2-(tert-butylamino)acetic acid hydrochloride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 November - 12 December 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF guidelines (2011)

Version / remarks:

including the most recent partial revisions

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-(tert-butylamino)acetic acid hydrochloride

EC Number:

614-959-3

Cas Number:

6939-23-7

Molecular formula:

C6H14ClNO2

IUPAC Name:

2-(tert-butylamino)acetic acid hydrochloride

Test material form:

solid: particulate/powder

Details on test material:

White powder
Test substance storage: In refrigerator (2-8°C) in the dark

Specific details on test material used for the study:

White powder
Test substance storage: In refrigerator (2-8°C) in the dark
Batch #: 11681004

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

3 male New Zealand White Albino rabbits from Charles River France, L’Arbresle Cedex, France. Recognized by international guidelines as the recommended test system. Animals used within the study were between 10-20 weeks old and body weights were between 2-4 kg. Identification earmark.
Conditions:
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0ºC (actual range: 18.5 – 20.4ºC), a relative humidity of 40-70% (actual range: 42 - 71%) and 12 hours artificial fluorescent light and 12 hours darkness per day. Cleaning procedures in the room might have caused the temporary fluctuations above the optimal
maximum level of 70% for relative humidity. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.
Accommodation:
Animals were individually housed in labeled cages with perforated floors and shelters. Acclimatization period was at least 5 days before start of treatment under laboratory conditions.
Diet:
Pelleted diet for rabbits approximately 100 grams per day. Hay and wooden sticks were available during the study period. Free access to tap water.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

Animals were treated by instillation of, on average, 28.9 mg (range 28.6 – 29.3 mg) of the test substance (a volume of approximately 0.1 mL) in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball.

Duration of treatment / exposure:

24 hrs.

Observation period (in vivo):

Observations were made 1, 24, 48 and 72 hours and 7 and 14 days after instillation.

Number of animals or in vitro replicates:

3 males

Details on study design:

The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 3 weeks later, after considering the degree of eye irritation observed in the first animal.
Treatment:
Animals were treated by instillation of, on average, 28.9 mg (range 28.6 – 29.3 mg) of the test substance (a volume of approximately 0.1 mL) in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control.
Immediately after the 24-hour observation, a solution of 2% fluorescein in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. This procedure was repeated in one animal to assess recovery. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.
After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20%.
Observations:
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Body Weight: Day of treatment (prior to instillation) and after the final observation.
Necropsy: No necropsy was performed according to protocol.
Irritation: The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7 and 14 days after instillation of the test substance. The
irritation scores and a description of all other (local) effects were recorded.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal: 155

Time point:

24/48/72 h

Score:

1

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal: 167

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible within: 1 hour

Remarks on result:

other: slight dulling of the normal luster of the cornea on Day 1

Results 3

Irritation parameter:

cornea opacity score

Basis:

animal: 170

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible within: 1 hour

Remarks on result:

other: slight dulling of the normal luster of the cornea on Day 1

Results 4

Irritation parameter:

iris score

Basis:

animal: 155

Time point:

24/48/72 h

Score:

1

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Results 5

Irritation parameter:

iris score

Basis:

animal: 167

Time point:

24/48/72 h

Score:

0

Remarks on result:

no indication of irritation

Results 6

Irritation parameter:

iris score

Basis:

animal: 170

Time point:

24/48/72 h

Score:

0

Remarks on result:

no indication of irritation

Results 7

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal: 155

Time point:

24/48/72 h

Score:

2.7

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Results 8

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal: 167

Time point:

24/48/72 h

Score:

2

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Results 9

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal: 170

Time point:

24/48/72 h

Score:

2

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Results 10

Irritation parameter:

conjunctivae score

Remarks:

Chemosis

Basis:

animal: 155

Time point:

24/48/72 h

Score:

1.7

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Results 11

Irritation parameter:

conjunctivae score

Remarks:

Chemosis

Basis:

animal: 167

Time point:

24/48/72 h

Score:

1.3

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Results 12

Irritation parameter:

conjunctivae score

Remarks:

Chemosis

Basis:

animal: 170

Time point:

24/48/72 h

Score:

1.3

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

There was no evidence of ocular corrosion.

Applicant's summary and conclusion

Interpretation of results:

Category 2A (irritating to eyes) based on GHS criteria

Conclusions:

Instillation of the test substance resulted in effects on the cornea, iris and/or conjunctivae for all animals. For one animal, the corneal injury consisted of opacity (maximum grade 1) and epithelial damage (maximum 35% of the corneal area). The corneal injury resolved within 7 days. The two other animals
showed slight dulling of the normal luster of the cornea on Day 1. The animal that showed corneal injury also showed iridial irritation (grade 1), which resolved within 7 days. No iridial irritation was observed for the other two animals. All animals showed irritation of the conjunctivae consisting of redness, chemosis and discharge. The irritation of the conjunctivae completely resolved within 14 days.

Executive summary:

Based on these results:

- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011), N-butylglycine HCl should be classified as: irritating to eyes (Category 2A).

- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, N-butylglycine HCl should be classified as Irritating to eyes (Category 2) and labeled as H319: Causes serious eye irritation.