Registration Dossier

Administrative data

Description of key information

Skin irritation (OECD 404): not irritating 
Eye irritation (OECD 405): not irritating

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:
23 - 27 Feb 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
Qualifier:
according to
Guideline:
other: JMAFF 12-Nousan-8147 (2000)
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Animals: New Zealand White rabbits
Source: Inhouse Bred (Outbred) New Zealand White rabbits Toxicology Department of Safety Assessment, Advinus Therapeutics Limited, Bangalore 560 058, INDIA
Number and sex: 3 Male rabbits
Age at treatment: 6-7 months
Date of birth: 30 July 2010
Body weight range at treatment: 2.77 to 2.94 kg
Accl imatization: One rabbit (RB8764) was acclimatized for five days while the other two (RB8765 and RB8766) rabbits were accl imatized for six days under laboratory conditions after physical examination.

Conditions
Animals were housed under standard laboratory conditions, air conditioned with adequate fresh air supply (12-15 air changes/hour). Environment: temperature 21 - 23°C, relative humidity 65 - 67%, with a 12 hour light and 12 hour dark cycle. The maximum and minimum temperature and relative humidity in the experimental room were recorded once daily. The relative humidity in the experimental room was calculated from dry and wet bulb temperature recordings.

Housing
The rabbits were housed in dividually in rabbit cages (approx. size: L 65 x B 65 x H 45 cm) with Noryl shallow cage body and facilities for pelleted food (Stainless steel feed hopper) and drinking water (750 mL markrolon bottle fitted with sipper tube). The litter collection tray (noryl waste tray) were washed daily with water (except on Sundays). Water was replenished daily. The water bottles and feed hoppers were changed once a week.

Diet: ad libitum
The animals were offered rabbit feed manufactured by Pranav agro Industries Ltd, Sangli.

Specific contaminants (heavy metals organochlorines and organophosphates) in the diet are analysed periodically and the reports are included in the raw data file.

Water: ad libitum
The animals were provided deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cumpurifier manufactured by Eureka Forbes Ltd. , Mumbai - 400 00 I, India. The water bottles were replenished once daiIy and the water bottles changed once a week.
Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
water
Controls:
other: each rabbit also served as it's own control with untreated areas of the skin.
Amount / concentration applied:
Sample preparation
A quantity of 0.5 g of test substance as received was made into paste by adding sufficient volume of water and mixed with a glass rod.

Preparation of Test Animals
Approximately 24 hours prior to treatment, the fur on the left side of the dorsolateral trunk surface [about 14 x 19 cm] was clipped with an electric clipper -Aesculap® - Germany. Care was taken to avoid abrading the skin. After clipping and prior to application, the animal 's skin (test site) was checked for any abnormalities according to the "Primary Skin Irritation Scoring System", and the test site was found to be normal.

Duration of treatment / exposure:
4 hours
Observation period:
3 days: skin reactions were evaluated 1 hour and then 24, 48 and 72 hours after removal of the patch
Number of animals:
3
Details on study design:
Testing Option
The study was initiated in a stepwise procedure (1 + 2 Rabbits).

Test substance Application
Preparation of Dose Site
Approximately 6 cm2 intact test site was selected on each animal. The adjacent untreated skin of each animal served as the control site.

Application of Test Substance
A quantity of 0.5 g of test substance as received was made into paste by adding sufficient volume of water and mixed with a glass rod. The test substance was applied directly to the skin of the test site and covered with a gauze pad. The entire trunk of each animal was then wrapped with non-irritating semiocclusive adhesive tape to avoid dislocation of the patch.

Exposure of Animals
In a stepwise manner (1 +2 rabbits) all the three rabbits were exposed to the test substance each with one test patch only for a four hour exposure period. The rabbits were restrained using Elizabethan collar for 72 hours post application of the test patch.

Toxic Signs and Pre-Terminal Deaths
Rabbits were observed for toxic signs and pre-terminal deaths for the first four hours (at hourly intervals) on the day of test substance application and once daily thereafter till the end of the observation period.

Body Weights
Individual body weights of the rabbits were recorded at the start of acclimatization, day 1 of treatment (just before test substance application) and
at the termination of observation.

Skin Scoring
Following the exposure period, test patches were removed and the application sites were washed with water and clean towels to remove any residual test substance. The 4 hour dose sites were examined for signs of erythema and edema within 60 minutes and at intervals of approximately 24, 48 and 72 hours after test patch removal accord ing to the "Primary Skin Irritation Scoring System" developed by Draize.

Individual skin scores were recorded for each rabbit.

Grading of skin reactions
The skin reaction was assessed according to the numerical scoring system of Draize et.al (1944).
1. ERYTHEMA AND ESCHAR FORMATION
No erythema.................... . ...................................................................................................................................................................... 0
Very slight erythema (barely perceptible)........................................................................................................................................... 1
Well-defined erythema........................................................................................................................................................................... 2
Moderate to severe erythema.......... .................................................................................................................................................... 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) .......................................................................... 4
MAXIMUM POSSIBLE SCORE - 4

2. EDEMA FORMATION
No edema.................................................................................................................................................................................................. 0
Very sl ight edema (barely perceptible) .............................................................................................................................................. 1
Slight edema (edges of area well defined by definite raising) ......................................................................................................... 2
Moderate edema (raised approximately 1 millimetre).... .................................................................................................................. 3
Severe edema (raised more than 1 millimetre and extending beyond area of exposure)........................................................... 4
MAXIMUM POSSIBLE SCORE - 4
Irritation parameter:
erythema score
Basis:
mean
Remarks:
out of all 3 animals
Time point:
other: 24, 48, 72 hours
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
edema score
Basis:
mean
Remarks:
out of all 3 animals
Time point:
other: 24, 48, 72 hours
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritant / corrosive response data:
There were no skin reactions observed In any of the rabbits through the observation period.
Other effects:
There were no toxic signs and pre-terminal deaths during the course of the experiment. The body weights of all the rabbits increased through the observation period.

Erythema and edema scores at 1, 24, 48 and 72 hours were 0 for each of the three animals tested.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified
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:
14 - 18 Mar 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Qualifier:
according to
Guideline:
other: JMAFF 12-Nousan-8147 (2000)
Qualifier:
according to
Guideline:
other: CounciI Regulation (EC) No. 440/2004
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Animals: New Zealand White Rabbits
Source: Inhouse Bred (Outbred), Toxicology, Department of Safety Assessment, Advinus Therapeutics Limited, Bangalore 560 058, INDIA
No. of rabbits: 3 male rabbits
Age at treatment: 7 - 8 months
Date of birth: 26 July 2010 to 30 July 2010
Body weight range at treatment: 2.61 to 2.94 kg
Accl imatization: One rabbit (RB8767) was acclimatized for five days while the other two (RB8768 and RB8769) rabbits were acclimatized for six days under laboratory conditions after physical examination.

Conditions
Animals were housed under standard laboratory conditions, air conditioned with adequate fresh air supply (12-15 air changes/hour). Environment: temperature 2 1-23°C, relative humidity 59-67%, with a 12 hour light and 12 hour dark cycle. The maximum and minimum temperature and relative humidity in the experimental room was recorded once dail y. The relative humidity in the experimental room was calculated from dry and wet bulb temperature recordings.

Housing
The rabbits were housed individually in rabbit cages (approx. size: L 65 x B 65 x H 45 cm) with noryl shallow cage body and faci lities for pelleted food (stainless steel feed hopper) and drinking water (750 mL markrolon bottle fitted with sipper tube). The litter collection tray (noryl waste tray) was changed daily (except on Sunday). The feed hoppers were changed once a week.

Diet ad libitum
The animals were offered rabbit feed manufactured by Pranav agro Industries Ltd, Sangli Maharashtra, India.

Water ad libitum
Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier, manufactured by Eureka Forbes Ltd., Mumbai-40000 I, India was provided to animals in Markrolon bottles with stainless steel sipper tubes. The water bottles were replenished once daily and the water bottles were changed once a week.
Vehicle:
unchanged (no vehicle)
Controls:
other: The second eye of an animal remained untreated serving as control
Amount / concentration applied:
0.1 g
Duration of treatment / exposure:
24 hours (until eyes were flushed following fluorescein administration)
Observation period (in vivo):
Toxic Signs and Pre-Terminal Deaths
The rabbits were observed for toxic signs and pre-terminal deaths four times (at hourly intervals) on the day of test substance instillation and once daily till 72 hours post instillation.

Body Weights
Body weights were recorded at the start of acclimatization, Day 1 of treatment (just before test substance instillation) and at study termination.

Eye Scoring
Ocular lesions were evaluated with the illumination of a white light source at intervals of approximately 1, 24, 48 and 72 hours post-instillation of the test substance according to the "Scale for Scoring Ocular Lesions" (Draize, 1977). The fluorescein dye evaluation procedure was used to examine the treated eye of one rabbit (# RB8767, which showed corneal opacity at 24 hours) at the 24- and 48-hour observation. At the 72-hour observation, this rabbit had no opacity and all its eye scores were 0. Hence, fluorescein was not used and the experiment was terminated. The other two rabbits had no corneal opacity, so their eyes were not observed using ophthalmic fluorescein.

Individual scores were recorded for each rabbit. All scores were calculated as described in the "Scale for Scoring Ocular Lesions", and the total possible points (out of 110) were tallied. In addition to the observation of the cornea, iris and conjunctivae, any other lesions that were observed were noted. To aid in the interpretation of data, the average score for all rabbits at each scoring period was calculated.

Reference:
Draize. J.H 1977. Dermal Toxicity In: Appraisal of the safety of chemicals in Foods, Drugs and Cosmetics. Association of Food and Drug Officials of the United States. Austin, Texas. p. 47.
Number of animals or in vitro replicates:
3
Details on study design:
Test Substance Instillation
Prior to instillation, both eyes of each rabbit were examined with the aid of a pen light for irritation and ocular defects. The eyes were then checked for opacity using ophthalmic fluorescein sodium strips. The eyes were then irrigated with enough physiological saline (0.9% NaCI) to remove any excess fluorescein (approximately 30 seconds). Using an ultraviolet lamp, the eyes were checked and evaluated for pre-existing corneal damage. No corneal defects were observed in any of the animals.

One hundred milligrams (0.1 g) of the finely ground test substance was placed in the everted lower lid (conjunctival sac) of the left eye of each rabbit.

The eye lids were gently held together for about one second, in order to minimize loss of the test substance. The right eye of each rabbit remained untreated and served as the reference control.

Animal Restraint
Each rabbit was restrained using an Elizabethan collar fixed around the neck. One rabbit RB8767 was restrained for 72 hours and the other two rabbits (RB8768 and RB8769) were restrained for 48 hours post-instillation.

Stepwise exposure of Animals
A single rabbit was initially exposed to the test substance. Since the test substance was not corrosive, the test was completed using two additional
animals.
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
other: mean over 24, 48 and 72 h
Score:
1.3
Max. score:
3
Reversibility:
not fully reversible within: 72 h
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
other: mean over 24, 48 and 72 h
Score:
0.3
Max. score:
3
Reversibility:
fully reversible within: 72 h
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
other: mean over 24, 48 and 72 h
Score:
0.3
Max. score:
3
Reversibility:
fully reversible within: 72 h
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: mean over 24, 48 and 72 h
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 72 h
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
out of animal #2 and animal #3
Time point:
other: mean over 24, 48 and 72 hrs
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
other: mean over 24, 48 and 72 hrs
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 72 h
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
out of animal #2 and animal #3
Time point:
other: mean over 24, 48 and 72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
iris score
Basis:
mean
Remarks:
out of all 3 animals
Time point:
other: mean over 24, 48 and 72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritant / corrosive response data:
The test material caused conjunctival irritation and corneal opacity in the rabbit eye below GHS and CLP C&L thresholds, which cleared by 72 hours.
Other effects:
There were no toxic signs or pre-terminal deaths. The body weights of all rabbits increased slightly through the observation period.

The individually determined mean irritation scores for each animal for corneal opacity, iris lesion, conjunctival redness and conjunctival chemosis for the 24, 48 and 72-hour intervals are:

 Animal No. (Sex)  Corneal Opacity  Iris Lesion  Conjunctival Redness  Conjunctival Chemosis
 RB8767 (M)  0.7  0  1.3  0.7
 RB8768 (M)  0  0  0.3  0
 RB8769 (M)  0  0  0.3  0
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Skin irritation

The acute dermal irritation study in New Zealand White Rabbits was conducted to evaluate the skin irritation potential of the test substance according to OECD guideline 404 and under GLP conditions (Prakash, 2011). Five hundred mg (0.5 g) of the test substance was made into a paste with sufficient volume of water and mixed with a glass rod and was applied to the dose site and covered with a gauze pad of size approximately 6 cm² (2 x 3 cm-6ply). The patches were secured to the body of the animal by a non-irritating semi-occlusive adhesive tape. After 4 hours contact period the bandage was removed and the dose sites were wiped with water. The degree of irritation was evaluated and scored by Draize’s evaluation method at 1, 24, 48 and 72 hours post removal of the test patch. Erythema and edema scores at 1, 24, 48 and 72 hours were 0 for each of the three animals tested. Under the experimental conditions of this test, the test substance is not irritating to skin.

Eye irritation

An in vivo eye irritation study in male New Zealand White rabbits was conducted to evaluate the eye irritation potential of the test substance according to OECD guideline 405 and under GLP conditions (Prakash, 2011). One hundred mg (0.1 g) of the test substance was instilled into the conjunctival sac of the left eye of the animal after gently pulling the lower lid away from the eyeball. The lids were gently held together for about one second, in order to minimize loss of the test substance. The right eye of each rabbit remained untreated and served as the reference control. The ocular lesions were evaluated at 1, 24, 48 and 72 hours according to the scale for scoring ocular lesions. Under the conditions of this study, the test substance caused slight conjunctival irritation and slight corneal opacity in the rabbit eye, which cleared by 72 hours in all animals. The mean conjunctivae, chemosis and iris scores after 24, 48, and 72 h were 1.3/0.3/0.3, 0.7/0/0 and 0.7/0/0 for the 3 individual animals, respectively. The mean scores for chemosis after 24, 48, and 72 h were 0 for all 3 individual animals. Therefore, the test substance is considered as not irritating to the skin. Under the experimental conditions of this in-vivo animal test, the test substance is considered not irritating to the eyes.

In addition, a GLP-compliant Hen's Egg Test on Chorioallantoic Membrane (HET-CAM) was performed according to the ICCVAM recommended HET-CAM Method Protocol (ICCVAM, 2006, updated draft: 11 May 2009) to assess the irritating potential of the test substance by measuring toxic effects on the blood vessels under the CAM of fertilised chicken eggs (Andres, 2012). In this in vitro assay, the vasculated CAM of the eggs was prepared and exposed to the undiluted test substance, the vehicle (physiological saline) or the positive control substances (1% sodium dodecyl sulphate and 1 N sodium hydroxide solution). During an observation period of 5 min, the blood vessels of each individual egg were monitored for the onset of haemorrhage, lysis and coagulation and the times required to induce these effects were measured. Based on the measured times, the irritation score (IS) was calculated. The positive and negative controls yielded the expected results. The undiluted test substance induced coagulation and lysis of the blood vessels in all eggs after 163 and 46 s, respectively. The calculated mean IS value for the test substance was 10.08, which corresponds to a classification as severely irritant (IS > 9). Under the experimental conditions of this HET-CAM test in-vitro, the test substance may be considered as corrosive to the eyes.

However, based on the outcome of the in vivo eye irritation study in rabbits, the substance is not considered as irritating to the eyes, and thus the result of the HET-CAM assay is not taken into account for classification and labelling purposes.

Respiratory tract

This information is not available.

 

References:

Schrage A, Gamer AO, van Ravenzwaay B, Landsiedel R. (2010). Experience with the HET-CAM method in the routine testing of a broad variety of chemicals and formulations. Altern Lab Anim. 38(1): 39-52

Justification for classification or non-classification

The available data on skin and eye irritation of the substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.