D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C18-C18 (unsaturated) acyl] derivs.

Endpoint summary

Description of key information

The skin irritant potential of the test substance (active content 80.4%) was investigated using the EPISKIN-Standard Model (EPISKIN-SM^TM), a reconstituted three-dimensional human epidermis model as a replacement for the Draize Skin Irritation Test (OECD 404). As a result, the mean relative tissue viability (% negative control) was > 50% after 15 minutes treatment and 42 hour post incubation. Glucamide OL is therefore classified as non-irritant in accordance with UN GHS and EU CLP "No Category".

The eye irritancy potential of the undiluted test substance was investigated in a GLP compliant bovine corneal opacity and permeability assay according to OECD TG 437 (BCOP). Based on the results of this study, a mean in vitro score (IVIS) of 3.47 was obtained. As the IVIS score is above the regulatory treshold of 3 for non-classification but below the treshold of 55 for classification , no C&L decision could be derived. Based hereupon, a confirmatory eye irritation test according to OECD TG 405 was performed. A single ocular application of the test item Glucamide OL to rabbits at a dose of 0.1 g produced very slight irritant effects, which were below the treshold for classification and which had been fully reversible within 3 to 7 days. Neither mortalities nor significant clinical signs of toxicity were observed. Based on the results of this study and in accordance with GHS (Globally Harmonized Classification System) the test item Glucamide OL has no obligatory labelling requirement for eye 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:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

other: EU Method B.46 (Skin Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Amount / concentration applied:

Solids: 10 mg + 5 µL aqua dest.]

Duration of treatment / exposure:

15 minutes

Observation period:

42 +/- 1 h

Details on study design:

The test was performed on EpiSkin, an organotypic reconstructed three-dimensional model of the human epidermis. 3 replicate tissues are dosed with the test item, the negative control (10µL PBS) and the positive control (10µL 5% SDS), respectively. After 15 minutes treatment period at room temperature the test item and the controls are rinsed off with PBS and the tissues are post-incubated for 42 +/- 1 h. Then the tissues are stained via MTT for 3 hours. Isopropanol extracts are measured photometrically at 550 nm.

Irritation / corrosion parameter:

other: other: mean relative tissue viability

Value:

>= 50

Remarks on result:

other:

Remarks:

Basis: other: mean tissue viability of negative control tissue. Time point: 15 minutes treatment followed by 42 h post-incubation. Reversibility: other: not measured in test system. Remarks: non-irritant, no category (EU CLP and UN GHS). (migrated information)

Irritation / corrosion parameter:

other: other: mean relative tissue viability

Value:

<= 50

Remarks on result:

other:

Remarks:

Basis: other: mean tissue viability of negative control tissue. Time point: 15 minute treatment followed by 42 hour post-incubation. Reversibility: other: not measured in test system. Remarks: irritant, category 2 (EU CLP and UN GHS). (migrated information)

Other effects:

The test item showed no direct MTT reducing capability and no colouring potential.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item is classified as "non-irritant" (No Category).

Executive summary:

The skin irritant potential of the test substance (active content 80.4%) was investigated using the EPISKIN-Standard Model (EPISKIN-SM^TM), a reconstituted three-dimensional human epidermis model as a replacement for the Draize Skin Irritation Test (OECD 404). The test compound was applied topically to the EPISKIN-SM tissue [10 mg test item + 5 µL A. dest.] for 15 minutes followed by a 42 hour post incubation period and immediate determination of cytotoxic effects via MTT reduction assay. Irritant potential was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with PBS. The test item showed no irritant effects. The mean relative tissue viability (% negative control) was >50% after 15 minutes treatment and 42 hour post incubation. The controls confirmed the validity of the study. C18/18 unsatd.-Glucamide therefore classified as non-irritant in accordance with UN GHS and EU CLP "No Category".

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-10-31 to 213-12-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, number 437 “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants” (adopted: 26 July 2013)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und dLebensmittelsicherheit, München, Germany)

Vehicle:

physiological saline

Amount / concentration applied:

The test item was diluted with physiological saline 0.9% NaCl to gain a 20% concentration.

Duration of treatment / exposure:

750 microL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method).
After 4 hours ± 5 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed and the epithelium washed
at least three times with MEM (containing phenol red).

Details on study design:

Test System

Preparation of the Corneas:
The assay uses isolated corneas obtained as a by-product from an abattoir from freshly slaughtered animals (from Attenberger Fleisch GmbH & Co. KG).
On the test day, fresh eyes were collected from the slautherhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (MC2, Clermont, France) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 +/- 1 °C in a water bath.

Calibration of the Opacitometer:
The opacitometer had been switched on 15 min before the calibration procedure was started. Empty cornea holders were placed into the opacitometer and the readout was adjusted to zero using the “BAL”-turning knob. For calibration the polyester foil no. 1 was introduced into the test chamber and the readout was adjusted to 75 using the “CAL”-turning knob. To test the linearity of the measurement, two additional calibration foils, polyester foil no. 2 and polyester foil no. 3, were measured. For these, the opacitometer was supposed to display 150 and 225, respectively (± 3%). If this had not been the case, the calibration procedure would have had to be repeated. The calibration procedure was performed before each test and was documented in the raw data.

Treatment of the Corneas:
After the equilibration period, the medium was removed from both chambers and replaced with fresh Complete RPMI. An initial opacity measurement was performed on each of the corneas using an opacitometer (MC2, Clermont, France). Three corneas with opacity readings approximately equivalent to the median opacity of all corneas were selected as negative-control corneas. The opacity of each cornea was read against an air-filled chamber and recorded. Corneas that have an initial opacity reading above 7 units were not dosed. The medium was removed from the anterior chamber and replaced with the test item or control.
750 microL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method).
After 4 hours ± 5 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed and the epithelium washed
at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an opacity measurement was performed.
After the opacity measurement the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes
at 32 +/- 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined,
using a spectrophotometer.

Test Groups:
3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive control treated with imidazole 20% in physiological saline 0.9% NaCl
The BCOP assay is considered to be valid if the in vitro score obtained with the positive control falls within the two standard deviations of the current historical mean.

Evaluation of Results:
The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were
corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each
treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.
The mean OD490 for the blank wells were calculated. The mean blank OD490 was subtracted from the OD490 of each well (corrected OD490).
Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were
taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive
control were calculated by subtracting the average corrected OD490 of the negative control corneas from the corrected OD490 value of each
treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas
for that treatment condition.
The following formula was used to determine the in vitro score:
In vitro score = mean opacity value + (15 x mean OD490 value)

Irritation parameter:

other: in vitro irritation score (IVIS)

Basis:

mean

Time point:

other: 240 minutes

Score:

3.47

Reversibility:

other: not measured in test system

Irritant / corrosive response data:

The following mean in vitro irritation score was calculated: 3.47

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

Interpretation of results:

other: mild irritant

Remarks:

Criteria used for interpretation of results: other: OECD

Conclusions:

Based on the results of this in vitro study and under the given conditions the test item C18/C18 unsatd.-Glucamide is considered to be a mild irritant to the eye. However, since the IVIS score 3.47 is slightly above the regulatory non-classification threshold, no C&L decision is based on these study results.

Executive summary:

The eye irritancy potential of the undiluted test substance was investigated in a GLP compliant bovine corneal opacity and permeability assay according to OECD TG 437 (BCOP). Evaluation of effects on the opacity and the permeability of the bovine corneas were performed on three corneas per test group. After 1 hour equilibration the corneas were exposed to a 20% (w/v) suspension in physiological saline for 240 minutes. After treatment the test item was rinsed off the corneas and the opacity was measured. Based on the results of this study a mean in vitro score of 3.47 was obtained. The test substance therefore is considered to be a mild eye irritant. However, as the IVIS score is slightly above the regulatory treshold for non-classification, no C&L decision is based on this outcome. The in vitro score obtained with the positive control was within the current historical mean and therefore this assay is considered to be valid.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-02-04 to 2014-05-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline 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

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Test System

Species/strain: healthy New Zealand White Rabbits, Crl: KBL (NZW)
Source: Charles River Deutschland, 97633 Sulzfeld, Germany
Sex: male
Body weight at the beginning of the study: > 2kg
Age at the beginning of the study: approximately 12 - 34 weeks old
Number of animals: 3
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on
Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions

- Semi barrier in an air-conditioned room
- Temperature: 18 +/- 3 °C (recommendations of TVT, GV-SOLAS)
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access to autoclaved hay and to Altromin 2123 maintenance diet for rabbits (lot no. 1351), rich in crude fibre
- Free access to tap water (drinking water, municipal residue control, microbiological controls at regular intervals)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Housed in ABS-plastic or Noryl rabbit cages, floor 4200 cm2
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

A dose of 0.1 g of the test item was applied to the test site.

Duration of treatment / exposure:

The treated eye of animal no. 1 was rinsed with physiological saline 0.9% NaCl 5 hours after the application,
while the treated eyes of animals no. 2 and 3 were not rinsed after the application.

Observation period (in vivo):

The animals were observed for 72 hours after dosing. To determine the reversibility of the observed effects, the observation period was extended up to 7 days after dosing.

Number of animals or in vitro replicates:

The in vivo test was performed initially using one animal.
The results of the initial test did not indicate the test item to be corrosive or a severe irritant to the eye using the procedure described. In order to
confirm the response, two additional animals were treated in the same manner.

Details on study design:

Preparation of the Animals
Approximately 24 hours before the test and immediately prior to the application both eyes of each animal were examined. A health inspection was
performed to ensure the good state of health of the animals.
Approximately 18 hours before the application the eyes were also examined with the aid of a fluorescein solution (Fluoreszein SE Thilo, Alcon Pharma GmbH, lot no. H 207, expiry date: 09/2014). The eyes were rinsed with physiological saline 0.9% NaCl (Diprom, lot no. 13001-4, expiry date: 12/2015)after the examination.
None of the animals showed eye irritation, ocular defects, or pre-existing corneal injury.

Application

One hour before the application of the test item, 0.01 mg/kg of buprenorphine (Reckitt Benckiser, lot no.: 5320, expiry date: 01/2017) was
administered subcutaneously in order to achieve a therapeutic level of systemic analgesia. Approximately 5 minutes prior to the application of the test item, 2-3 drops of an ocular anaesthetic (proparacaine hydrochloride ophtalmic 0.5% solution, Ursapharm, lot no.: 277457, expiry date:
08/2014) were administered in both the treated and the control eye of each animal.
The test item was applied at a single dose in the conjunctival sac of one eye of each test animal after pulling the lower lid away from the eyeball. The lids were then gently held together for about 1 second in order to prevent loss of the material. The untreated contralateral eye served as control.
The treated eye of animal no. 1 was rinsed with physiological saline 0.9% NaCl (see 10.5) 5 hours after the application, while the treated eyes of
animals no. 2 and 3 were not rinsed after the application.
All animals showed pain and distress after the application of the test item and were treated subcutaneously with buprenorphine (0.01 mg/kg bw,
see above) and meloxicam (0.5 mg/kg bw, Boehringer Ingelheim, lot no. E20801A-06, expiry date: 01/2015) until up to 1 day post-application.

Clinical Observation

The eyes were examined for signs of irritation throughout the observation period. The eye irritation was scored and recorded.
For the calculation only the 24, 48 and 72-hour readings were used.
72 hours post-application as well as at the end of the prolonged observation period the treated eyes were examined with the aid of a fluorescein
solution. The eyes were rinsed with physiological saline 0.9% NaCl after the examination.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no effects

Irritation parameter:

iris score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

other: no effects

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

0.77

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

all animals

Time point:

24/48/72 h

Score:

0.11

Max. score:

1

Reversibility:

fully reversible

Irritant / corrosive response data:

After the application into the eyes of three male NZW rabbits the test item produced irritant but no corrosive ocular effects.
Neither mortalities nor significant clinical signs of toxicity were observed.
The eyes were not rinsed after the application, except for animal no. 1.
Upon fluorescein examinations at the end of the observation period of 72 hours no corneal lesions were found in any animal.
At the end of the prolonged observation period no corneal lesions were found in any animal.
Conjunctival redness, chemosis and discharge were observed in all animals and iris lesion was observed in animals no. 1 and no. 2.

Other effects:

There were no significant body weight changes during the observation period.

Interpretation of results:

slightly irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

Under the conditions of the present study, a single ocular application of the test item C18/C18 unsatd.-Glucamide to rabbits at a dose of 0.1 g
produced irritant effects, which were fully reversible within up to 7 days. Neither mortalities nor significant clinical signs of toxicity were observed.
In conformity with the EC criteria for classification and labelling requirements for dangerous substances and preparations according to Annex VI
of Commission Directive 2001/59/EC, the test item C18/C18 unsatd.-Glucamide has no obligatory labelling requirement for eye irritation.
According to Annex I of Regulation (EC) 1272/2008, the test item C18/C18 unsatd.-Glucamide has no obligatory labelling requirement for eye
irritation.
According to GHS (Globally Harmonized Classification System) the test item C18/C18 unsatd.-Glucamide has no obligatory labelling requirement
for eye irritation.

Executive summary:

Summary Results

On the basis of the test results given below and in conformity with the criteria given in Annex VI to Commission Directive 2001/59/EC, the substance should be assigned the following risk phrase:

R36 Irritating to eyes
R41 Risk of serious damage to eyes
no phrase		X

On the basis of the test results given below and in conformity with the criteria given inAnnex I of Regulation (EC) 1272/2008, the substance should be:

classified into category 1
classified into category 2
not classified		X

On the basis of the test results given below and in conformity with the criteria given in GHS (Globally Harmonized System of Classification and Labelling of Chemicals), the substance should be:

classified into category 1
classified into category 2A
classified into category 2B
not classified		X

Species/strain:                   New Zealand White Rabbits Crl: KBL (NZW)

Number of animals:           3

Amount of substance:       0.1 g per test site

First time of effects:          animal no. 1:1 hour post-application redness grade 2, chemosis grade 2, discharge grade 2 and iris lesion grade 1
animal no. 2:1 hour post-application redness grade 2, chemosis grade 1, discharge grade 2 and iris lesion grade 1
animal no. 3:1 hour post-application redness grade 1, chemosis grade 1 and discharge grade 2

Last time of effects:          animal no. 1:6 days post-application redness grade 1
animal no. 2:4 days post-application redness grade 1
animal no. 3:1 hour post-application redness grade 1, chemosis grade 1 and discharge grade 2

Reversibility of the observed effects:      animal no. 1:fully reversible within 7 days post-application
animal no. 2:fully reversible within 5 days post-application
animal no. 3:fully reversible within 24 hours post-application

Method:                            OECD 405; EC 440/2008, Method B.5; OPPTS 870.2400

The calculated mean scores did not exceed the limit values according to Directive 2001/59/EC, to Regulation (EC) 1272/2008 and to GHS.

Mean Values of Eye Irritation Scores – (24, 48, 72 Hour Reading)

Animal No.	Sex	Conjunctival Redness	Conjunctival Chemosis	Iris	Cornea Opacity
1	male	1.33	0.33	0.00	0.00
2	male	1.00	0.00	0.00	0.00
3	male	0.00	0.00	0.00	0.00

Conclusion

Under the conditions of the present study, a single ocular application of the test item C18/C18 unsatd.-Glucamide to rabbits at a dose of 0.1 g produced irritant effects, which were fully reversible within up to 7 days. Neither mortalities nor significant clinical signs of toxicity were observed.

In conformity with the EC criteria for classification and labelling requirements for dangerous substances and preparations according toAnnex VI of Commission Directive 2001/59/EC, the test item C18/C18 unsatd.-Glucamidehas no obligatory labelling requirement for eye irritation.

According to Annex I of Regulation (EC) 1272/2008, the test item C18/C18 unsatd.-Glucamide has no obligatory labelling requirement for eye irritation.

According to GHS (Globally Harmonized Classification System) the test item C18/C18 unsatd.-Glucamide has no obligatory labelling requirement for eye irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of a guideline and GLP compliant OECD TG 439 study (Episkin^TM), no classification of Glucamide OL with regard to skin irritation/corrosion is warranted according to the criteria of the EU Classification, Labelling and Packaging Regulation 1272/2008/EC (CLP).

Based on the results of a guideline and GLP compliant OECD TG 437 study (BCOP), no firm conclusion regarding the eye irritation potential of Glucamide OL could be derived. However, a confirmatory OECD TG 405 compliant test revealed that Glucamide OL is not irritating to eyes and no classification is warranted according to the criteria of the EU Classification, Labelling and Packaging Regulation 1272/2008/EC (CLP).