Methyl 3-aminopyrazinecarboxylate

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12-28 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF guidelines (2011) including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 3-aminopyrazinecarboxylate

EC Number:

240-387-5

EC Name:

Methyl 3-aminopyrazinecarboxylate

Cas Number:

16298-03-6

Molecular formula:

C6H7N3O2

IUPAC Name:

methyl 3-aminopyrazine-2-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Amiloride Compound 6
- Description: Tan powder
- Storage condition of test material: In refrigerator (2-8°C) protected from light, desiccated
- Other: Reactive to light, moisture and oxygen

Test animals

Species:

rat

Strain:

other: Wistar strain Crl:WI (Han) (outbred, SPF-Quality).

Sex:

female

Details on test animals or test system and environmental conditions:

Species: Rat, Wistar strain Crl:WI (Han) (outbred, SPF-Quality). Recognized by international guidelines as the recommended test system (e.g. OECD,
EC). Source: Charles River Deutschland, Sulzfeld, Germany.
Number of animals: 6 Females (nulliparous and non-pregnant). Each dose group consisted of 3 animals.
Age and body weight: Young adult animals (approx. 8 weeks old) were selected. Body weight variation did not exceed +/- 20% of the sex mean.
Identification: Earmark and tail mark
Health inspection: At least prior to dosing. It is ensured that the animals were healthy and without any abnormality that might affect the study integrity

Animal husbandry
Conditions
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation
Group housing of 3 animals per cage in labeled Makrolon cages (MIV type; height 18 cm.) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cageenrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
Acclimatization period was at least 5 days before start of treatment under laboratory conditions.

Diet

Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water

Free access to tap water.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed
according to facility standard procedures. There were no findings that could interfere with the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Treatment
Method: Oral gavage, using plastic feeding tubes.
Fasting: Animals were deprived of food overnight prior to dosing and until 3-4 hours after administration of the test substance. Water was available adlibitum.
Frequency: Single dosage on Day 1.
Dose level (volume): 2000 mg/kg (10 mL/kg) body weight.

Doses:

The toxicity of the test substance was assessed by stepwise treatment of groups of 3 females. The
first group was treated at a dose level of 2000 mg/kg. The absence or presence of mortality of animals
dosed at one step determined the next step, based on the test procedure defined in the guidelines.
The onset, duration and severity of the signs of toxicity were taken into account for determination of
the time interval between the dose groups.

No. of animals per sex per dose:

2 groups of 3 per sex per dose

Control animals:

no

Details on study design:

Observations

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 acco rding to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 4: grading slight (1) to very severe (4)
Maximum grade 3: grading slight (1) to severe (3)
Maximum grade 1: presence is scored (1).

Necropsy: At the end of the observation period, all animals were sacrificed by oxygen/carbon dioxide procedure and subjected to necropsy.
Descriptions of all internal macroscopic abnormalities were recorded.

Electronic data capture

Observations/measurements in the study were recorded electronically using the following programs:
REES Centron Environmental Monitoring system version SQL 2.0 (REES scientific, Trenton, NJ,
USA); TOXDATA version 8.0 (WIL Research Europe B.V., ‘s-Hertogenbosch, The Netherlands):
Clinical signs, Body weights.

Statistics:

The oral LD50 value of the test substance was ranked within the following ranges: 0-5, 5-50, 50-300 or
300-2000 mg/kg b.w. or as exceeding 2000 mg/kg b.w. The LD50 cut-off value was established based
on OECD guideline 423. 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

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occurred.

Clinical signs:

other: Lethargy, hunched posture, uncoordinated movements, piloerection and/or ptosis were noted in the animals on Day 1. Hunched posture was also noted in all animals on Day 2.

Gross pathology:

No abnormalities were found at macroscopic post mortem examination of the animals.

Any other information on results incl. tables

Table 1: Mortality

TESTDAY	1	1	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
HOURSAFTERTREATMENT	0	2	4
FEMALES 2000MG/KG	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
FEMALES 2000MG/KG	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-

Table 2: clinical Signs:

TESTDAY		1	1	1	2	3	4	5	6	7	8	9	101112131415
HOURS AFTER TREATMENT	MAXGRADE	0	2	4
FEMALES 2000 MG/KG
ANIMAL1 Behavior

Lethargy                                         (3)        -   -   1  -   -   -   -   -   -   -   -   -   -   -   -   -   -

Posture

Hunched posture                              (1)       1  1  1  1  -   -   -   -   -   -   -   -   -   -   -   -   -

Gait/motility

Uncoordinatedmovements                     (3)       -   -   1  -   -   -   -   -   -   -   -   -   -   -   -   -   -

Skin/fur

Piloerection                                 (1)        -   -   1  -   -   -   -   -   -   -   -   -   -   -   -   -   -

Various

Ptosis                                           (3)         -   -   1  -   -   -   -   -   -   -   -   -   -   -   -   -   -

ANIMAL2

Behavior

Lethargy                                       (3)        -   -   1  -   -   -   -   -   -   -   -   -   -   -   -   -   -

Posture

Hunched posture                               (1)        1  1  1  1  -   -   -   -   -   -   -   -   -   -   -   -   -

Gait/motility

Uncoordinatedmovements                    (3)         -   -   1  -   -   -   -   -   -   -   -   -   -   -   -   -   -

Skin/fur

Piloerection                                    (1)         -   -   1  -   -   -   -   -   -   -   -   -   -   -   -   -   -

Various

Ptosis                                           (3)         -   -   1  -   -   -   -   -   -   -   -   -   -   -   -   -   -

ANIMAL3

Behavior

Lethargy                                      (3)          -   -   1  -   -   -   -   -   -   -   -   -   -   -   -   -   -

Posture

Hunched posture                               (1)          -   1  1  1  -   -   -   -   -   -   -   -   -   -   -   -   -

Gait/motility

Uncoordinatedmovements                    (3)         -   -   1  -   -   -   -   -   -   -   -   -   -   -   -   -   -

Skin/fur

Piloerection                                    (1)          -   -   1  -   -   -   -   -   -   -   -   -   -   -   -   -   -

Various

Ptosis                                            (3)          -   -   1  -   -   -   -   -   -   -   -   -   -   -   -   -   -

 

FEMALES2000MG/KG

ANIMAL4

Posture

Hunched posture                              (1)          1  1  1  1  -   -   -   -   -   -   -   -   -   -   -   -   -

Skin/fur

Piloerection                                  (1)          -   1  -   -   -   -   -   -   -   -   -   -   -   -   -   -   -

ANIMAL5

Posture

Hunched posture                               (1)         1  1  1  1  -   -   -   -   -   -   -   -   -   -   -   -   -

Skin/fur

Piloerection                                   (1)          -   1  -   -   -   -   -   -   -   -   -   -   -   -   -   -   -

ANIMAL6

Posture

Hunched posture                               (1)            1  1  1  1  -   -   -   -   -   -   -   -   -   -   -   -   -

Skin/fur

Piloerection                                   (1)            -   1  -   -   -   -   -   -   -   -   -   -   -   -   -   -   -

Table 3 : Body Weight

SEX/DOSE LEVEL       ANIMAL  DAY1    DAY8   DAY 15

 

FEMALES 2000MG/KG	1	159	193	208
	2	155	185	196
	3	150	187	203
	MEAN	155	188	202
	ST.DEV.	5	4	6
	N	3	3	3

 

FEMALES 2000MG/KG	4	164	193	207
	5	155	179	194
	6	152	180	195
	MEAN	157	184	199
	ST.DEV.	6	8	7
	N	3	3	3

Table 4: Macroscopic Findings

ANIMAL   ORGAN                            FINDING                       DAY OF DEATH

 

FEMALES 2000MG/KG 1	No	findingsnoted	Scheduled necropsy
2	No	findingsnoted	Day15 after treatment Scheduled necropsy
3	No	findingsnoted	Day15 after treatment Scheduled necropsy
			Day15 after treatment
FEMALES2000MG/KG 4	No	findingsnoted	Scheduled necropsy
5	No	findingsnoted	Day15 after treatment Scheduled necropsy
6	No	findingsnoted	Day15 after treatment Scheduled necropsy
			Day15 after treatment

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The oral LD50 value of Amiloride Compound 6 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 exceed 5000
mg/kg body weight.
Based on these results, Amiloride Compound 6 does not have to be classified and has no obligatory
labelling requirement for acute oral toxicity according to the Globally Harmonized System of
Classification and Labelling of Chemicals (GHS) of the United Nations (2011) and Regulation (EC) No
1272/2008 on classification, labelling and packaging of substances and mixtures, including all
amendments.

Executive summary:

Assessment of acute oral toxicity with Amiloride Compound 6 in the rat (Acute Toxic Class Method).

The study was carried out based on the guidelines described in: OECD No.423 (2001) "Acute Oral Toxicity, Acute Toxic Class Method"

Commission Regulation (EC) No 440/2008, B1 tris: "Acute Oral Toxicity, Acute Toxic Class Method" EPA,

OPPTS 870.1100 (2002), "Acute Oral Toxicity"

JMAFF guidelines (2011) including the most recent partial revisions.

Amiloride Compound 6 was administered by oral gavage to two subsequent groups of three female Wistar rats at 2000 mg/kg body weight. Animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed after terminal sacrifice (Day 15).

No mortality occurred.

Lethargy, hunched posture, uncoordinated movements, piloerection and/or ptosis were noted in the animals on Day 1. Hunched posture was also noted in all animals on Day 2.

The body weight gain shown by the animals over the study period was considered to be normal.

No abnormalities were found at macroscopic post mortem examination of the animals.

The oral LD50 value of Amiloride Compound 6 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 exceed 5000 mg/kg body weight.

Based on these results, Amiloride Compound 6 does not have to be classified and has no obligatory labelling requirement for acute oral toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, including all amendments.