Reaction product of D-Glucopyranoside, methyl; esterified with oleic acid, methyl ester

Endpoint summary

Description of key information

Skin irritation: OECD Guideline 404; two male New Zealand White rabbits; the test item produced a primary irritation index of 0.0 and was considered to be non-irritant to rabbit skin according to the Draize scheme. No corrosive effects were noted.

Eye irritation: OECD 405; Two male rabbits New Zealand White rabbits; The test item produced a maximum group mean score of 8.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system. The substance is not an eye irritant in accordance with the CLP Regulation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-04-21 - 2015-04-24

Reliability:

1 (reliable without restriction)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The department of health of the Government of the United Kingdom

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hsdlf:NZW

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Age at study initiation: 12 to 20 weeks old
- Weight at study initiation: 2.75 or 3.0 kg
- Housing: individually in suspended cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 hrs continuous light (06:00 to 18:00) and 12 hrs darkness.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL / site

Duration of treatment / exposure:

4 hrs

Observation period:

approximately 1, 24, 48 and 72 hrs after removal of the patches

Number of animals:

Two males.
The number of animals used was the minimum required to achieve the objectives of the study. Testing was conducted in two animals and the response in those animals was such that exposure of a third animal would not affect classification of the test item, no further testing was needed.

Details on study design:

TEST SITE
On the day before the test two rabbits were clipped free of fur from the dorsal/flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.
- Area of exposure: 2.5 cm x 2.5 cm on backs
- Type of wrap if used: 2.5 cm x 2.5 cm cotton gauze patch

REMOVAL OF TEST SUBSTANCE
- Washing: corset and patches were removed from each animal and any residual test item removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: 4 hrs

OBSERVATION TIME POINTS
approximately 1, 24, 48 and 72 hrs after removal of the patches

SCORING SYSTEM:
- Examination for evidence of primary irritation and scored according to the following scale:

EVALUATION OF SKIN REACTIONS
Erythema and Eschar Formation Value
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to eschar 4
formation preventing grading of erythema

Edema Formation
No edema 0
Very slight edema (barely perceptible) 1
Slight edema (edges of area well-defined by 2
definite raising)
Moderate edema (raised approximately 1 millimeter) 3
Severe edema (raised more than 1 millimeter and 4
extending beyond the area of exposure)

Any other skin reactions and clinical signs of toxicity, if present, were also recorded.
Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Method of calculation:
Calculation of Primary Irritation Index and Grading of Irritancy Potential Using the Draize Scheme
The scores for erythema and edema at the 24 and 72-hour readings were totaled for the two test rabbits (8 values) and this total was divided by 4 to give the primary irritation index of the test item. The test item was graded according to the following scheme devised by Draize, J.H. (1959):

Primary Irritation Index Grading of Irritancy
0 Non-irritant
> 0 to 2 Mild irritant
>2 to 5 Moderate irritant
> 5 to 8 Severe irritant

If irreversible alteration of the dermal tissue is noted in any rabbit, as judged by the Study Director, which include ulceration and clear necrosis or signs of scar tissue, the test item is considered to be corrosive to rabbit skin. Grading according to Draize may, therefore, not be applicable.

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Time point:

other: 24/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Irritant / corrosive response data:

Very slight erythema and very slight edema were noted at both treated skin sites 1 hour after patch removal.
The treated skin sites appeared normal at the 24-Hour observation.

Other effects:

Both animals showed expected gain in body weight during the study.

Table 1: Individual Skin Reactions

Skin Reaction	Observation Time (following patch removal)	Individual Scores		Total
		Rabbit number and Sex
		75038 Male	75039 Male
Erythema/Eschar Formation	Immediately	0	0	0
	1 Hour	1	1	(2)
	24 Hours	0	0	0
	48 Hours	0	0	(0)
	72 Hours	0	0	0
Edema Formation	Immediately	0	0	0
	1 Hour	1	1	(2)
	24 Hours	0	0	0
	48 Hours	0	0	(0)
	72 Hours	0	0	0
Sum of 24 and 72-Hour Readings (S): 0	0
Pimary Irritation Index (S/4): 0/4 =	0.0
Grading:	NON-IRRITANT

( )= Total values not required for calculation of ptimary irritation index

Table 2: Individual Body Weights and Body Weight Change

Rabbit Number and Sex	Individual Body Weight (kg)		Body Weight Change (kg)
	Day 0	Day 3
75038 Male	3.00	3.02	0.02
75039 Male	2.75	2.80	0.05

Interpretation of results:

GHS criteria not met

Conclusions:

The test item produced a primary irritation index of 0.0 and was considered to be non-irritant to rabbit skin according to the Draize scheme. No corrosive effects were noted.

Executive summary:

The primary dermal irritation potential of the test item was evaluated in this study with New Zealand White rabbits (2M) in accordance with OECD Guideline 404 and GLP requirements. There was one group of two male albino rabbits that received a single, four-hour, semioccluded exposure. Each 0.5 mL dose of the test article was applied to the clipped, unabraded skin. At completion of exposure, the bandages were removed and the sites washed. Application sites were evaluated in accordance with the method of Draize at approximately 1 and 24, 48 and 72 hours after patch removal. There were no remarkable body weight changes during the study period. The test article induced very slight erythema and edema on all animals at 1 hrs after removal of the patch. The skin appeared normal again at 24 hrs. No corrosive effects were noted. The Primary Irritation Index was calculated to be 0.0. The test article received a descriptive rating classification of non-irritating.

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:

2015-04-27 - 205-05-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to 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

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Specific details on test material used for the study:

The pH of the test item was determined prior to commencement of the study and found to be as follows:

Preparation:
Undiluted as Supplied: immediately: 5, after 10 minutes: not applicable
90% v/v aqueous preparation of the test item: immediately: 6, after 10 minutes: 6

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Two New Zealand White (Hsdlf:NZW) strain rabbits (male) were supplied by Harlan Laboratories UK Ltd., Leicestershire, UK. At the start of the study the animals weighed 2.09 or 2.85 kg and were 12 to 20 weeks old. After an acclimatization period of at least 5 days each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study. The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light (06:00 to 18:00) and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

For the purpose of the study the test item was used as supplied. 0.1 mL of the test item was placed into the conjunctiva! sac of the right eye.

Duration of treatment / exposure:

A subcutaneous injection ofbuprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre-dose anesthesia of ocular anesthetic (two drops of 0.5% tetracaine hydrochloride) was applied to each eye. A volume of 0.1 mL of the test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale.

Observation period (in vivo):

Eight hours after test item application, a subcutaneous injection of post-dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required.

Number of animals or in vitro replicates:

The rabbit is the preferred species of choice as historically used for irritation studies and is specified in the appropriate test guidelines. The number of animals used was the minimum required to achieve the objectives of the study. Testing was conducted in two animals and the response in those animals was such that exposure of a third animal would not affect classification of the test item, therefore, no further testing was needed.

Details on study design:

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope. Any clinical signs of toxicity, if present, were also recorded. Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24 h

Score:

6

Max. score:

6

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

48 h

Score:

2

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

72 h

Score:

0

Max. score:

0

Irritant / corrosive response data:

A single application of the test item to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. Moderate conjunctiva! irritation was noted in both treated eyes 1 hour after treatment with minimal conjunctival irritation noted at the 24 and 48-Hour observations.Both treated eyes appeared normal at the 72-hour observation. No corneal or iridial effects were noted during the study.

Other effects:

No other effect were reported.

Table 1: Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	75050 Male				75060 Male
Time After Treatment	1 hour	24 hours	48 hours	72 hours	1 hour	24 hours	48 hours	72 hours
CORNEA
E= degree of Opacity	0	0	0	0	0	0	0	0
F= Area of Cornea Involved	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0
IRIS
D	0	0	0	0	0	0	0	0
Score (D x 5)	0	0	0	0	0	0	0	0
CONJUNCTIVAE
A = Redness	1	1	1	0	1	1	1	0
B = Chemosis	1	1	0	0	1	1	0	0
C = Discharge	2	1	0	0	2	1	0	0
Score (A + B + C) x 2	8	6	2	0	8	6	2	0
Total Score	8	6	2	0	8	6	2	0

Table 2: Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number and Sex	Individual Total Scores At:
	1 hour	24 hours	48 hours	72 hours
75050 Male	8	6	2	0
75060 Male	8	6	2	0
Group Mean Score	8.0	6.0	2.0	0.0

Interpretation of results:

GHS criteria not met

Conclusions:

The test item produced a maximum group mean score of 8.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

Executive summary:

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit in accordance with OECD 405 and GLP compliance.

A volume of 0.1 mL of the test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale. Eight hours after test item application, a subcutaneous injection of post-dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope. Any clinical signs of toxicity, if present, were also recorded. Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

No corneal or iridial effects were noted during the study.

Moderate conjunctival irritation was noted in both treated eyes 1 hour after treatment with minimal conjunctival irritation noted at the 24 and 48-hour observations. Both treated eyes appeared normal at the 72-hour observation. Both animals showed expected gain in body weight during the study.

The test item produced a maximum group mean score of 8.0 and was considered to be a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

In the key study (Sanders, 2015 (A)), the primary dermal irritation potential of the test item was evaluated in this study with New Zealand White rabbits (2M) in accordance with OECD Guideline 404 and GLP requirements. There was one group of two male albino rabbits that received a single, four-hour, semioccluded exposure. Each 0.5 mL dose of the test article was applied to the clipped, unabraded skin. At completion of exposure, the bandages were removed and the sites washed. Application sites were evaluated in accordance with the method of Draize at approximately 1 and 24, 48 and 72 hours after patch removal. There were no remarkable body weight changes during the study period. The test article induced very slight erythema and edema on all animals at 1 hour after removal of the patch. The skin appeared normal again at 24 hours. No corrosive effects were noted. The Primary Irritation Index was calculated to be 0.0. The test article received a descriptive rating classification of non-irritating.

In a supporting study (Smolin, 1983), a primary dermal irritation study in compliance with GLP was conducted, following principal national standard methods with acceptable restrictions. Six (6) New Zealand white rabbits each received a single dermal application of 0.5 mL of the test article on two test sites, one abraded and one intact. The test sites were occluded for 24 hours and were observed individually for erythema, edema, and other effects 24 and 72 hours after application. Mean scores from the 24 and 72 hour reading were averaged to determine the primary irritation index. The test article was used as a 20% gravimetric mineral oil suspension.

The primary irritation index was 2.1. This test article is not a primary dermal irritant to rabbits under conditions of this test.

Eye irritation

The key study (Sanders, 2015 (B)) was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit in accordance with OECD 405 and GLP compliance.

A volume of 0.1 mL of the test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale. Eight hours after test item application, a subcutaneous injection of post-dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope. Any clinical signs of toxicity, if present, were also recorded. Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

No corneal or iridial effects were noted during the study.

Moderate conjunctival irritation was noted in both treated eyes 1 hour after treatment with minimal conjunctival irritation noted at the 24 and 48-hour observations. Both treated eyes appeared normal at the 72-hour observation. Both animals showed expected gain in body weight during the study.

The test item produced a maximum group mean score of 8.0 and was considered to be a mild irritant (Class 4 on a I to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

In a supporting study (Smolin, 1983) the ocular irritation potential of the test substance was determined in rabbits in accordance with GLP and national standard methods with acceptable restrictions.

Six (6) New Zealand white rabbits, free from visible ocular defects, each received a single intraocular application of 0.1 milliliter of the test article in one eye. The contralateral eye, remaining untreated, served as a control. The eyes of all animals remained unwashed for 24 hours. Observations of corneal opacity, iritis, and conjunctivitis were recorded 24, 48, and 72 hours after treatment, and at 4 and 7 days if irritation persisted. The test article was used as a 20% gravimetric mineral oil suspension.

The total mean Draize scores were 0.3 at 24 hours and 0.0 at 48 and 72 hours.

This test article is not an ocular irritant to rabbits under conditions of this test.

Justification for classification or non-classification

Skin irritation

According to the classification criteria outlined in the section 3.2.2.3.2.2. (Guidance on the Application of CLP criteria, 2015), if the mean value of≥2.3≤4.0 was reached for erythema/eschar or for oedema in at least two of three tested animals from gradings at 24, 48 and 72 hours after patch removal, the classification as Skin Irritant would be assigned.

The key study (Sanders, 2015 (A)) revealed a very slight erythema/eschar formation with an individual score of 1 and edema formation with an individual score of 1 on all two animals at 1 hour after patch removal.

The skin appeared normal again at 24 hours and the Primary Irritation Index was calculated to be 0.0.

Based on this knowledge, the test item does not meet criteria of Regulation (EC) No. 1272/2008 for classification and labelling as skin irritant. The results of the supporting skin irritation study (Smolin, 1983) support the negative result of the key study. The study was conducted according to the USA Federal Hazardous Substances Act (US-FHSA). The results for erythema and edema for six rabbits of the intact site showed very slight to slight formation with Draize scores from 0 to 2 after 72 hours with a primary irritation index of 2.1 and does therefore not meet the classification criteria of Regulation (EC) No 1272/2008.

Eye irritation

According to the classification criteria outlined in the section 3.3.2.3.2.2. (Guidance on the Application of CLP criteria, 2015), if a substance cause positive but reversible reactions such as corneal opacity (score ≥ 1), iritis (score ≥ 1), conjunctival redness (score ≥ 2) or conjunctival oedema (chemosis) (score ≥ 2) in at least 2 of 3 tested animals from gradings at 24, 48 and 72 hours after application, the classification as Eye Irritant (Category 2) is assigned.

In the key study (Sanders, 2015 (B)) the scores for corneal opacity and iritis were 0 at all time points for both animals. The scores for conjunctival redness and chemosis were between 0 and 1 for 1 hour, 24 and 48 hours. At 72 hours all signs of irritation were fully reversible.

Based on this observation, the test item does not meet the criteria of Regulation (EC) No 1272/2008 for classification and labelling as an eye irritant.

The results of the supporting eye irritation study (Smolin, 1983) support the negative result of the key study. From six tested animals only one animal showed any signs of eye irritation such as minimal conjunctivae redness (score 1) at 24 hours that was fully reversible after 48 hours. The total mean Draize scores were 0.3 at 24 hours and 0.0 at 48 and 72 hours.