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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June-July 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Qualifier:
according to guideline
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxyethanesulphonic acid, compound with 4,4'-[hexane-1,6-diylbis(oxy)]bis[benzenecarboxamidine] (2:1)
EC Number:
211-533-5
EC Name:
2-hydroxyethanesulphonic acid, compound with 4,4'-[hexane-1,6-diylbis(oxy)]bis[benzenecarboxamidine] (2:1)
Cas Number:
659-40-5
Molecular formula:
C20H26N4O2.2C2H6O4S
IUPAC Name:
4-[6-(4-carbamimidoylphenoxy)hexoxy]benzenecarboximidamide;2-hydroxyethanesulfonic acid
Test material form:
solid: particulate/powder
Details on test material:
Appearance: White to slightly yellow powder
Specific details on test material used for the study:
Hexamidine diisethionate batch 42964
test item identified by the code number PH-17/0372

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals or test system and environmental conditions:
nine rats supplied by Elevage JANVIER LABS (France) used after an acclimatization period of at least 5 days. The animals were nulliparous adn non-pregnant. At the beginning of the study, the animals were 8-week old.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
DMSO
Details on oral exposure:
In the first step of the study, 2.0008g of teh test item were weighed and DMSO was added to a 10 mL volumetric flask. Just before administration, the preparation was stirred by vortex to obtain a colorless solution.
In the second and third steps of the study, 0.3023g and 0.3004g of the test item were weighed and DMSO was added to a 10 mL volumetric flask. Just before administration, the preparations were stirred by vortex for the second step and manually for the third step, to obtain a colorless solutions.
Each preparation wad administered under a volume of 10 mL/kg body weight using a quitable syringe graduated fitted with an oesophageal metal canula.
Doses:
As no information regarding the acute toxicity of the test item was available and in accordance with the animal welfare, the first tested dose was 2000mg/kg body weight. The animals of the treated groups received an effective dose of 2000mg/kg or 300 mg/kg body weight of test item.
No. of animals per sex per dose:
3 animals for tested dose 2000mg/kg body weight and 6 animals for tested dose 300 mg/kg body weight
Control animals:
yes
Details on study design:
PRINCIPLE
The acute toxic class method set out in this test is a stepwise procedure with the use of 3 animals of a single sex per step. Depending on the mortality and/or the moribund status of the animals, on average 2-4 steps may be necessary to allow judgement on the acute toxicity of the test item. This procedure is reproducible, uses very few animals and is able to rank items in a similar manner to the other acute toxicity testing methods.

The objective of this study is not intended to allow the calculation of a precise lethal dose (LD50), following the oral administration of an item, but does allow for the determination of defined exposures range where lethality is expected since death of a proportion of the animals is still the major endpoint of this test. The method allows for the determination of a LD50 value only when at least two doses result in mortality higher than 0 % and lower than 100 %.

The method uses pre-defined doses and the results allow a substance to be ranked and classified according to the Globally Harmonised System for the classification of chemicals which cause acute toxicity.

It is the principle of the test that, based on a stepwise procedure with the use of a minimum number of animals per step, sufficient information is obtained on the acute toxicity of the test item to enable its classification. The item is administered orally to a group of experimental animals at one of the defined doses. The item is tested using a stepwise procedure, each step using three animals of a single sex (normally females). Absence or presence of compound-related mortality of the animals dosed at one step will determine the next step ,i.e.;
- no further testing is needed,
- dosing of three additional animals, with the same dose
- dosing of three additional animals at the next higher or the next lower dose level.

5 - EXAMINATION OF THE ANIMALS

5.1 – Clinical examinations

Systematic examinations are carried out to identify any behavioural or toxic effects on the major physiological functions 30 minutes, 1 hour, 3 hours, 4 hours, 24 hours and 48 hours after administration of the item and continued on the following days if necessary.

This examination focused particularly on a list of symptoms, recorded as "present" or "absent" on the observation sheet.
These observations are compared to control data.

Observations and a mortality check are then carried out every day during 14 days. The time of death is noted individually in days and hours (if possible).


5.2 - Weight evolutions

The animals are weighed on day D0 (just before administering the item) then on D2, D7, and D14.
Weight changes should be calculated and recorded.


5.3 - Necropsies

Necropsies are carried out on animals which died during the test. Macroscopic observations are entered on individual autopsy sheets.
On D14, the animals are sacrificed with sodium pentobarbital (Dolethal®). Observations are entered on an autopsy sheet.

Only those organs likely to be modified in cases of acute toxicity are examined. Those presenting macroscopic anomalies can be removed and preserved in view to microscopic exanimations.

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 cut-off
Effect level:
500 mg/kg bw
Based on:
test mat.
Mortality:
Three mortalities were noted in animals treated at the of 2000 mg/kg body weight (3/3), two at 24 hours post dose and the last one at 48 hours post-dose.
One mortality was noted in animals treated at the dose of 300 mg/kg body weight (1/6), at 48 hours post-dose.
Clinical signs:
other: see summary
Other findings:
see summary

Any other information on results incl. tables

Three mortalities were noted in animals treated at the of 2000 mg/kg body weight (3/3), two at 24 hours post dose and the last one at 48 hours post-dose. The mortalities were preceded by a decrease in spontaneous activity (3/3), muscle tones (3/3), righting reflex (2/3), mydriasis (3/3), bradypnea (1/3), and eyes partly closed (1/3). Rigor mortis was noted before the necropsy (3/3). The macroscopic examination of the animals revealed a thinning of forestomach and a white corpus (2/3), and a thinning of red forestomach (1/3) and a thickening of white corpus with brown spots (1/3).

One mortality was noted in animals treated at the dose of 300 mg/kg body weight (1/6), at 48 hours post-dose. The mortality was preceded by a decrease in spontaneous activity, muscle tones, righting reflex, Preyer's reflex, associated with an increase of salivation, piloerection, mydriasis, bradypnea, and eyes partly closed. Rigor mortis was noted before the necropsy.

The macroscopic examination of the animal revealed a thinning of forestomach and red spots on the corpus. In the surviving animals (5/6), a decrease in spontaneous activity (3/5), muscle tones (3/5), righting's reflex (3/5) associated with an increase of salivation (4/5) were noted during the first hours of the test. The animals recovered a normal activity between days 1 and 2. The body weight evolution of the animals treated at the dose of 300 mg/kg body weight revealed an absence of body weight gain at D2 versus D0. Then, the body weight evolution was normal.

The macroscopic examination of the animals treated at the dose of 300 mg/kg body weight at the end of the study did not reveal treatment related changes.

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
The LD50 of the test item Hexamidine diisethionate is higher than 300 mg/kg body weight and lower than 2000 mg/kg body weight by oral route in the rat.
In accordance with the OECD test Guideline No423, the LD50 cut-off of the test item may be considered as 500 mg/kg body weight by oral route in the rat.
According to the criteria for classification, packaging and labelling of dangerous substances and preparations in accordance with the Regulation EC No. 1272/2008, the test item Hexamidine diisethionate has to be classified in category 4. The signal xord "Warningé and hazard statement H302 "Harmful if swallowed" are required.
Executive summary:

The test item Hexamidine diisethionate was administered to a groups of 3 female Sprague Dawley rats at the dose of 2000 mg/kg body weight and then to a group of 6 female Sprague Dawley rats at the dose of 300 mg/kg body weight. The experimental protocol was established according to the official method as defined in the OECD Test Guideline No. 423 dated December 17th, 2001 and the test method B1 tris of the Council regulation No. 440/2008.

Three mortalities were noted in animals treated at the of 2000 mg/kg body weight (3/3), two at 24 hours post dose and the last one at 48 hours post-dose. The mortalities were preceded by a decrease in spontaneous activity (3/3), muscle tones (3/3), righting reflex (2/3), mydriasis (3/3), bradypnea (1/3), and eyes partly closed (1/3). Rigor mortis was noted before the necropsy (3/3). The macroscopic examination of the animals revealed a thinning of forestomach and a white corpus (2/3), and a thinning of red forestomach (1/3) and a thickening of white corpus with brown spots (1/3).

One mortality was noted in animals treated at the dose of 300 mg/kg body weight (1/6), at 48 hours post-dose. The mortality was preceded by a decrease in spontaneous activity, muscle tones, righting reflex, Preyer's reflex, associated with an increase of salivation, piloerection, mydriasis, bradypnea, and eyes partly closed. Rigor mortis was noted before the necropsy.

The macroscopic examination of the animal revealed a thinning of forestomach and red spots on the corpus. In the surviving animals (5/6), a decrease in spontaneous activity (3/5), muscle tones (3/5), righting's reflex (3/5) associated with an increase of salivation (4/5) were noted during the first hours of the test. The animals recovered a normal activity between days 1 and 2. The body weight evolution of the animals treated at the dose of 300 mg/kg body weight revealed an absence of body weight gain at D2 versus D0. Then, the body weight evolution was normal.

The macroscopic examination of the animals treated at the dose of 300 mg/kg body weight at the end of the study did not reveal treatment related changes.