Ammonium 3-nitrobenzoate

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-07-20 - 2017-08-16

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 420 (2001): „Acute oral toxicity – fixed dose method”

Deviations:

yes

Remarks:

During the experiment, relative air humidity exceed 70% a few times. These changes were temporary and did not influence the study course and results.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)

Version / remarks:

EU Method B.1.BIS.: „Acute oral toxicity – fixed dose procedure”

Deviations:

yes

Remarks:

During the experiment, relative air humidity exceed 70% a few times. These changes were temporary and did not influence the study course and results.

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium 3-nitrobenzoate

EC Number:

242-965-2

EC Name:

Ammonium 3-nitrobenzoate

Cas Number:

19328-56-4

Molecular formula:

C7H5NO4.H3N

IUPAC Name:

ammonium 3-nitrobenzoate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Cmdb: WI; outbred

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: the husbandry of laboratory animals of the Experimental Medicine Centre at the Medical University in Białystok, kept behind the breeding barrier (number in the register of units entitled to the husbandry of laboratory animals: 0043). The Health Certificate issued by the Experimental Medicine Centre at the Medical University in Białystok confirmed good health of the animals.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation / Weight at study initiation: One 9-week-old animal weighing 211.0 g (dose of 300 mg/kg b.w.), and one 9-week-old animal weighing 213.0 g (dose of 2000 mg/kg b.w.) was used in the preliminary experiment. Four 10-week-old animals whose average body weight was 197.3 g (dose of 300 mg/kg b.w.) were used in the main experiment.
- Fasting period before study: yes, about 16 hours before the administration of the test item
- Housing:The animals were kept in plastic cages covered with wire bar lids. The dimensions of the cages were 58 x 37 x 21 cm (length x width x height). In the preliminary experiment, the animals were caged individually. In the main experiment, there were four animals in one cage. Autoclaved, and additionally UV-irradiated wood chips were used as bedding [SOP/T/16, SOP/T/48]. The environment of the animals was enriched by placing wooden blocks, tunnels and nesting materials for laboratory animals in the cages.
- Diet (e.g. ad libitum): The animals were given ad libitum access to "Labofeed H Standard" standard laboratory fodder (lot numbers: 1/17, and 2/17) produced by Zofia Potczyhska Wytwôrnia Pasz "Morawski", Kcynia
- Water (e.g. ad libitum): drinking tap water, ad libitum
- Acclimation period: The animals were quarantined and observed daily for 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 25 °C
- Humidity (%): 47 - 82%
- Air changes (per hr): about 10-20 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 60 mg test item/ml
- Amount of vehicle (if gavage): 0.5 mL/100 g bw

MAXIMUM DOSE VOLUME APPLIED: 0.5 mL/100 g b.w.

Doses:

300, 2000 mg/kg bw

No. of animals per sex per dose:

5 (300 mg(kg bw) resp. 1 (2000 mg/kg)
(300 mg/kg bw was administered to four animals. The animal from the preliminary experiment which had received the dose of 300 mg/kg b.w. was included in the main experiment.)

Control animals:

no

Remarks:

not required

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The evaluation of general condition of the animals, i.e. the observation of all animals for morbidity and mortality was conducted twice a day or once a day (on days off) during the 14-day experiment. The detailed clinical observations were performed on the day of the test item administration (day 0), i.e. 10, 30, and 60 minutes after the administration and then at hourly intervals up to the 5th hour after the administration. From the 1st to the 14th day of the experiment, the detailed clinical observations were performed once a day. Body weights of the animals were determined on days 0 (directly before the administration of the test item), 7, and 14 (before euthanasia).
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Results and discussion

Preliminary study:

Clinical signs
Following single administration of the test item at a dose of 300 mg/kg b.w. to one animal used in the preliminary experiment, no signs of toxicity were noticed. The animal survived the experiment. Following single administration of the test item at a dose of 2000 mg/kg b.w. to the next animal used in the preliminary experiment, the following changes were observed on the administration day: slight decrease of locomotor activity, dejection, and bristled coat. Animal died within 24 hours after administration the test item.

Body weights of the animals
During the 14-day experiment, body weight gain was observed in the animal.
Gross examinations
The animal did not exhibit any pathological changes.

Effect levels

Key result

Sex:

female

Dose descriptor:

discriminating dose

Effect level:

300 mg/kg bw

Based on:

test mat.

Mortality:

The animals survived the experiment.

Clinical signs:

Following single administration of the test item at a dose of 300 mg/kg b.w. to four animals used in the main experiment, no signs of toxicity were noticed. The animals survived the experiment.

Body weight:

During the 14-day experiment, body weight gain was observed in the animals.

Gross pathology:

The animals did not exhibit any pathological changes.

Other findings:

none stated

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The study was conducted under GLP according to OECD guideline 420 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations. Hence, the results can be considered as reliable to assess the acute toxicity of the test substance in rats.
On the grounds of the obtained results, the test item, i.e. Ammonium-3-nitrobenzoate can be classified to the following categories:
- category 4 - according to the Globally Harmonized System (GHS),
- category 4 - according to the Commission Regulation (EU) No. 286/2011 of March 10, 2011 amending, for the purposes of its adaptation to technical and scientific progress, Regulation (EC) No. 1272/2008 of the European Parliament and of the Council on classification, labelling, and packaging of substances and mixtures.

Executive summary:

The acute oral toxicity study (OECD 420, GLP) based on the fixed dose method was performed in order to obtain information on health hazards likely to arise from a single oral exposure to the test item, i.e. Ammonium-3-nitrobenzoate.

The experiment commenced with a preliminary experiment. Since no data were available, the preliminary experiment started with the administration of the test item at a dose of 300 mg/kg b.w. to one animal, Wistar female rats (Cmdb: WI; outbred).

Because the animal survived after the administration of the test item at a dose of 300 mg/kg b.w., the second animal was treated with the test item at a dose of 2000 mg/kg b.w.

On the grounds of the preliminary experiment results, four animals used in the main experiment were given the test item at a dose of 300 mg/kg b.w. (the animal from the preliminary experiment which had been given the dose of 300 mg/kg b.w. was included in the main experiment).

Before the administration, the animals had been fasted for 16 hours. The test item in the form of an aqueous solution in a volume of 0.5 mL/100 g b.w. was administered with a metal stomach tube.

After the administration of the test item, the animals were observed for 14 days. General and detailed clinical observations were conducted daily during the entire experiment.

Body weights of the animals were determined on days 0 (directly before the administration of the test item), 7, and 14.

After the 14-day observation period, the animals were euthanized and subjected to a detailed gross examination.

Following single administration of the test item at a dose of 300 mg/kg b.w. to one animal used in the preliminary experiment, no signs of toxicity were noticed. The animal survived the experiment. Following single administration of the test item at a dose of 2000 mg/kg b.w. to the next animal used in the preliminary experiment, changes in the locomotor activity, reaction to sound stimuli, and bristled coat were observed. The animal died within 24 hours after administration the test item.

Following single administration of the test item at a dose of 300 mg/kg b.w. to four animals used in the main experiment, no signs of toxicity were noticed. The animals survived the experiment.

During the 14-day experiment, body weight of the animals increased.

The gross examinations light red lungs with dark red areas, hyperaemia of the blood vessels of the stomach and intestines, especially the caecum, and autolytic changes in the small intestine in the female (no. 1) which died within 24 hours after administration the test item. No pathological changes were observed in the animals, which survived the experiment.

Because female no. 1 (dose of 2000 mg/kg b.w.) died within 24 hours after the administration of the test item, and there were no macroscopic changes in the remaining animals, a microscopic examination was not performed.

Dose of the test item(mg/kg b.w.)	2000	300
	Preliminary experiment	Preliminary experiment	Main experiment
Mortality	1/1	0/1	0/4
Clinical signs	slight decrease of locomotor activity, dejection, bristled coat	no changes	no changes

On the grounds of the obtained results, the test item, i.e. Ammonium-3-nitrobenzoate can be classified to the following categories:

- category 4 - according to the Globally Harmonized System (GHS),

- category 4 - according to the Commission Regulation (EU) No. 286/2011 of March 10, 2011 amending, for the purposes of its adaptation to technical and scientific progress, Regulation (EC) No. 1272/2008 of the European Parliament and of the Council on classification, labelling, and packaging of substances and mixtures.