Ammonium 3-nitrobenzoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-07-08 - 2017-07-13 (experimental phase)

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 430 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER))

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 430 (2015): “In vitro skin corrosion: transcutaneous electrical resistance test (TER)”

Deviations:

yes

Remarks:

During quarantine and period before experimental phase the relative air humidity exceed 70% a few times. These changes had no impact on the course and results of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

EU Method B.40.: “In vitro skin corrosion: transcutaneous electrical resistance test (TER)”. Council Regulation (EC) No. 440/2008 of May 30, 2008

Deviations:

yes

Remarks:

During quarantine and period before experimental phase the relative air humidity exceed 70% a few times. These changes had no impact on the course and results of the study.

GLP compliance:

yes (incl. QA statement)

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

In vitro test system

Test system:

isolated skin discs

Source species:

rat

Cell type:

other: whole skin

Cell source:

other: dorso-lateral skin of rats

Source strain:

Wistar

Details on animal used as source of test system:

SOURCE ANIMAL
- Source: Conventional husbandry of laboratory animals of the Centre for Experimental Medicine at the Medical University in Katowice (number in the register of units entitled to the husbandry of laboratory animals: 0053)
- Sex: female
- Age at study initiation (in days): 21 days (importation) / 29 days (day of skin removal)
- Housing: Both rats were kept in a plastic cage covered with a wire bar lid. The dimensions of the cage were 58 x 37 x 21 cm. UV-sterilized, autoclaved, dust-free wood chips were used as bedding. 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 had ad libitum access to the "Labofeed H Standard" (lot numbers: 1/17) standard laboratory fodder produced by Zofia Połczyńska Wytwórnia Pasz “Morawski”, Kcynia.
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25°C
- Humidity (%): 49 – 82%
- Air changes (per hr): 10-20 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Justification for test system used:

As stipulated in the guideline / 1st study in a top down approach as no information on the skin irritating / corrosive properties of the test item was available.

Vehicle:

unchanged (no vehicle)

Remarks:

0.02 g of the test item (ground to a powder) was applied evenly to the disc, 150 µl deionized water was added on top of the solid

Details on test system:

SKIN DISC PREPARATION
- Procedure used: Skin discs used in the experiment were collected from two WISTAR female rats (outbred). At the day of the importation of the animals, the animals were 21 days old. They were quarantined and observed daily for 3 days. A general medical-veterinary examination was performed at the beginning of the quarantine period, whereas a detailed medical-veterinary examination was performed prior to the beginning of the experiment . All skin discs used in the experiment were obtained from two 29-day-old animals.
The animals used in the experiment did not exhibit any pathological symptoms. According to the animal health monitoring and veterinary care programme, all animals were examined every day from the admission to the quarantine until the end of the experimental part of the study. The infectious disease monitoring programme was implemented in the animal facility. It provides information on animal health and hygiene in the animal facility.
Preparation of the animals: After the quarantine, the dorsal and flank hair from young, 25-day-old, female rats was carefully removed with a shaver. Then, the shaven area was washed with an antibiotic solution containing streptomycin (10 mg/mL), penicillin (10 µg/mL), and amphotericin (0.25 mg/mL) to inhibit bacterial growth. The animals were washed with antibiotics again on the third day after the first wash and used in the study within one day of the second wash
Preparation of the skin discs: The animals were euthanized by intraperitoneal administration of morbital at a dose of 200 mg/kg b.w. After euthanasia, the dorso-lateral skin of each animal was removed and stripped of excess subcutaneous fat by carefully peeling it away from the skin using a paper towel. The skin discs were cut out using a scalpel. As many as 11 skin discs with a diameter of 20-mm each were obtained from a single rat skin.
- Quality control for skin discs: Two of them were used to control the quality of the procedure, whereas the remaining nine were used for the purpose of the experiment. The age of the animals was particularly important. The age of 29 days ensures that the hair follicles are in the dormant phase before adult hair growth begins.
Before the test, the electrical resistance of two skin discs was measured as a quality control procedure for each animal skin. Both discs should give resistance values greater than 10 kOhm for the remainder of the discs to be used in the test.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The test item and the control items were applied for 24 hours at 20-22.5°C.
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: After application for 24h they were removed by washing with a jet of tap water at up to 30°C
- Observable damage in the tissue due to washing: After the transcutaneous electrical resistance test (TER), the skin discs were subjected to a detailed gross examination in order to reveal possible damage. No damage was stated.
DYE BINDING METHOD
The dye binding procedure was not necessary in this case since all TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: Two independent animals, each 3 skin disks for each test item / control
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
The test item is considered to be non-corrosive to skin if:
- the mean TER value obtained for the test item is greater than 5 kΩ, or
- the mean TER value is less than or equal to 5 kΩ, and the skin disc shows no obvious damage.
The test item is considered to be corrosive to skin if:
- the mean TER value obtained for the test item is less than or equal to 5 kΩ and
- the skin disc is obviously damaged.

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): A sufficient amount (0.02 g) of the test item (ground to a powder) was applied evenly to the disc to ensure that the whole surface of the epidermis is covered.
- Concentration (if solution): After adding 150 μL deionized water on top of the solid, the tube will be gently agitated.

NEGATIVE CONTROL
distilled water

POSITIVE CONTROL
10M hydrochloric acid

Duration of treatment / exposure:

The test item and the control items were applied for 24 hours at 20-22.5°C.

Duration of post-treatment incubation (if applicable):

none

Number of replicates:

Three skin discs obtained from each animal were used for the test item and three for each control item.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

transcutaneous electrical resistance (in kΩ)

Run / experiment:

1st animal, mean of three disks

Value:

10.72

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

transcutaneous electrical resistance (in kΩ)

Run / experiment:

2nd animal, mean of three disks

Value:

12.91

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no, besides gross changes induced by the positive control

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

not corrosive

Conclusions:

The study was conducted under GLP according to OECD guideline 430 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation and only one minor deviation to the study plan concerning humidity (see "Overall remarks, attachments"), the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the corrosive potential of the test substance to the skin in vitro.
On the grounds of the study, it may be stated that the test item, i.e. Ammonium-3-nitrobenzoate belongs to a group of substances which do not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.
In a tiered in vitro testing strategy, this result (non-corrosive) may however only give an indication of the skin-damaging potential, but does not allow a definitive classification according to Regulation 1272/2008. So, an additional skin irritation test should be conducted.

Executive summary:

The in vitro skin corrosion: transcutaneous electrical resistance test (TER) was performed according to OECD TG 430 (GLP) in order to obtain information on health hazards resulting from skin contact with the test item, i.e. Ammonium-3-nitrobenzoate.

Study course: Skin discs used in the experiment were obtained from two 29-day-old animals.

In order to control the procedure quality, the electrical resistance of two skin discs obtained from each test animal was measured before the start of the experiment. In each case, the skin disc resistance values were greater than 10 kΩ; therefore, the remainder of the animals’ skin discs could have been used in the experiment.

The test item (ground to a powder) was uniformly applied to the epidermal surface of the skin disc placed inside a tube and wetted. Concurrent positive (10M hydrochloric acid) and negative (distilled water) controls were used. Three skin discs obtained from each animal were used for the test item and three for each control item.

The test item and the control items were evenly applied to the discs for 24 hours and kept at 20-22.5°C. Then, they were removed by washing with a jet of tap water. Prior to measuring the electrical resistance, the surface tension of the skin was reduced by adding a sufficient volume of 70% ethanol to cover the epidermis. After a few seconds, the ethanol was removed from the tube, whereas the tissue was hydrated by the addition of 3 mL of a solution of MgSO4 (154 mM). A LCR 6401 low-voltage, alternating current databridge was used to measure the electrical resistance of the skin in kΩ by placing the databridge electrodes on either side of the skin disc.

After the transcutaneous electrical resistance test (TER), the MgSO4 solution was removed from the tube, whereas the skin discs were subjected to a gross examination in order to reveal possible damage.

The dye binding procedure was not necessary in this case since all TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

Study results: The experiment was performed in duplicate, because the difference of TER means were below 5 ± 0,5 kΩ.

The mean TER results for the skin discs treated with the test item were equal to 10.72 kΩ (animal no. 1) and 12.91 kΩ (animal no. 2). They can be accepted because the concurrent positive and negative control values fell within the acceptable ranges for the method.

Gross examinations of the skin discs treated with the test item did not reveal any pathological changes.

Interpretation of the study results: On the grounds of the study, it may be stated that the test item, i.e. Ammonium-3-nitrobenzoate belongs to a group of substances which do not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.