Ammonium formate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-06-27 to 2019-12-18 (provisional draft final report)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium formate

EC Number:

208-753-9

EC Name:

Ammonium formate

Cas Number:

540-69-2

Molecular formula:

CH2O2.H3N

IUPAC Name:

ammonium formate

Test material form:

solid: crystalline

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was performed by electric current). The heads were transported to the testing facility at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was (19.3 ºC to 20.4 ºC) during the transport which is considered optimal. All eyes used in the assay were from the same groups of eyes collected on one specific day.
After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box).
- Time interval prior to initiating testing: minimum 3h
- indication of any existing defects or lesions in ocular tissue samples: No, after removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, i.e. not obvious damage on the head or eye, the eyeball was carefully removed from the orbit.
- Indication of any antibiotics used: No

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 g

Duration of treatment / exposure:

10 sec

Duration of post- treatment incubation (in vitro):

240 min

Number of animals or in vitro replicates:

3 eyes for treatment, positive and negative control

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 (w/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then, and prior to excision from the head, the fluorescein-treated cornea was examined with a hand-held slit lamp or a slit lamp microscope to ensure that it was not damaged (i.e., fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit.

The eyeballs were carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short, i.e. could not be held with surgical forceps. The procedure avoided pressure on the eyes in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eyes were placed onto paper moisted with saline and the nictitating membrane and connective tissue were cut away. The prepared eyes were kept on moisted paper in a closed box to maintain humidity and to prevent the eyes from becoming dry.

EQUILIBRATION AND BASELINE RECORDINGS
The prepared eyes were placed in steel clamps with the cornea positioned vertically with the eyes in the correct relative position (same position as in the chicken head), again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied to fix the eyes properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 4 drops/minutes. The door of the chamber was kept closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate numbers of eyes were selected and after being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to assess their condition. For this purpose, the focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface.
Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected and replaced.
The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equaling 0.095 mm. Any eye with cornea thickness deviating more than 10 % from the mean value of all eyes, or eyes that showed any other signs of damage, were rejected and replaced.
For the eyes selected for the test because they satisfied the suitability criteria, acclimatization started and was maintained for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization and treatment periods.

NUMBER OF REPLICATES
3
NEGATIVE CONTROL USED
physiological saline (NaCl 0.9%)

POSITIVE CONTROL USED
Imidazole (0.03g)

APPLICATION DOSE AND EXPOSURE TIME
0.03 g of the test item for 10 sec

OBSERVATION PERIOD
240 min

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The time of application was monitored and, after an exposure period of 10 seconds from the end of the application, the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.
- Indicate any deviation from test procedure in the Guideline : No differences reported

METHODS FOR MEASURED ENDPOINTS:
The control and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable. The cornea thickness and cornea opacity were measured at all time points. Fluorescein retention was determined at baseline (t=0) and 30 minutes after the post-treatment rinse.
- Corneal opacity:
Corneal opacity was measured according to the following formulae:
ΔCO at time t = CO at time t – CO at t=0
Mean CO(at time t) = FEΔCO (at time t)+ SEΔCO(at time t) + TEΔCO(at time t)/3
with
CO = Cornea opacity
ΔCO = Difference between cornea opacity and cornea opacity reference value
FEΔCO = Difference between first eye cornea opacity and first eye cornea opacity reference value
SEΔCO = Difference between second eye cornea opacity and second eye cornea opacity reference value
TEΔCO = Difference between third eye cornea opacity and third eye cornea opacity reference value
Mean CO = The mean corneal opacity value
at time t = Observation time at 30, 75, 120, 180 or 240 minutes after the post-treatment rinse
at t=0 = Reference value

- Damage to epithelium based on fluorescein retention:
Fluorescein retention was calculated according to the following formulae:
ΔFR at time t = FR at time t – FR at t=0

Mean FR = FEΔFR (at time t) + SEΔFR(at time t) + TEΔFR at time t)/3
with:
FR = Fluorescein retention
ΔFR = Difference between fluorescein retention and fluorescein retention reference value
FEΔFR = Difference between first eye fluorescein retention and first eye fluorescein retention reference value
SEΔFR = Difference between second eye fluorescein retention and second eye fluorescein retention reference value
TEΔFR = Difference between third eye fluorescein retention and third eye fluorescein retention reference value
Mean FR = The mean fluorescein retention value
at time t = Observation time at 30 minutes after the post-treatment rinse
at t=0 = Reference value
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting:
Cornea swelling was calculated according to the following formulae
CS at time t = CT at time t – CT at t=0 X100/CT at t=0
Mean CS at time t = FECS(at time t)+ SECS (at time t) + TECS (at time t)/3
with
CS = Cornea swelling
CT = Cornea thickness
FECS = First eye cornea swelling
SECS = Second eye cornea swelling
TECS = Third eye cornea swelling
Mean CS = The mean percentage of corneal swelling
at time t = Observation time at 30, 75, 120, 180 or 240 minutes after the post-treatment rinse
at t=0 = Reference value
- Others (e.g, histopathology): After consultation with the Sponsor no histopathology evaluation was performed. Corneas are discarded 2 months after the final report.

DECISION CRITERIA: Decision criteria from OECD Guideline 438 was used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Remarks:

Mean maximum corneal swelling at up to 75 min

Run / experiment:

Mean of three treated eyes

Value:

17

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

3% (Mean maximum corneal swelling at up to 75 min)

Positive controls validity:

valid

Remarks:

30% (Mean maximum corneal swelling at up to 75 min)

Results 2

Irritation parameter:

percent corneal swelling

Remarks:

Mean maximum corneal swelling at up to 240 min

Run / experiment:

Mean of three treated eyes

Value:

19

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

3% (Mean maximum corneal swelling at up to 240 min)

Positive controls validity:

valid

Remarks:

37% (Mean maximum corneal swelling at up to 240 min)

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

Mean of three treated eyes

Value:

2.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

0.5 (Mean maximum corneal opacity)

Positive controls validity:

valid

Remarks:

4.0 (Mean maximum corneal opacity)

Results 4

Irritation parameter:

fluorescein retention score

Remarks:

Mean fluorescein retention

Run / experiment:

Mean of three treated eyes

Value:

1.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

0.0 (Mean fluorescein retention)

Positive controls validity:

valid

Remarks:

2.8 (Mean fluorescein retention)

Results 5

Irritation parameter:

morphological effects

Run / experiment:

In all three replicates

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Corneal opacity score 4 was observed in three eyes at 30 minutes after the post-treatment rinse.

Remarks on result:

other: no effects observed in the eyes treated with the test item

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Corneal opacity score 4 was observed in three eyes at 30 minutes after the post-treatment rinse in the3 positive control.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method the test facility demonstrated the technical proficiency in a separate study using the ten Proficiency Chemicals according to OECD Test Guideline No. 438.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
According to OECD guideline 438 A test is considered acceptable if the concurrent negative or vehicle/solvent control and the concurrent positive control are identified as GHS Non-Classified and GHS Category 1, respectively.
- Range of historical values if different from the ones specified in the test guideline: please refer to any other information on results incl. tables for the historical data range

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In this ICET, Ammonium Formate did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE classes were twice III (based on the corneal swelling of 19% within 240 minutes and fluorescein retention of 1.7) and once IV (based on the corneal opacity score of 2.7).
Positive and negative controls showed the expected results. The experiment was considered to be valid.
According to the guideline OECD 438, Ammonium Formate's overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made”.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of Ammonium Formate by means of the ICE assay using fresh chicken corneas according to OECD guideline 438, adopted 25 June 2018.

The corneas were incubated with the test substance and controls for 10 sec. After rinsing with saline, the corneas were incubated for another 2 h. The test was performed in triplicates. Corneal opacity and swelling as well as fluorescein retention were determined.The overall ICE classes were once II (based on the fluorescein retention of 1.3) and twice III (based on the corneal swelling of 13% within 75 minutes and corneal opacity score of 1.7). 

The positive control was classified as corrosive/severely irritating, UN GHS Classification: Category 1 and the negative control had no significant effects on the chicken eye in this study.

Since the overall ICE classes of the test substance were neither specified for Category 1 nor for Non-Classification, Ammonium Formate could not be classified asUN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Categoryunder the experimental conditions described in this report.On the basis of the results obtained in this experiment, the test item is categorized as “No prediction can be made”.