Registration Dossier

Administrative data

Description of key information

Skin irritation in vitro: Irritating, based on the results of irritation studies performed in rabbits, guinea-pigs and humans and on the results of  the TER assay (WoE).
Eye irritation: Irritating, based on the BCOP results (K, rel.1) and on the skin irritancy potential.
Respiratory irritation: Irritating, based on the results of the repeated dose toxicity study via inhalation in rats (K, rel.1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From October 29 to December 08, 1979
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Pre-GLP and pre-guideline study. Deviations from OECD test guideline No 404: no recovery period although irritation still present at the 72-h observation time, draize scale not used.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
yes
Remarks:
no recovery period although irritation still present at the 72-h observation time, draize scale not used, no details on housing of animals and environmental conditions of animal room; body weights not recorded
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Remarks:
pre-GLP
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 9-12 weeks
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
Duration of treatment / exposure:
4 h
Observation period:
24, 48 and 72 h after treatment
Number of animals:
7
Details on study design:
TEST SITE
- Area of exposure: Dorsal area
- % coverage: No data
- Type of wrap if used: Occlusive patches prepared by heat-sealing 1ʺ x 1ʺ 24 ply gauze pads on to 1.25ʺ x 1.25ʺ squares of polythene sheeting which is then attached to 3.5ʺ x 1ʺ strips of adhesive tape were used.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Treatment sites were wiped clean of excess test material.
- Time after start of exposure: 4 h

SCORING SYSTEM: Using 8-point anchored ordinate scale ranging from "a" (very slight) to "h" (severe).
Irritation parameter:
overall irritation score
Basis:
other: total score of 7 animals
Time point:
24/48/72 h
Score:
164
Reversibility:
not reversible
Remarks on result:
other: mean score/site: 23.43; mean score/site/day: 5.86
Irritation parameter:
erythema score
Time point:
24/48/72 h
Remarks on result:
not determinable
Irritation parameter:
edema score
Time point:
24/48/72 h
Remarks on result:
not determinable
Irritant / corrosive response data:
- Very slight to quite distinct erythema and very slight to well-developed oedema were observed.
- Very slight to slight cracking was observed in 2/7 and 5/7 animals at 48 and 72 h, respectively.
- Very slight to slight scaling was observed in 2/7 and 3/7 animals at 48 and 72 h, respectively.
- Diffuse ‘pin-prick’ spots of haemorrhage were observed in 3/7 animals at 4 h.
- Very slight to slight pale brown discoloration of skin over 1/4 of test site was observed in one animal at 24 and 48 h.
- Test material produced a variable reaction from marginal to distinct with suspected necrosis in one animal and haemorrhaged areas in two animals after 72 h.
Other effects:
None

Tentative conversion to Draize scale:

Reaction grade a & b <=> Score 1

Reactions grade c & d <=> Score 2

Reactions grade e & f <=> Score 3

Reactions grade g & h <=> Score 4

Table 7.3.1/1: Mean irritant/corrosive response data for each animal at each observation time up to removal of animals from the test (intact skin)

 

Score at time point / Reversibility

Erythema

Max. score 4

Oedema

Max. score 4

4 h

1 / 2 / 2 / 2 / 1 / 2 / 0

1 / 2 / 2 / 2 / 0 / 2 / 0

24 h

2 / 2 / 2 / 2 / 1 / 2 / 1

3 / 3 / 2 / 3 /0 / 3 / 0

48 h

2 / 2ab / 2 / 2 / 1 / 2 / 1ab

2 / 3 / 3 / 3 / 0 / 2 / 0

72 h

2ab / 2ab / 2a / 2 / 1 / 1a / 0ab

1 / 2 / 2 / 2 / 0 / 1 / 0

Average 24h, 48, 72h

2 / 2 / 2 / 2 / 1 / 1.67 / 0.67

2 / 2.67 / 2.33 / 2.67 / 0 / 2 / 0

Reversibility*)

nc.

nc.

*) Reversibility: c. = completely reversible; n.c. = not completely reversible; n. = not reversible

a) Cracking

b) Scaling

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the test condition, test material is classified as irritant to the rabbit skin (Category 2) according to Regulation (EC) No.1272/2008 (CLP) and to the GHS.
Executive summary:

In a dermal irritation study, 0.5 mL of undiluted test material was dermally applied on the clipped dorsum of 10 New Zealand White rabbits for 4 h under semi-occlusive dressing. Skin irritation was assessed and scored at 24, 48 and 72 h after the removal of the patch.

Very slight to quite distinct erythema and very slight to well-developed oedema were observed. Very slight to slight cracking was observed in 2/7 and 5/7 animals at 48 and 72 h, respectively. Very slight to slight scaling was observed in 2/7 and 3/7 animals at 48 and 72 h, respectively. Diffuse ‘pin-prick’ spots of haemorrhage were observed in 3/7 animals at 4 h. Very slight to slight pale brown discoloration of skin over 1/4 of test site was observed in one animal at 24 and 48 h. Test material produced a variable reaction from marginal to distinct with suspected necrosis in one animal and haemorrhaged areas in two animals after 72 h.

Under the test condition, test material is classified as irritant to the rabbit skin (Category 2) according to Regulation (EC) No.1272/2008 (CLP) and to the GHS.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
No data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Study was conducted according to a non-standard method. However results were deemed reliable (consistent with other existing studies).
Reason / purpose:
reference to same study
Principles of method if other than guideline:
In comparative studies on the irritancy of different materials to the skin of different species, undiluted isoeugenol was applied under occlusion to the dorsal skin of albino angora rabbits and guinea-pigs for 24 hours, then the patches were removed, and a second application of undiluted isoeugenol was made 30 minutes later.
GLP compliance:
no
Remarks:
pre-GLP
Species:
other: rabbit and guinea pig
Strain:
other: rabbit (Angora) and guinea pig (Hartley)
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: rabbit: 2.3-3.0 kg (average weight 2.6 kg); guinea pig: 350-500 g

Type of coverage:
occlusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
other: the opposite site was left untreated
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g
- Concentration (if solution): 100 %
Duration of treatment / exposure:
Two exposures of 24 h each (sequence of application was rotated)
Observation period:
Skin reactions were recorded at 24 h after each application.
Number of animals:
6 rabbits and 6 guinea pigs
Details on study design:
TEST SITE
- Area of exposure: Dorsal surface (rabbits); dorsal, mid-lumbar region (guinea pigs)
- Type of wrap if used: plastic collar
- The test areas measured 3 x 3 cm

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No

SCORING SYSTEM: Scoring system used was:
1. On the living skin
Reddening rate (erythema)
- : No reddening
± : Very slight reddening (barely perceptible)
+ : Well defined reddening
++ : Moderate reddening
+++ : Severe reddening (beet redness)
Others: scaling, crust formation, loss of elasticity and fissures.
2. On the removed skin: Dilating rate, swelling rate (oedema), bluing rate (increased capillary permeability) and bleeding rate.
3. Total score:
Total score (72 h reading) = dilating rate + swelling rate + bluing rate + redding rate
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Remarks:
all rabbits
Time point:
72 h
Score:
3
Max. score:
3
Reversibility:
no data
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Remarks:
all guinea-pigs
Time point:
72 h
Score:
3
Max. score:
3
Reversibility:
no data
Irritation parameter:
erythema score
Time point:
24/48/72 h
Remarks on result:
not determinable
Irritation parameter:
edema score
Time point:
24/48/72 h
Remarks on result:
not determinable
Irritant / corrosive response data:
- Test material produced severe irritating reactions on rabbit and guinea pig skin.
Other effects:
None

None

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the test condition, test material produced severe irritating reactions on rabbit and guinea pig skin.
Executive summary:

In a primary dermal irritation study, a group of 6 angora rabbits and 6 hartley guinea pigs were dermally exposed to test material (0.1 g) on the dorsal surface (3 x 3 cm). The opposite site was left untreated. Test material was kept in contact with the animals for 24 h. After 24 h reading, the hair on the test area was clipped and the test compound was again applied 30 minutes later. A second set of reading and application was made 48 h later (= 72 h reading). After the 72 h reading, animals were killed, the dorsal skin was removed and evaluation of skin reactions was performed using Evans blue solution. Removed skin was also processed for histopathological examinations.

 

Test material induced severe skin irritation in rabbits and guinea pigs and the primary irritation index was 3.

Under the test conditions, topical application of test material induced severe skin irritation in rabbits and guinea pigs. 

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
02-03 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 430 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER))
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on June 17, 2015 / signed on September 24, 2015)
Test system:
isolated skin discs
Source species:
rat
Source strain:
Wistar
Details on animal used as source of test system:
SOURCE ANIMAL
- Strain: Wistar (RccHan™:WIST)
- Source: Envigo RMS (UK) Limited, Oxon, UK.
- Sex: female
- Age at the start of pelt preparation: 21-23 days
- Housing: Animal was housed in a suspended solid-floor polypropylene cage furnished with woodflakes.
- Diet: Food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK), ad libitum
- Water: Mains drinking water, ad libitum
- Acclimatization period: 2 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: At least fifteen changes per hour
- Photoperiod: 12 h dark / 12 h light
Justification for test system used:
Following the REACH bottom-up strategy, the Transcutaneous Electrical Resistance Assay was used to assess skin corrosion as recommended in the OECD test guideline No. 439. This method was chosen because the registered substance was considered incompatible with the EpiDerm™ skin corrosivity test due to non-specific reduction of MTT by the test substance.
Vehicle:
unchanged (no vehicle)
Details on test system:
SKIN DISC PREPARATION
- Procedure used: Following an acclimatization period of 2 days the animal was shaved to remove hair from the dorsal surface and skin area was washed using an antibiotic wash. After 3 days a second antibiotic wash was performed. Two days later the animal was killed using ascending concentrations of carbon dioxide followed by cervical dislocation. The dorsal skin was removed from the rat as a single pelt. Excess fat was removed and the pelt mounted, epidermal side uppermost, onto a polytetrafluoroethylene (PTFE) tube. The tissue was secured in place using a rubber “O” ring. Excess tissue was trimmed away and the “O” ring/PTFE interface sealed with soft paraffin wax. The tube was supported by a clamp inside a labelled 30 mL glass receptacle containing 10 mL electrolyte solution (154 mM MgSO4).
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was greater than 10 kΩ in order for the remainder of the pelt to be used in the assay

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 20 to 23°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: not reported. At the end of the exposure period, the test item was removed by washing the skin disc with a jet of warm tap water until no further test item could be removed
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none reported

DYE BINDING METHOD
- Dye used in the dye-binding assay: None (Dye penetration is assessed only if the mean TER value of the test item is less than or equal to 5 kΩ)

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the mean TER value is less than or equal to 5 kΩ and the skin disk is obviously damaged, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage, but the mean disc dye content is greater than or equal to the mean disc dye content of the 10M HCl positive control obtained concurrently.
- The test substance is considered to be non-corrosive to skin if the mean TER value obtained for the test substance is greater than 5 kΩ, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage, and the mean disc dye content is well below the mean disc dye content of the 10M HCl positive control obtained concurrently.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 150 μL of test item was applied to the inner epidermal surface of skin discs using an automatic pipettor
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 150 μL
- Concentration (if solution): water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 150 μL
- Concentration (if solution): 10M hydrochloric acid
Duration of treatment / exposure:
24 hours
Number of replicates:
Total: 9 skin discs - 3 skin discs/group for test item, negative and positive controls
Irritation / corrosion parameter:
transcutaneous electrical resistance (in kΩ)
Run / experiment:
24 hours exposure period
Value:
21.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
No indication of corrosion
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 7.3.1/1: Individual and mean transcutaneous electrical resistance measurements

 

Treatment

 

Tissue Number

 

TER ()

 

Mean TER ± SD (kΩ)

 

Test Item

 

1

23.7

21.5 ± 7.9

2

12.8

3

28.1

Positive Control

 

4

$

0.887.5* ± 0.0361

5

0.862

6

0.913

Negative Control

 

7

9.2

16.2 ± 6.4

8

21.8

9

17.5

 

$ = Reading from skin disc unobtainable due to perforation (considered indicative of corrosion)

= Mean TER based on two skin discs due to perforation of one skin disc

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, the test item was considered unlikely to have the potential to cause corrosion in vivo.
Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 430 and in compliance with GLP, using the Transcutaneous Electrical Resistance (TER) Assay. The test item (150 μL) was applied to the epidermal surface of three skin discs for a contact period of 24 hours. At the end of the exposure period, the test item was removed by washing the skin disc with a jet of warm tap water until no further test item could be removed. The positive control was 10M hydrochloric acid (approximately 36%) and the negative control was sterile distilled water. The contact period for the positive and negative controls was 24 hours. The TER was measured using a Wheatstone Bridge with a low voltage alternating current and mean TER for the skin discs was calculated.

The mean TER recorded for the test item was 21.5 kΩ. The mean TER recorded for the positive and negative control discs were as follows: Positive control disc, 10M Hydrochloric acid (approximately 36%): 887.5 Ω Negative control disc, Sterile distilled water: 16.2 kΩ The mean TER values for the positive and negative control discs were within the acceptable range for the method, and the variability between skin disc replicates was considered to be acceptable, therefore the test results were considered to be valid.

Following assessment of the data the test item was considered unlikely to have the potential to cause corrosion in vivo.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20-21 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on July 01, 2014 / signed on October 07, 2015)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: supplied by Joseph Morris Abattoir, were excised by an abattoir employee and collected as soon after slaughter as possible (last excised at 12:30 hours, 20 October 2015).
- Number of animals: not reported
- Characteristics of donor animals (e.g. age, sex, weight): cattle aged under 30 months.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Excised eyes were maintained and transported to the laboratory, at ambient temperature, in sufficient HBSS, containing 1% (v/v) penicillin/streptomycin solution, to cover all the eyes in the receptacle.
- Time interval prior to initiating testing: The eyes were used within 4 hours of slaughter (incubation of mounted corneas commenced at 14:36 hours, 20 October 2015).
- indication of any existing defects or lesions in ocular tissue samples: none; Instructions were given to avoid damaging the corneas during excision.
- Indication of any antibiotics used: HBSS containing 1% (v/v) penicillin/streptomycin solution
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL of test item was applied on each cornea.
- Concentration (if solution): The test item was tested undiluted.
- The pH of the test substance was approximately 7.0.
Duration of treatment / exposure:
10 minutes (± 30 seconds) at 32 ± 1 °C in horizontal position.
Duration of post- treatment incubation (in vitro):
2 hours ±10 minutes at 32 ± 1 °C in a vertical position.
Number of animals or in vitro replicates:
Total: 9 corneas - 3 corneas/group for test item, negative and positive controls
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The corneas were mounted in the cornea holders with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was then positioned on top of the cornea and secured with screws. Both compartments of the holder were filled with HBSS plus 1% (v/v) penicillin/streptomycin, using a syringe. The posterior compartment was always filled first to return the cornea to its natural shape. The holders were then plugged and incubated, in an upright position, overnight at room temperature (approximately 20 °C). After overnight incubation at room temperature (approximately 20 °C) the HBSS plus 1% (v/v) penicillin/streptomycin was removed from both chambers. Both chambers were filled with cMEM (posterior chamber first), taking care to exclude air bubbles and plugged. For equilibration, the corneas in the holder were incubated in the upright position for 60 minutes ± 5 minutes at 32 ± 1 °C in a water-bath. At the end of the 60 minutes incubation period, the medium was removed and refilled with fresh cMEM and then, the basal opacity was determined.

QUALITY CHECK OF THE ISOLATED CORNEAS : Throughout the assay the corneas were examined for opaque spots or other irregularities.

NUMBER OF REPLICATES : 3 corneas/group

NEGATIVE CONTROL USED : 0.9% sodium chloride solution

POSITIVE CONTROL USED : Ethanol

APPLICATION DOSE AND EXPOSURE TIME :
The anterior compartment received the test item or negative or positive control at a volume of 750 μL on the surface of the corneas. The corneas were incubated in a horizontal position for 10 minutes (± 30 seconds) at 32 ± 1 °C in the water-bath.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times or until the wash medium (EMEM with phenol red) was clear and there was no discolouration. The test substance required four washes.
- POST-EXPOSURE INCUBATION: yes, after the test item was rinsed off from the application site by washing, the corneas were incubated at 32 ± 1 °C for 2 hours ± 10 minutes in an upright position.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacitometer measured the light transmission through the centre of each mounted cornea, displaying a numerical opacity value (arbitrary unit). The opacity of each cornea was measured by reading each holder in the right-hand chamber of the calibrated opacitometer. Corneas with an opacity value greater than 7 units were discarded. The mean basal opacity value of the corneas was then calculated and three corneas with opacity values close to the mean value were chosen for use as negative control corneas.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490). Following the final opacity measurement, the incubation medium was removed from the anterior compartment of the holder and replaced by 1 mL of sodium fluorescein solution (4 mg/mL). Corneas were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment was removed, well mixed and transferred to a 1 cm path length cuvette and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
- Others (e.g, pertinent visual observations, histopathology): Throughout the assay the corneas were examined for opaque spots or other irregularities. Moreover an estimate of the pH of the test substance as 10% v/v solution in 0.9% sodium chloride solution, was determined using pH test sticks and recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: the decision criteria as indicated in the TG was used.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean/3 corneas incubated 2 hours
Value:
39.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Remarks:
39.4 ± 8.2
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Following treatment with test substance, the corneas were noted as clear. The corneas treated with the positive control, ethanol, were opaque and the corneas, treated with the negative control, 0.9% sodium chloride solution, were clear. It was noted during the post opacity measurement that cornea number 1, treated with the negative control, 0.9% sodium chloride solution, had an opaque line in the centre of the cornea. As cornea no. 1 was visibly damaged the opacity and permeability results were not included in the calculation of the mean In Vitro Irritancy Score for test substance and the positive control ethanol.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline:
The positive control should elicit an In Vitro Irritancy Score that falls within two standard deviations of the historical mean, 2 x SD = 25.5 – 50.3, for the laboratory.
The negative control mean opacity change value should be ≤3.0 and the permeability mean value ≤0.1 (historical period August 2010 to August 2015).

Table 7.3.2/1: Results of the BCOP assay

 

Sample

Opacity ± SD

Permeability ± SD

In vitro irritancy Score ± SD

In vitro

classification

Test substance

18.667 ± 4.619

1.382 ± 0.349

39.4 ± 8.2

No prediction can be

made

Ethanol

16.667 ± 1.528

0.713 ± 0.240

27.4 ± 2.7

No prediction can be

made

0.9% Sodium

chloride solution

2.000 ± 0.000

0.015 ± 0.001

Not applicable

Not applicable

 

 

ASSAY VALIDITY

The positive control, ethanol, elicited an In Vitro Irritancy Score of 27.4. This value was within the historical range (mean ± 2 x SD = 25.5 – 50.3) for the assays performed to date.

The negative control, 0.9% sodium chloride solution, opacity mean change value was 2.0 which was below the maximum acceptance value of 3.0. The permeability mean of the negative control was 0.015, which was below the maximum acceptance value of 0.1.

Interpretation of results:
other: No prediction can be made
Conclusions:
With an IVIS of 39.4, no prediction can be made as the result is outside the decision criteria (i.e.≤3 or >55).
Executive summary:

An in vitro eye irritation study was performed according to the OECD Guideline 437 and in compliance with GLP to assess the corneal damage potential of test substance by means of the BCOP assay using fresh bovine cornea.  750 µL of test item was applied to cornea for 10 minutes followed by an incubation period of 2 hours at 32 ± 1 °C and corneal opacity was measured. Three corneas were used for each treated series (undiluted test item; negative control; positive control: ethanol). Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. Two endpoints, corneal opacity and permeability, were measured and combined to give an In Vitro Irritancy Score.   Following treatment with test substance, the corneas were noted as clear. The corneas treated with the positive control, ethanol, were opaque and the corneas, treated with the negative control, 0.9% sodium chloride solution, were clear. It was noted during the post opacity measurement that cornea number 1, treated with the negative control, 0.9% sodium chloride solution, had an opaque line in the centre of the cornea. As cornea no. 1 was visibly damaged the opacity and permeability results were not included in the calculation of the mean In Vitro Irritancy Score for test substance and the positive control ethanol. The negative and positive controls met the acceptance criteria for this assay.   The calculated mean in vitro irritancy score for test substance and positive control were 39.4 ± 8.2 and 27.4 ± 2.7, respectively.  

With an IVIS of 39.4, no prediction can be made as the result is outside the decision criteria (i.e.≤3 or >55)

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Skin irritation:

No in vivo skin irritation studies were identified on the registered substance (trans-Isoeugenol). However, several data are available on the supporting substance (Isoeugenol, mixture of cis- and trans-Isoeugenol, see Iuclid section 13 for read-across justification). They are summarized below.

Animal data:

- In comparative studies on the irritancy of different materials to the skin of different species, undiluted Isoeugenol was applied under occlusion to the dorsal skin of albino angora rabbits and guinea-pigs for 24 hours, then the patches were removed, and a second application of undiluted Isoeugenol was made 30 minutes later. Readings were made visually and by examination of excised skin following intravenous injection of saline Evans blue. Under these conditions, it was concluded that undiluted Isoeugenol was severely irritating to the skin of rabbit and guinea-pig (Motoyoshi, 1979a, Rel.4, WoE).

- 48-h occlusive patch testing of miniature swine with undiluted Isoeugenol, was reported to give no signs of irritant effects (Motoyoshi, 1979b, Rel.3, disregarded). It should be pointed out that this study was conducted according to a non-standard method.

- In a pre-GLP and pre-guideline study, but performed similarly to the OECD Test Guideline No. 404, very slight to quite distinct erythema and very slight to well-developed oedema were observed. Isoeugenol produced a variable reaction from marginal to distinct with suspected necrosis in one animal and haemorrhaged areas in two animals after 72 h (URL, 1979, rel.4, WoE).

- Haemorrhaged areas were also observed in an acute dermal toxicity study (CSE, 1979).

Human data:

- In humans, a solution of 32% Isoeugenol in acetone was found to be moderately irritating when applied to the skin of 50 adult males in occlusive patches over 48 hours (Motoyoshi, 1979c, Rel.4, WoE).

Available data indicate that undiluted Isoeugenol is a severe irritant to animal skin. Results in human volunteers mirror those seen in animal studies.

However, due to the suspected corrosive reactions in the animal studies, an in vitro test for corrosivity was performed on trans-Isoeugenol (Envigo, 2015, Rel.1, WoE). In this Transcutaneous Electrical Resistance (TER) Assay, the mean TER value was 21.5 kΩ, i.e. greater than the limit of 5 kΩ fixed in the OECD Test Guideline No. 430. Trans-Isoeugenol is considered to be non-corrosive to skin.

In conclusion, Trans-Isoeugenol does not meet the criteria of corrosivity but should be classified as skin irritant. No key study was selected since the in vitro and in vivo studies performed on trans-Isoeugenol and on Isoeugenol were complementary.

Eye irritation:

A study was identified (Envigo, 2016, Rel.1). This study was performed to assess the ocular irritancy potential of the undiluted test item to the isolated bovine cornea. The study was conducted according to the OECD guideline No. 437 and in compliance with GLP. The quality criteria required for acceptance of results in the test were satisfied. The In Vitro Irritancy Score of the test item and positive control were 39.4 ± 8.2 and 27.4 ± 2.7, respectively, after the 10 -minute exposure period followed by 120 -minute incubation period. Therefore with an IVIS between 3 and 55, it was not possible to conclude on the ocular irritancy potential of test substance in this assay.

However, based on the results of this BCOP and on the skin irritancy potential of the substance as well as its respiratory tract irritancy potential, the substance can be considered to be an ocular irritant.

Therefore, Trans-Isoeugenol does not meet the criteria of corrosivity but should be classified as eye irritant.

Respiratory irritation:

A key study was identified (WIL, 2012). In this repeated dose toxicity study conducted according to the OECD Guideline 412 and in compliance with GLP, test material was administered by inhalation-aerosol to groups of Sprague-Dawley rats (10 animals/sex/dose) at the concentrations of 1, 10 and 100 mg/m3 (0.15, 1.5, and 14.9 ppm, respectively) for 6 hours per day, 5 days per week for 2 weeks (10 total exposures). A concurrent control group was exposed to humidified, filtered air on a comparable regimen. Examinations during the study included: mortality, clinical observation, body weight change, food consumption, laboratory investigations: haematology, blood clinical chemistry, Bronchoalveolar lavage fluid (BALF) clinical pathology and serum and BALF cytokine evaluation, gross pathology, organ weights and histopathology.

 

Portal of entry effects suggestive of irritation were present in the nasal cavity of males and females exposed to test material. Epithelial inflammation and degeneration of the nasal cavity were noted in the 1, 10, and 100 mg/m3 group males and females. A dose-relationship was evident as higher incidences and severity of degeneration and subacute inflammation in males and females affecting transitional epithelium (nasal level II) at the 10 and 100 mg/m3 exposure levels, compared with effects at the 1 mg/m3 exposure. Based on severity and combined incidences for males and females, a similar test substance-relationship was evident for subacute inflammation of the respiratory epithelium at nasal level III. The findings were most prominent at nasal levels II and III and showed reduced incidence and severity in the posterior nasal levels (IV-VI), and were generally more frequent in males than in females at all exposure and nasal cavity levels.

All nasal cavity findings were minimal to mild in severity and would be considered reversible with removal of the irritant. 

Therefore Trans-Isoeugenol should be classified as respiratory tract irritant.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information, Trans-Isoeugenol should be classified as Skin irr. Category 2 (H315: Causes skin irritation), Eye irr. Category 2 (H319: Causes serious eye irritation) and STOT-SE Category 3 (H335: May cause respiratory irritation) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.