 6

 50

-

0 / 6

0 / 6

n/a

-

0 / 6

0 / 6

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
Based of the experimental conditions of this study, the test article was classified as Category 4 in respect of its acute oral toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).
Executive summary:

OECD 423 (2017) - This study was conducted to assess the acute toxicity of the test article (Reaction Mass of N,N,N',N-tetrabutylmethylenediamine and dibutylamine), following a single oral administration to small groups of rats. The study design provides information for hazard assessment and classification and enables a chemical to be assigned to toxicity classes but severely restricts animal usage.

 

A group of three female fasted rats was given the test article as a single dose by oral gavage at a dose level of 2000 mg/kg. Based on the results from this dose level, further groups of three female fasted rats were treated at dose levels of 50 mg/kg,


300 mg/kg and 2000 mg/kg. Treatment of animals was sequential. Sufficient time was allowed between each group to confirm the survival of the previously dosed animals.

 

The test article was used undiluted for the 300 and 2000 mg/kg dose levels and was dispersed in corn oil for the 50 mg/kg dose level. Surviving animals were killed on Day 15. All animals underwent a full necropsy.

 

Two animals treated at 2000 mg/kg were found dead on Day 2. One other animal was humanely killed due to the severity of the clinical signs on this day.

 

There were no deaths at dose levels of 50 or 300 mg/kg.

 

Decreased activity and hunched posture were noted 3 and 4 hours after dosing, in one animal treated at 2000 mg/kg. Hypothermia, ptosis, piloerection, laboured breathing and prone posture were noted in the animal that was killed in extremis. No clinical signs were seen prior to death in the two animals that were found dead.

No clinical signs were seen in animals treated at 50 or 300 mg/kg.

 

All surviving rats achieved body weight gains during the first and second weeks of the study.

No abnormalities were noted at necropsy of animals that died or were killed in extremis during the study or at necropsy of those that were killed at the end of the study.

Based of the experimental conditions of this study, the test article was classified as Category 4 in respect of its acute oral toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS). Based on the observed effects the LD50 using the cut-off value was estimated to be 500 mg/kg bw.