Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-10-23 - 2018-11-29
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Molecular Formula:
HOCH2C(Br)CH2OH
Molecular Weight:
245.91
Characteristics (Physical Appearance):
White crystalline powder
CAS No.:
3234-02-4
Batch Number:
1088
Purity:
100%

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
Species and justification for the selection of the species:
Rat (Rattus norvegicus); The regulatory guidelines for this test has preferred rat among the species of rodents.
Strain:
Wistar; the strain was selected due to its availability in requisite numbers.
Sex:
Female only. It has been observed that females are generally slightly more sensitive than males to toxic effects. The selected females were nulliparous and non-pregnant.
Source:
INTOX PVT. LTD.
Age at start of treatment:
8 to 9 weeks
Weight range at start of the treatment:
129 g to 150 g
Environmental conditions:
The experimental animal room was supplied with fresh and filtered air, with 10 to 15 air changes per hour. The room was air conditioned with temperature between 19 to 25 °C, relative humidity 30 to 70% and illumination cycle set to 12 hours light and 12 hours dark.
Accommodation:
Animals were housed in room number AR-10, in the experimental animal facility of INTOX PVT. LTD., maintained under appropriate barriers.
Animals were housed in sterilised solid bottom polypropylene cages [size: 42 cm (L) x 29 cm (W) x 19 cm (H)] with stainless steel grill tops, facilities for food and water bottle, and with bedding of clean and sterilised paddy husk. Cages were suspended on movable stainless steel racks.
Animals were group housed, with three animals of same dose group being housed in one cage.
Diet:
‘Altromin' brand pelleted rat feed manufactured by M/s Altromin Spezialfutter GmbH & Co. KG, Germany, and supplied by ATNT Laboratories, Mumbai was provided ad libitum. Certificates of nutrient analysis for each batch of diet used have been retained in the facility records. The diet has been tested and certified to be free from undesired levels of contaminants.
Water:
Potable water passed through 'Aquaguard' water filter was provided ad libitum in sterilized bottles with stainless steel sipper tubes.
The drinking water has been tested and certified for potability and the water source has been verified to be free from undesired levels of contaminants.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: analytical grade water with 0.2% Tween 80
Details on oral exposure:
The test item was administered by oral gavage to each rat as a single dose using a suitably graduated syringe and a stainless steel intubation needle (16G). The dose administered to individual rat was adjusted according to its body weight that was recorded just before dosing to give a constant dosage volume of 10 ml/kg body weight.
Doses:
Three: Step 1: 300 mg/kg; Step 2 and 3: 50 mg/kg body weight
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
The toxicity of the test item was assessed by stepwise treatment of animals. Three female rats were used per step. Absence or presence of compound-related mortality of the animals dosed at one step determined the next step, i.e. either no further testing is needed or dosing of three additional animals with the same dose or dosing of three additional animals at the next higher / lower dose level.
OBSERVATIONS
Following observations were made during the course of this study.
MORTALITY AND CLINICAL SIGNS
On the day of dosing, all animals were observed for signs of toxicity and death, periodically during the first 24 hours with special attention given during the first 4 hours (i.e. at 10, 30 minutes, 1 hour, 2 and 4 hours following dosing) and thereafter they were observed once a day for 14 days after treatment.
Cage side observations included changes in the skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern. The appearance, progress and disappearance of the signs were recorded.
BODY WEIGHTS
The body weights of rats were individually recorded at one day prior to dosing (day 0), on the day of dosing (day 1, fasting body weight), on day 7 and at termination on day 14. Weight gain and group mean values were computed (over day -1 body weight).
NECROPSY AND HISTOPATHOLOGY
At end of the study, all surviving animals were weighed and humanely sacrificed by carbon dioxide asphyxiation. All animals in the study were subjected to a complete necropsy and the gross pathological changes were recorded. Histopathological examination was not carried out in the absence of treatment related gross pathological changes.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 50 - <= 300 mg/kg bw
Based on:
test mat.
Mortality:
STEP 1: STARTING DOSE - GROUP G1: 300 mg/kg BODY WEIGHT
When tested on three female rats at the dose level of 300 mg/kg body weight, trans-2,3-Dibromo-
2-Butene-1,4-Diol induced abnormal clinical signs of hypoactivity in two females on day 1 after treatment. Two female rats died during the first day of the observation, whereas one female rat died
during the second day of the observation.
STEP 2: GROUP G2: 50 mg/kg BODY WEIGHT
In step 2, when tested on three female rats at the dose level of 50 mg/kg body weight, trans-2,3-
Dibromo-2-Butene-1,4-Diol did not induce any abnormal clinical signs and mortality in treated rats.
STEP 3: GROUP G3: 50 mg/kg BODY WEIGHT
In step 3, when tested on three female rats at the dose level of 50 mg/kg body weight, trans-2,3-
Dibromo-2-Butene-1,4-Diol did not induce any abnormal clinical signs and mortality in treated rats.
Body weight:
STEP 1: STARTING DOSE - GROUP G1: 300 mg/kg BODY WEIGHT
--
STEP 2: GROUP G2: 50 mg/kg BODY WEIGHT
The body weight gain by treated rats was not adversely affected during the 14 days observation
period after treatment.
STEP 3: GROUP G3: 50 mg/kg BODY WEIGHT
The body weight gain by treated rats was not adversely affected during the 14 days observation
period after treatment.
Gross pathology:
STEP 1: STARTING DOSE - GROUP G1: 300 mg/kg BODY WEIGHT:
Gross pathological alterations of the female rat (Rk2071) was observed during necropsy when
found dead on day 1 after treatment. The necropsy findings were 1) Liver was enlarged and mottled.
2) Stomach (Forestomach) showed focal reddish discolouration.
HISTOPATHOLOGY
Upon histopathological observation of the liver and stomach from one dead animal RK2071 showed
that liver had centrilobular necrosis with inflammatory cell infiltration, midzonal hepatocellular
hypertrophy, angiectasia, and congestion in central vein. These were of moderate severity. The
non-glandular (forestomach) showed mild congestion whereas glandular stomach too exhibited mild
congestion and hemorrhage.
STEP 2: GROUP G2: 50 mg/kg BODY WEIGHT:
No gross pathological alterations were encountered in any of the female rats when sacrificed at termination of the study.
STEP 3: GROUP G3: 50 mg/kg BODY WEIGHT:
No gross pathological alterations were encountered in any of the female rats when sacrificed at termination of the study.

Applicant's summary and conclusion

Interpretation of results:
Category 3 based on GHS criteria
Conclusions:
Acute oral toxicity study of trans-2,3-Dibromo-2-Butene-1,4-Diol in Wistar rats was performed as per OECD Guidelines for Testing of Chemicals, Section 4, No. 423 - Acute Oral Toxicity - Acute Toxic Class Method, adopted by the council on 17 December 2001.
Based on these results, and according to the "Globally Harmonised System (GHS) for classification of chemicals which cause acute toxicity; Seventh Revised Edition (ST/SG/AC.10/30/Rev.7); United Nations, New York and Geneva, 2017; eISBN 978-92-1-060457-4, United Nations, 2017 and the EC Requirements for Classification and Labelling of Dangerous Substances (Commission Directive 2001/59/EC of 6th August 2001 adapting to technical progress for the 28th time Council Directive
67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the Classification, Packaging and Labelling of Dangerous Substances. Official Journal of the European Communities OJL 225 pp. 1-333), the test item, trans-2,3-Dibromo-2-Butene-1,4-Diol, is classified in GHS Category 3 for the obligatory labelling requirement for oral toxicity. This category corresponds to an LD50 value lying between 50 < ATE ≤ 300 mg/kg body weight, while the corresponding LD50 cut-off value for this product was observed to be 200 mg/kg body weight.
Executive summary:

Acute oral toxicity study of trans-2,3-Dibromo-2-Butene-1,4-Diol in Wistar rats was performed as per Organization for Economic Co-operation and Development (OECD) Guidelines for Testing of

Chemicals, Section 4, No. 423 - Acute Oral Toxicity - Acute Toxic Class Method, adopted by the council on 17 December, 2001. The method uses pre-defined doses and the results allow a

substance to be ranked and classified according to the Globally Harmonised System (GHS) for classification of chemicals which cause acute toxicity. In this study, single oral administration of trans-2,3-Dibromo-2-Butene-1,4-Diol was made to groups of three female Wistar rats in step-wise manner to assess its acute toxicity. The test item was formulated in analytical grade water with 0.2% Tween 80 to obtain concentrations of 30 mg/ml and 5 mg/ml. In step-1 of the study, when three female rats were administered the test item, at the dose of 300 mg/kg, test item induced abnormal clinical signs of hypoactivity in two females on day 1 after treatment. Two female rats died during the first day of the observation, whereas one female rat died during the second day of the observation. No gross pathological changes were observed except one female rat. Therefore histological examination was carried out of the same. Analysis of the sample of the dose formulated for Step-1 treatment revealed that the average

measured concentration of trans-2,3-Dibromo-2-Butene-1,4-Diol was 30.62 mg/ml against the nominal concentration of 30 mg/ml. The small observed difference of 2.07 confirmed that the rats

received an adequate dose of the test item. In step-2 and step-3, when further tested at the dose of 50 mg/kg body weight, trans-2,3-Dibromo-2-Butene-1,4-Diol did not induce any abnormal clinical signs and mortality in treated rats. The body weight gain of female rats was not affected during the observation period. No gross pathological changes were observed in any of the rats, as evident at terminal necropsy.

Based on these results, and according to the "Globally Harmonised System (GHS) for classification of chemicals which cause acute toxicity; Seventh Revised Edition (ST/SG/AC.10/30/Rev.7); United

Nations, New York and Geneva, 2017; eISBN 978-92-1-060457-4, United Nations, 2017 and the EC Requirements for Classification and Labelling of Dangerous Substances (Commission Directive

2001/59/EC of 6th August 2001 adapting to technical progress for the 28th time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to

the Classification, Packaging and Labelling of Dangerous Substances. Official Journal of the European Communities OJL 225 pp. 1-333), the test item, trans-2,3-Dibromo-2-Butene-1,4-Diol,

is classified in GHS Category 3 for the obligatory labelling requirement for oral toxicity. This category corresponds to an LD50 value lying between 50 < ATE ≤ 300 mg/kg body weight, while the corresponding LD50 cut-off value for this product was observed to be 200 mg/kg body weight.