Registration Dossier

Administrative data

Description of key information

Skin irritation, rabbit primary skin irritation/corrosion test, 4 hours, semi-occlusive dressing. Result: primary skin irritation index 0.02, practically non irritating to the skin.

Eye irritation, rabbit primary eye irritation/corrosion test, 24 hours, Result: calculated Draize score of 6.3 (24 hour), mildly irritating (scheme of Kay and Calandra)

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:
1987-11-17 - 1987-11-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPP 81-5 (Acute Dermal Irritation)
Version / remarks:
revised Nov. 1984
Deviations:
no
GLP compliance:
yes
Remarks:
UD FDA (21 CFR 58) and uS ePA (40 CFR 160 and 40 CFR 792)
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
Six young adult albino rabbits of the New Zealand White strain, SPF-quality, were obtained from The Broekman Institute, Someren, The Netherlands. The animals arrived at the animal house November 9, 1987 and were approximately 10 weeks old at the start of the study. The animals were identified by means of ear labels and were individually housed in plastic cages with perforated floors. An Acclimatisation period of seven days was observed. The body weights of the animals, were measured one day before test substance administration. The animals were fed standard laboratory animal diet (100 g per day), obtained from Hope Farms, Woerden (LK-01, pellet diameter 4 mm), and had free access to decalcified tap-water.

ENVIRONMENTAL CONDITIONS
The animal room temperature was 20 - 21°C and the relative humidity 60 - 70 per cent. The artificial light sequence was 12 hours light, 12 hours dark.
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.5 g
Duration of treatment / exposure:
4 hours
Observation period:
after 1, 24, 48 and 72 hours
Number of animals:
6
Details on study design:
TEST SYSTEM
One day before dose administration, the fur was removed from the dorsal flanks of the animals by clipping, exposing an area of the skin of approximately 10 cm x 10 cm. Prior to administration of the test substance the skin of all animals was inspected and found to be intact and normal.
The day of test substance administration was designated as day 0. Six portions of 0.5 gram of the test substance were transferred into glass vials; prior to administration each portion was moinstened with 0.5 mL Milli-RO water (Millipore corp., Bedford, Mass., USA). Immediately thereafter, each portion of the prepared test substance was evenly spread on a 6 cm2 gauze patsch of Metalline (Lohmann, FRG) and mounted on permeable tabe (Micropore, 3M, St. Paul, USA). This was applied to the left flank of each animal, the right flank being covered with the same dressing without test substance. Finally, the animals were wrapped in flexible bandage (Coban, 3M, St. Paul, USA). Exposure duration was 4 hours after which period the remaining test substance was removed, using a dry tissue and subsequently a tissue moistened with tap-water. However not all the test substance could remove from the skin: the skin looked slightly black.

OBSERVATION TIME POINTS
The exposed skin areas were examined for signs of erythema and oedema and the responses were scored approximately 60 minutes, and 24, 48 and 72 hours after removal of the patches. For reference the control site on the contralateral flank was used.

SCORING SYSTEM:
The following scoring system was used for grading of the skin irritation:
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 eschar formation
(injuries in depth) 4
Oedema formation
No oedema 0
Very slight oedema (barely perceptible) 1
Slight oedema (edges of area well defined by definite
raising) 2
Moderate oedema (raised approximately 1 millimetre) 3
Severe oedema (raised more than 1 millimetre and
extending beyond area of exposure) 4

Interpretatiton of the skin reactions
Primary irritation index Degree
0 not irriitating
< 2.0 slightly irritating
2.0 - 5.0 moderately irritating
> 5.0 severely irritating
The primary irritation index of a test material is calculated by combining the average skin irritation scores after 60 min, 24, 48 and 72 hours.
Note: if the interpretation based on the primary irritation index is not in accordance with the clinical appearance, the latter is taken for interpreting the skin reactions.
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
other: 60 min
Score:
0.02
Reversibility:
fully reversible within: 24 hrs
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
animal #6
Time point:
other: 60 min
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 24 hrs
Remarks on result:
probability of weak irritation
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
>= 0 - <= 1
Max. score:
1
Reversibility:
fully reversible within:
Remarks:
24 h
Irritant / corrosive response data:
The test substance did not not cause any reaction to the skin, except with one animal. The skin of this animal showed very skight erythema 60 min after removal of the test substance only.
Other effects:
Signs of systemic intoxication were not observed in any of the rabibits.

The treated skin of all six animals was scored for erythema and oedema; the results are shown in Table 1.

Table 1: Primary Skin irritation scores in the rabbit (intact skin)
Test item
Rabbit #, sex and body weight Erythema / Eschar formation Oedema Values
Observations ST / 8*
60 min 24 hrs 48 hrs 72 hrs 7 d 14 d 21 d 60 min 24 hrs 48 hrs 72 hrs 7 d 14 d 21 d
3230
F
2386 g
0 0 0 0 - - - 0 0 0 0 - - - 0/8 = 0.00
3232
F
2248 g
0 0 0 0 - - - 0 0 0 0 - - - 0/8 = 0.00
3234
F
2427 g
0 0 0 0 - - - 0 0 0 0 - - - 0/8 = 0.00
3236
F
2370 g
0 0 0 0 - - - 0 0 0 0 - - - 0/8 = 0.00
3214
M
2086 g
0 0 0 0 - - - 0 0 0 0 - - - 0/8 = 0.00
3216
M
2190
1 0 0 0 - - - 0 0 0 0 - - - 1/8 = 0.13
Total = 0.13
Primary Irritation Index ( P. I. I.) = 0.13/6 = 0.02
Notes:
* Subtotal of scores for erythema and oedema of the first four observations according to the EPA calculatioon method.
- not measured
Interpretation of results:
other: EU GHS criteria not met
Conclusions:
Based on the calculated primary irritation index of 0.02, the test substance would have to be considered as slightly irritating to the skin. However, from the very brief reaction in only one animal the interpretation 'practically non irritating' is preferred.
Executive summary:

The purpose of the study was to evaluate local irritating or corrosive effects of the test item on the rabbit skin following a single application of the test substance in an in vivo rabbit primary skin irritation/corrosion test according to US EPA FIFRA Guideline 81 -5 and in compliance with GLP.

The flank skin of 6 adult rabbits was exposed to 0.5 g of the test substance moistened with Willi-RO water for 4 hours using semi-occlusive dressing. The test substance did not cause any reaction to the skin, except for very slight erythema in one animal 60 min after removal of the dressings and disappearing within 24 hours. The primary skin irritation index amounted to 0.02. Based on these results the test substance should be considered as practically non irritating to the skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
1987-11-05 - 1987-12-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPP 81-4 (Acute Eye Irritation)
Version / remarks:
revised Nov. 1984
Deviations:
no
GLP compliance:
yes
Remarks:
US FDA (21 CFR 58) and US EPA (40 CFR 160 and 40 CFR 792)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
Six adult female albino rabbits of the New Zealand White strain, SPF quality, were obtained from The Broekman Institute, Someren, The Netherlands. The animals arrived at the animal house August 31, 1987 and were at least 10 week old at the start of the study. The animals were identified by means of ear labels and were individually housed in plastic cages with perforated floors. An acclimatisation period of at least 5 days was allowed. Both eyes were inspected on the day of exposure and found to be intact and normal. The body weights of the animals were measured on the day of treatment. The animals were fed standard laboratory animal diet (100 g per day), obtained from Hope Farms, Woerden (LK-01, pellet diameter 4 mm), and had free access to decalcified tap-water.

ENVIRONMENTAL CONDITIONS
The animal room temperature was 20 - 21°C and the relative humidity 55 - 65 per cent. The artificial light sequence was 12 hours light, 12 hours dark.

The New Zealand White rabbit is the species and strain of choice because it is readily available and easy to handle, house and treat. The absence of pigmentation of the iris facilitates the evaluation of induced eye reactions, and there is a considerable amount of published literature on eye irritation available on this species to assist in the assessment of the significance of the effects observed.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
21 +/- 2 mg (equivalent to 20.1 mL)
Duration of treatment / exposure:
24 hrs
Observation period (in vivo):
10 days
Number of animals or in vitro replicates:
6
Details on study design:
Dose administration
The day of test substance administration was designated as day 0. The test substance was ground to a fine powder using a mortar and pestle. On the cay of dose administration, six portions of 21 + 2 mg (equivalent to 0.1 ml) of the test powder were dispensed in glass containers with screw caps. Each portion of the dispensed amount of test substance was instilled in the conjunctival sac of the left eye of each animal using a spatula. The lids were then neld gently together for two seconds and released. Immediately after treatment, the animals were transferred to their cages. The right eye, remaining untreated, served as a control.

Observations
Immediately after instillation of the test substance, the animals were observed and abnormalities were recorded. In addition, the eyes were examined approximately 60 min, 24, 48 and 72 hours, and 7 and 10 days after instillation of the test substance.

TOOL USED TO ASSESS SCORE: fluorescein (24 h after exposure of the test substance)
2% fluorescein in water (pH adjusted to 7.0 and sterilized) was applied to both eyes of each animal to quantitatively examine the potential for corneal injury. The brightly green staining area indicating epithelial damage was estimated as a percentage of the total corneal area. This procedure was repeated 3 days after dose administration.
Observed local (or systemic) effects other than those covered by the scoring system, were also recorded.

SCORING SYSTEM:
The following scoring system was used for grading of the ocular lesions:

CORNEA:
Opacity: degree of density (area most dense taken for reading) - No ulceration or opacity 0
Scattered or diffuse areas of opacity (other than slight dulling of normal lustre),details of iris clearly visible 1*
Easily discernible translucent areas, details of iris slightly obscured 2*
Nacreous areas, no details of iris visible, size of pupil barely discernible 3*
Opaque cornea, iris not discernible through the opacity 4*
CORNEA:
Area of cornea involved:
No ulceration or opacity 0
One quarter or less but not zero 1*
Greater than one quater, but less than half 2*
Greater than half, but less than three quarters 3*
Greater than three quarters, up to whole area 4*
IRIS:
Normal 0
Markedly deepened rugae, congestion, swelling,
moderate circumcorneal hyperaemia or injection,
any of these or any combination thereof, iris still
reacting to light (sluggish reaction is positive) 1*
No reaction to light, haemorrhage, gross
destruction (any or all of these) 2*
CONJUNCTIVAE:
Redness:(refers to palpebral and bulbar conjunctivae, excl. cornea and iris)
Blood vessels normal 0
Some blood vessels definitely hyperaemic or injected 1
Diffuse, crimson colour, individual vessels not easily discernible 2*
Diffuse beefy red 3*
Chemosis: lids and/or nictating membranes
No swelling 0
Any swelling above normal (includes nictating membranes) 1
Obvious swelling with partial eversion of lids 2*
Swelling with lids about half closed 3*
Swelling with lids more than half closed 4*
Discharge:
No discharge 0
Any amount different from normal (does not include small
amounts observed in inner canthus of normal animals 1
Discharge with moistening of the lids and hairs just
adjacent to lids 2
Discharge with moistening of the lids and hairs and
considerable area around the eye 3
* Starred figures indicate positive effect.

Interpretation of the results The results of the study were expressed according to the Draize scoring system. This score was calculated with the following formula: (5 x corneal opacity grade x area of opacity grade) + (5 x iridial injury grade) + 2 x (conjunctival redness grade + chemosis grade + di scharge grade).
The calculated Draize score was interpreted according to the scheme of Kay and Calandra (see below).
Draize score Classification according to Kay and Calandra
0 - 0.5 not irritating
0.5 - 2.5 practically not irritating
2.5 - 15 minimally irritating
15 - 25 mildly irritating
25 - 50 moderately irritating
50 - 80 severely irritating
80 - 100 extremely irritating
100 - 110 maximally irritating
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
>= 0 - <= 3
Max. score:
3
Reversibility:
fully reversible within:
Remarks:
7 days
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
>= 1 - <= 2
Max. score:
2
Reversibility:
fully reversible within:
Remarks:
7 days
Irritation parameter:
other: Draize caclulation
Basis:
mean
Time point:
other: 1 h
Score:
5.7
Reversibility:
fully reversible
Irritation parameter:
other: Draize calculation
Basis:
mean
Time point:
24 h
Score:
6.3
Reversibility:
fully reversible
Irritation parameter:
other: Draize calculation
Basis:
mean
Time point:
48 h
Score:
4.7
Reversibility:
fully reversible
Irritation parameter:
other: Draize calculation
Basis:
mean
Time point:
72 h
Score:
5.3
Reversibility:
fully reversible
Irritation parameter:
other: Draize calculation
Basis:
mean
Time point:
7 d
Score:
0.7
Reversibility:
fully reversible
Irritation parameter:
other: Draize calculation
Basis:
mean
Time point:
10 d
Score:
0
Irritant / corrosive response data:
Instillation of the test substance into one of the eyes of each of six female rabbits affected the conjunctivae only. All six animals showed very slight conjunctival redness and very slight to obvious chemosis 60 minutes after exposure. The capillary injection of the scleral conjunctivae in animal 2298 and of the nictating membrane in animal 2308 increased briefly to diffuse beefy red on day 3. The conjunctival redness had disappeared in four animals by day 7 and in the others by day 10. The obvious chemosis decreased within 48 hours to very slight and had disappeared completely in all animals between study day 3 and day 7. Adverse effects on the cornea were not observed in any of the rabbits during the entire observation period. Treatment of the eyes with fluorescein 24 and 72 hours after instillation of the test substance revealed no epithelial damage. Twenty-four hours after instillation of the test substance all six rabbits showed a considerable amount of tne test material in the eyes and therefore the eyes of all six animals were washed out with tepid tap-water. It proved impractical to remove all the test material due to its adhesion to the eyebols.
Four animals showed some lacrimation or an increased amount of discharge during study days 1 and 2.
Other effects:
No signs of systemic intoxication were observed in any of the rabbits during the entire observation period.
Table 1: Eye irritation Scores in the rabbit
Rabbit #, Sex and Body Weight Obser-vations Cornea Iris Conjunctivae Draize total score Fluorescence area % Remarks
opacity area redness chemosis discharge
(0-4) (0-4) (0-2) (0-3) (0-4) (0-3)
2296
F
3560 g
1 hr 0 0 0 1 2 0 6 chemosis score 2 for lower eyelid only
1 day 0 0 0 1 2 0 6 0 chemosis score 2 for lower eyelid only
2 day 0 0 0 1 1 0 4
3 day 0 0 0 1 1 0 4 0
7 day 0 0 0 0 0 0 0
10 day 0 0 0 0 0 0 0
2298
F
3057 g
1 hr 0 0 0 1 2 0 6

chemosis score 2 for lower eyelid only

1 day 0 0 0 1 1 2 8 0
2 day 0 0 0 1 1 1 6
3 day 0 0 0 3 1 0 8 0

redness score 3 for scleral conjunctivae only

7 day 0 0 0 1 0 0 2

redness score 1 for scleral conjunctivae only

10 day 0 0 0 0 0 0 0
2300
F
3212 g
1 hr 0 0 0 1 2 0 6 chemosis score 2 for lower eyelid only
1 day 0 0 0 1 1 1 6 0
2 day 0 0 0 1 1 0 4
3 day 0 0 0 1 1 0 4 0
7 day 0 0 0 0 0 0 0
10 day 0 0 0 0 0 0 0
2306
F
3112 g
1 hr 0 0 0 1 1 0 4
1 day 0 0 0 1 1 0 4 0
2 day 0 0 0 1 1 0 4
3 day 0 0 0 1 1 0 4 0
7 day 0 0 0 1 0 0 2 redness score 1 for scleral conjunctivae only
10 day 0 0 0 0 0 0 0
2308
F
3462 g
1 hr 0 0 0 1 2 0 6 chemosis score 2 for lower eyelid only
1 day 0 0 0 1 2 1 8 0
2 day 0 0 0 1 1 1 6
3 day 0 0 0 3 1 0 8 0 redness score 3 for nictating membrane only
7 day 0 0 0 0 0 0 0
10 day 0 0 0 0 0 0 0
2310
F
3229 g
1 hr 0 0 0 1 2 0 6 chemosis score 2 for nictating membrane only
1 day 0 0 0 1 1 1 6 0
2 day 0 0 0 1 1 1 4
3 day 0 0 0 1 1 0 4 0 chemosis score 1 for eyelids only
7 day 0 0 0 0 0 0 0
10 day 0 0 0 0 0 0 0
Draize calculation 1 hr 1 day 2 day 3 day 7 day 10 day
Mean total score 5.7 6.3 4.7 5.3 0.7 0.0
Kay and Calandra interpretation: mildly irritating
Interpretation of results:
other: Category 2 (irritating to eyes) based on EU GHS criteria
Conclusions:
Based on the calculated Draize score of 6.3 (24 hours) the test substance should be classified as mildly irritationg according to the scheme of Kay and Calandra.
Executive summary:

A sample of the test material was tested in the in vivo rabbit primary eye irritation/corrosion test according to EPA OPP 81-4 (Acute Eye Irritation) and in compliance with GLP to determine its possible irritating or corrosive effects. A single instillation of approximately 21 mg (equivalent to 0.1 ml) of the test substance in one of the eyes of each of six female rabbits resulted in effects on the conjunctivae only. Very slight conjunctival redness was observed in all six animals with a short increasing to diffuse redness in two animals on day 3. The redness had disappeared on day 10. Five of the six animals showed obvious chemosis, the sixth showed very slight chemosis. Chemosis was not noted after day 7 in any rabbit.

Some lacrimation or an increased amount of discharge was observed during days 1 and 2 in four of the six animals. No signs of systemic intoxication were observed in any of the rabbits.

Based on the calculated Draize score of 6.3 (24 hour) the test substance should be classified as mildly irritating according to the scheme of Kay and Calandra.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion

The purpose of the study was to evaluate local irritating or corrosive effects of the test item on the rabbit skin following a single application of the test substance in an in vivo rabbit primary skin irritation/corrosion test according to US EPA FIFRA Guideline 81 -5 and in compliance with GLP.

The flank skin of 6 adult rabbits was exposed to 0.5 g of the test substance moistened with Willi-RO water for 4 hours using semi-occlusive dressing. The test substance did not cause any reaction to the skin, except for very slight erythema in one animal 60 min after removal of the dressings and disappearing within 24 hours. The primary skin irritation index amounted to 0.02. Based on these results the test substance should be considered as practically non irritating to the skin.

Eye irritation

A sample of the test material was tested in the in vivo rabbit primary eye irritation/corrosion test (Weterings, 1987b) according to EPA OPP 81-4 (Acute Eye Irritation) and in compliance with GLP to determine its possible irritating or corrosive effects. A single instillation of approximately 21 mg (equivalent to 0.1 ml) of the test substance in one of the eyes of each of six female rabbits resulted in effects on the conjunctivae only. Very slight conjunctival redness was observed in all six animals with a short increasing to diffuse redness in two animals on day 3. The redness had disappeared on day 10. Five of the six animals showed obvious chemosis, the sixth showed very slight chemosis. Chemosis was not noted after day 7 in any rabbit.

Some lacrimation or an increased amount of discharge was observed during days 1 and 2 in four of the six animals. No signs of systemic intoxication were observed in any of the rabbits.

Based on the calculated Draize score of 6.3 (24 hour) the test substance should be classified as mildly irritating according to the scheme of Kay and Calandra.

Respiratory irritation

There are currently no validated animal tests that deal specifically with RTI, however, useful information may be obtained from the single and repeated inhalation toxicity tests. The acute inhalation toxicity studies (McDonald, 1988 and Hardy, 1988) serve as weight of evidence evaluation. In the key inhalation toxicity study (McDonald, 1988) four groups each of 5 males and 5 females were exposed to dust concentrations of 0.15, 0.31, 0.52 and 1.01 mg/L test material by snout only for 4 hours. Respiratory depression was observed during exposure at all concentrations. Gross post mortem examinations of the animals that died following exposure to the test material revealed extensive haemorrhage in the lungs and resultant pulmonary oedema.

In the supporting acute inhalation toxicity study (Hardy, 1988) 4 groups each consisting of 5 male and 5 female rats were exposed to a dust test atmosphere (whole body) of ferbam for 4 hours at concentrations of 0.336, 0.414, 0.548 and 1.21 mg/L. During exposure signs consistent with exposure to an irritant dust including partial closing of the eyes, fluid discharge from the mouth or nose, exaggerated respiratory movements and the adoption of a hunched posture were recorded. The lungs of rats that died as a result of exposure were congested and the trachaea contained frothy fluid.

No further studies on inhalation hazards of the test material are available.

Taking these findings into consideration irritating effect on the respiratory system are likely.

Justification for classification or non-classification

In vivo key studies (Weterings, 1987a,b) with the test item showed the substance to be practically non irritating to the skin and mildly irritating to the eyes (Kay and Calandra). A short increasing to diffuse conjunctival redness in two animals on day 3 was observed. The conjunctivae chemosis score was up to 2 for 5 animals and only slight for one animal and declined to 1 during 24 hours. Chemosis was not noted after day 7 in any rabbit. Indications of an irritating effect of the test item on the respiratory systems in rats are recorded in two acute inhalation toxicity studies (McDonald, 1988, Hardy, 1988). During exposure signs consistent with exposure to an irritant dust. In addition the substance is classified as irritant to the respiratory tract (H335) in Annex VI of Regulation (EC) No 1272/2008.

Therefore, the test item does not meet the criteria for classification and labelling for skin irritation in accordance with Regulation (EC) No 1272/2008 based on available data, but classification as skin irritant Cat. 2 (H315) is still warranted due to harmonised classification. The test item does meet the criteria for classification and labelling for eye irritation category 2 in accordance with Regulation (EC) No 1272/2008 (CLP Regulation). The test item does meet the criteria for classification and labelling for respiratory irritation category 3 ( STOT Single Exp. 3) in accordance with Regulation (EC) No 1272/2008 (CLP Regulation).