N,N-bis(carboxymethyl)-L-glutamic acid

Endpoint summary

Description of key information

GLDA-H4 was not irritating in an in vitro skin irritation test which was supported by the fact that GLDA-Na4 was not irritating to the rabbit skin. 
GLDA-H4 was irritating in two in vitro tests (BCOP and ICE) and in an in vivo eye irritation test; GLDA-Na4 was not irritating to the eyes. The commercial product was also tested in vitro (ICE test) and showed only slight eye irritation.
No data are available to conclude about respiratory tract irritation. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17-Jun-2014 to 23-Jun-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

other: EU method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10.3 to 10.4 mg moistened with 5 µl water

NEGATIVE CONTOL:
- Amount(s) applied (volume or weight with unit): 25 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 25 µl
- Concentration (if solution): 5% (aq) Sodium dodecyl sulphate

Duration of treatment / exposure:

Exposure: 15 minutes
Post incubation period: 42 hours

Details on study design:

TEST SITE
- Area of exposure: human epidermis model
- % coverage: 0.38 cm2

REMOVAL OF TEST SUBSTANCE
- Washing (if done): phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.

Irritation / corrosion parameter:

other: other: percentage viability

Value:

113

Remarks on result:

other:

Remarks:

Basis: other: percentage of control. Time point: 15 minutes. (migrated information)

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

It is concluded that this test is valid and that GLDA-H4 is non-irritant in the in vitro skin irritation test.

Executive summary:

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with GLDA-H4 compared to the negative control tissues was 113%. Since the mean relative tissue viability for GLDA-H4 was above 50% after 15 minutes treatment GLDA-H4 is considered to be non-irritant.

The positive control had a mean cell viability of 35% after 15 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 8%, indicating that the test system functioned properly.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 August - 08 September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nohsan, Notification No 8147, April 2011, including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

- Source: Charles River France, L’Arbresle Cedex, France.
- Age at study initiation: Animalused within the study was 14 weeks old.
- Weight at study initiation: Body weights was 2991 grams.
- Housing: Individually housed in cages with perforated floors.
- Diet: Free access to pelleted diet for rabbits (Global Diet 2030 Harlan Teklad, Italy). Hay and wooden sticks were available during the study period.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

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.

IN-LIFE DATES: From: 18 August - 08 September 2014

Vehicle:

unchanged (no vehicle)

Controls:

other: The other eye of the animal remained untreated and served as the reference control.

Amount / concentration applied:

TEST MATERIAL- Amount applied: 41.2 mg

Duration of treatment / exposure:

Single instillation on Day 1.

Observation period (in vivo):

The eyes of the animal were examined approximately 1, 24, 48 and 72 hours and 7, 14 and 21 days after instillation of the test substance.

Number of animals or in vitro replicates:

1 male

Details on study design:

STUDY DESIGN
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). Based on the results of the first animal the conclusion of the study was clear, therefore the two further rabbits assigned to the study were not treated.

PREEMPTIVE PAIN MANAGEMENT
One hour prior to instillation of the test substance, buprenorphine (Buprenodale®, Dechra Ltd., Stokeon-Trent, United Kingdom) 0.01 mg/kg was administered by subcutaneous injection in order to provide a therapeutic level of systemic analgesia. Five minutes prior to instillation of the test substance, two drops of the topical anesthetic alcaine 0.5% (SA Alcon-Couvreur NV, Puurs, Belgium) were applied to both eyes.

TREATMENT
The animal was treated by instillation of 41.2 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. One hour after treatment, the treated eye was not rinsed with water since no residual test substance was visible. Additional injections of buprenorphine 0.01 mg/kg were administered immediately after the 1-hour observation and at the end of the first day to reduce pain and distress. Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) 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 to assess recovery. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area. Immediately after fluorescein examination on Day 2, in order to provide a continued level of systemic analgesia, buprenorphine 0.01 mg/kg and meloxicam (Metacam®, Boehringer Vetmed GmbH, Ingelheim/Rhein, Germany) 0.5 mg/kg were administered by subcutaneous injection. An additional injection of buprenorphine 0.03 mg/kg was supplied at the end of the day to reduce pain and distress. Additional injections of buprenorphine 0.03 mg/kg and meloxicam 0.5 mg/kg were administered again immediately after the 48-hour observation. After the final observation, the animal was sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

REMOVAL OF TEST SUBSTANCE
-Washing: No

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 the animal were examined approximately 1, 24, 48 and 72 hours and 7, 14 and 21 days after instillation of the test substance. The irritation scores and a description of all other (local) effects wererecorded.

SCORING SYSTEM:
The irritation was assessed according to the numerical scoring system according to OECD 405.

Irritation parameter:

cornea opacity score

Remarks:

(opacity)

Basis:

mean

Time point:

other: 24, 48 and 72 hrs.

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 21 Days

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24, 48 and 72 hrs.

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 7 Days

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

mean

Time point:

other: 24, 48 and 72 hrs.

Score:

2.7

Max. score:

3

Reversibility:

not fully reversible within: 21 Days

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24, 48 and 72 hrs.

Score:

3.3

Max. score:

4

Reversibility:

not fully reversible within: 21 Days

Remarks on result:

other: No chemosis was noted at 14 Days after instillation

Irritant / corrosive response data:

Irritation: Instillation of 41.2 mg of GLDA-H4 (a volume of approximately 0.1 mL) into an eye of one rabbit resulted in severe effects on the cornea, iris and conjunctivae. The corneal injury consisted of opacity and epithelial damage. As a result of the corneal injury, pannus (neovascularization of the cornea) was apparent at 7 and 14 days after instillation. The corneal injury resolved within 21 days. Iridial irritation was observed and resolved within 7 days. The irritation of the conjunctivae consisted of redness, chemosis and watery discharge. The effects were not fully recovered at termination (21 days after exposure). Reduced elasticity of the eyelids was noted from 48 hours after installation onwards and had resolved within 21 days after installation.
Corrosion: There was no evidence of ocular corrosion.

Other effects:

Colouration/Remnants: No staining of (peri) ocular tissues by the test substance was observed and no test substance remnants were seen.
Toxicity/Mortality: No symptoms of systemic toxicity were observed in the animal during the test period and no mortality occurred.

Interpretation of results:

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

Remarks:

Migrated information: No interpretation of the data was done.

Conclusions:

Instillation of GLDA-H4 into a rabbit eye resulted in severe effects on the cornea, iris and conjunctivae. Effects on cornea and iris disappeared within the 21-day period; redness and chemosis were not fully recovered and watery discharge was still seen at termination (21 days after exposure). However, at 21 day the score was only slight (1 in a range of 0-3 or 0-4) and it was expected that these changes would also have disappeared in a few more days.

Executive summary:

The study was carried out based on the guidelines described in:

OECD No.405 (2012) "Acute Eye Irritation / Corrosion"

EC, No 440/2008, B5: "Acute Toxicity: Eye Irritation/Corrosion"

EPA, OPPTS 870.2400 (1998): "Acute Eye Irritation"

JMAFF Guidelines (2000), including the most recent revisions.

A single sample of 41.2 mg of GLDA-H4 (a volume of approximately 0.1 mL) was instilled into an eye of one rabbit. Observations were made 1, 24, 48 and 72 hours and 7, 14 and 21 days after instillation.

Instillation of the test substance resulted in severe effects on the cornea, iris and conjunctivae. The corneal injury consisted of opacity and epithelial damage. As a result of the corneal injury, pannus (neovascularization of the cornea) was apparent at 7 and 14 days after instillation. The corneal injury resolved within 21 days. Iridial irritation was observed and resolved within 7 days. The irritation of the conjunctivae consisted of redness, chemosis and watery discharge. Reduced elasticity of the eyelids was noted 7 and 14 days after installation. Redness and chemosis were not fully recovered and watery discharge was still seen at termination (21 days after exposure). However, at 21 day the score was only slight (1 in a range of 0-3 or 0-4) and it was expected that these changes would also have disappeared in a few more days.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a standard tests for in vitro skin irritation, GLDA-H4 was not irritating which was supported by the fact that GLDA-Na4 was not irritating to the rabbit skin in vivo.

In a standard test for in in vitro eye irritation (BCOP) GLDA-H4 was irritating but no conclusion could be drawn and an in vivo study was performed in one rabbit. Effects on cornea and iris disappeared within the 21 -day period; redness and chemosis were not fully recovered and watery discharge was still seen at termination (21 days after exposure). However, at 21 day the score was only slight (1 in a range of 0-3 or 0-4) and it was expected that these changes would also have disappeared in a few more days. Finally, an in vitro ICE test was carried out with GLDA-H4 and with Dissolvine StimWell HTF (the commercial product). The ICE test was chosen as the results of the in vitro BCOP test with GLDA-H4 required an in vivo study, whereas a BCOP study followed by an in vivo study with Stimwell HTF would not be possible as a commercial product should not be tested in vivo. Secondly, the ICE test uses the complete eye (not only the cornea) and has additional parameters included such as histopathology. The results of the ICE test with GLDA-H4 again showed eye irritation requiring classification Cat. 2 whereas the ICE test with Stimwell HTF showed slight eye irritation but it would not need classification for this endpoint. The commercial product has a higher pH than GLDA-H4 (is less acidic).

In an acute inhalation toxicity test in which rats were exposed to the technically highest attainable concentration of GLDA-Na4, clinical signs of toxicity included slightly decreased breathing rate during exposure and sniffing in a few animals shortly after exposure up to 2 days after exposure. These signs only point at very slight respiratory irritation at a very high concentration.

Justification for selection of skin irritation / corrosion endpoint:
Well performed and reported study in vitro study supported by the results of an in vivo study with the read across substance GLDA-Na4.

Justification for selection of eye irritation endpoint:
Well performed and reported in vivo study (one animal) based on the results of an in vitro study (BCOP); supported by the results of two other in vitro studies (ICE), one with GLDA-H4 and one with the commercial product.

Effects on eye irritation: irritating

Justification for classification or non-classification

No classification is needed for skin irritation. GLDA-H4 induced eye irritation in vivo most probably due to its low pH as GLDA-Na4 was not irritating to the eyes. Although slight effects on the eyes were still seen in the tested animal 21 days after treatment, it was expected that these would have fully disappeared in a few more days. Therefore, because the score of corneal opacity was below 3 and the score for iritis was below 1.5, and because the effects observed are considered to be transient and were expected to have reversed in a few more days, and because GHS Cat. 2A is applicable when effects fully reverse within an observation period of normally 21 days (and no fixed period of 21 days), GLDA-H4 should be classified as an eye irritant Cat. 2A, which would be Cat. 2 in Europe. Another in vitro study with GLDA-H4 (ICE test) confirmed classification as eye irritant Cat. 2. The commercial product Dissolvine StimWell HTF did only induce slight eye irritation in the ICE test and would therefore not need classification.