Registration Dossier

Administrative data

Description of key information

Skin Irritation

0.1% aqueous test chemical was irritating to human skin.

Eye Irritation

Four hours after instillation of the test chemical, mild conjunctival hyperemia.  At day 1, eyes treated with the test chemical all returned to normal aspects without significant differences.

The Draize scores after 4 hours of instillation were 3±0.6.

Based on the scores and observations, 0.002% test chemical solution was considered to be irritating to rabbit eyes.

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:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
data is from peer reviewed journals
Qualifier:
according to guideline
Guideline:
other: Patch tests with the European standard series (ICDRG)
Principles of method if other than guideline:
Patch test were performed to determine the irritation and allergenic potential of the test chemical
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material (as cited in study report): Cetalkonium chloride
- Molecular formula : C25H46ClN
- Molecular weight : 396.098 g/mole
- Substance type: Organic
- Physical state: solid
Species:
other: humans
Strain:
not specified
Details on test animals or test system and environmental conditions:
- Sex: Male
- Source: Department of Dermatology, Free University Academic Hospital, Netherlands
- Age at study initiation: 28-year-old
Type of coverage:
occlusive
Preparation of test site:
other: intact skin
Vehicle:
water
Controls:
not specified
Amount / concentration applied:
0.1%
Duration of treatment / exposure:
72 hours
Observation period:
48 and 72 hours
Number of animals:
1
Details on study design:
No data available
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
72 h
Reversibility:
not specified
Remarks on result:
positive indication of irritation
Irritant / corrosive response data:
Signs of irritation observed
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
0.1% aqueous test chemical was irritating to human skin.
Executive summary:

This study reports of case of allergic dermal reactions observed in humans after exposure to a hair conditioner.

A 28-year-old male had recurrent itchy dermatitis on the left side of the chest and adjacent area of the arm, where his wife usually rested her head when sleeping. He had noted that the eruption always developed after his wife had used a hair conditioner.

Patch tests with the European standard sense (ICDRG) and the conditioner (tested undiluted) showed a positive reaction only to the conditioner. Later its ingredients and additional quaternary ammonium salts were also tested separately.

 

0.1% aqueous test chemical was irritating to human skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
data is from peer reviewed journals
Qualifier:
according to guideline
Guideline:
other: Draize test
Principles of method if other than guideline:
Draize test was performed to assess the extent of ocular damage caused to rabbits eyes when exposed to the test chemical
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material (as cited in study report): Cetalkonium chloride
- Molecular formula : C25H46ClN
- Molecular weight : 396.098 g/mole
- Substance type: Organic
- Physical state: Solid
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Sex: Male
- Weight at study initiation: 2-3 kgs
Vehicle:
other: PBS
Controls:
yes, concurrent vehicle
Amount / concentration applied:
50 microliters of PBS containing 0.002% Solution
Duration of treatment / exposure:
15 times at 5 minute intervals
Observation period (in vivo):
4 hours, 1 day and 4 days
Duration of post- treatment incubation (in vitro):
no data available
Number of animals or in vitro replicates:
seven rabbits – 5 for study and 2 were sacrificed on day 1 for immunohistopathological observations
Details on study design:
Scoring: The maximum possible total score was 110 (conjunctiva=20, cornea=80, iris=10).
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 4 hours
Score:
3
Reversibility:
fully reversible within: 1 day
Remarks on result:
positive indication of irritation
Irritant / corrosive response data:
Four hours after instillation of the test chemical, mild conjunctival hyperemia. At day 1, eyes treated with Cetalkonium chloride all returned to normal aspects without significant differences.
Mild conjunctival irritation was observed in all rabbits
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Four hours after instillation of the test chemical, mild conjunctival hyperemia. At day 1, eyes treated with the test chemical all returned to normal aspects without significant differences.
The Draize scores after 4 hours of instillation were 3±0.6.
Based on the scores and observations, 0.002% test chemical solution was considered to be irritating to rabbit eyes.
Executive summary:

Draize test was performed to assess the extent of ocular damage caused to rabbits eyes when exposed to the test chemical.

 50 microliters of PBS containing 0.002% test chemical Solution was applied to the eyes of seven male New Zealand White rabbits15 times at 5-min intervals.The first instillation was chosen as time zero (T0). During the instillations, the time when conjunctival redness appeared was recorded. At H4, D1, and D4, the eyes were examined using slit lamp microscopy for ocular irritation and scored according to a weighted scale for grading the severity of ocular lesions modified from the Draize Test. The degree of redness, swelling (chemosis), and tearing of the conjunctiva; the degree and area of cornea opacity; and the increased.The maximum possible total score was 110 (conjunctiva=20, cornea=80, iris=10).

Four hours after instillation of the test chemical, mild conjunctival hyperemia. At day 1, eyes treated with test chemical all returned to normal aspects without significant differences.

The Draize scores after 4 hours of instillation were3±0.6.

Based on the scores and observations, 0.002% test chemical solution was considered to be irritating to rabbit eyes.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation:

Various studies have been summarized to ascertain the degree of dermal irritation caused by test chemical. These studies include in vivo studies in rabbits, mice, humans for the target chemical along with its structurally similar read across chemicals.

The dermal irritation potential of test chemical was assessed in humans.150 micrograms of test chemical was applied to human skin for 3 days and observed for effects. Mild irritation was observed after 3 days of exposure to human skin. Hence, test chemical can be considered to be irritating to skin.

This is supported by the experimental study which reports of case of allergic dermal reactions observed in humans after exposure to a hair conditioner. A 28-year-old male had recurrent itchy dermatitis on the left side of the chest and adjacent area of the arm, where his wife usually rested her head when sleeping. He had noted that the eruption always developed after his wife had used a hair conditioner.

Patch tests with the European standard sense (ICDRG) and the conditioner (tested undiluted) showed a positive reaction only to the conditioner. Later its ingredients and additional quaternary ammonium salts were also tested separately. 0.1% aqueous Test chemical was irritating to human skin.

These studies are supported by the results of dermal irritation study performed to assess the irritation potential of the structurally similar chemical. 500 mg of undiluted test chemical was applied to the intact and abraded skin of each rabbit and exposed for 4 hours. The test animals were observed for dermal reactions.

The animals showed strong skin irritating reactions caused due to the test chemical. Hence, the test chemical was considered to be severely irritating to the rabbits’ skin.

The above studies are further supported by the experimental study which reports of case of allergic dermal reactions observed in humans after exposure to a hair conditioner. A 28-year-old male had recurrent itchy dermatitis on the left side of the chest and adjacent area of the arm, where his wife usually rested her head when sleeping. He had noted that the eruption always developed after his wife had used a hair conditioner. A patch test was conducted on a 28-year-old male to determine the test chemical according to the European standard series (ICDRG) after he had recurrent itchy dermatitis on the left side of the chest and adjacent area of the arm at concentration of 0.1% for 48 and 72-hrs. Of 23 controls tested with 0.1% aq. test chemical, 16 were negative, 6 had a? +, and 1 a+ reaction. A positive reaction was observed to 0.1% aq. test chemical at 48 h (+) and 72 h (+ +). Therefore, the test chemical was considered to be irritating to the human skin.

These studies are also supported by the results of another dermal irritation study performed to assess the irritation potential of the structurally similar chemical in mice. Single dermal applications of 0.05 mL of 13 or 50% aqueous solutions were applied to the skin of 48 mice. The treated mice were observed for signs of irritation(duration of observation not mentioned).

Single dermal applications of 0.05 mL of 13 or 50% aqueous solutions have respectively caused death of 9 of 48 and 20 of 48 mice. The remaining mice developed skin inflammation and necrosis, and alopecia.

Hence, the test chemical can be considered to corrosive to skin.

Based on the available studies for the target as well its read across chemicals and applying the weight of evidence approach, test chemical can be considered to be irritating to skin. Since Test chemical was applied to the skin as a solution in suitable solvent in very low concentrations, hence there is slight possibility that it can be corrosive to the skin at higher concentrations and when tested in undiluted form. Comparing the above annotations with criteria in CLP regulation, test chemical can be classified under the category “Category 1”.

Eye Irritation:

Various studies have been summarized to ascertain the extent of ocular damage caused by test chemical. These studies include in vivo studies in rabbits, humans, guinea pigs for the target chemical and its structurally similar chemical along with in vitro studies for the target chemical.

Draize test was performed to assess the extent of ocular damage caused to rabbit’s eyes when exposed to test chemical. 50 microliters of PBS containing 0.002% Test chemical Solution was applied to the eyes of seven male New Zealand White rabbits 15 times at 5-min intervals. The first instillation was chosen as time zero (T0). During the instillations, the time when conjunctival redness appeared was recorded. At H4, D1, and D4, the eyes were examined using slit lamp microscopy for ocular irritation and scored according to a weighted scale for grading the severity of ocular lesions modified from the Draize Test. The degree of redness, swelling (chemosis), and tearing of the conjunctiva; the degree and area of cornea opacity were evaluated and scored. The maximum possible total score was 110 (conjunctiva=20, cornea=80, iris=10).

Four hours after instillation of the test chemical, mild conjunctival hyperemia. At day 1, eyes treated with Test chemical all returned to normal aspects without significant differences.

The Draize scores after 4 hours of instillation were 3±0.6.

Based on the scores and observations, 0.002% Test chemical solution was considered to be irritating to rabbit eyes.

This is supported by the experimental study conducted using 0.002% CBAC solution.

0.002% CBAC solution (in phosphate buffered saline)was applied 15 times at intervals of 5 minutes and observed for signs of irritation.

Slight conjunctival irritation was observed in all rabbits. Hence, test chemical was considered to be irritating to rabbit eyes.

The above results are supported by the experimental study performed in rabbits. 150 mg of the test chemical was instilled in the eyes of rabbits and observed for signs of irritation. Mild conjunctival irritation was observed in all rabbits.

Hence, test chemical can be considered to be irritating to rabbit eyes.

These results are further supported by the experimental study conducted in guinea pigs.500 mg of the test chemical was instilled in the eyes of guinea pigs and observed for signs of irritation.

Moderate irritation was observed in all the guinea pigs. Hence, test chemical can be considered to be irritating to eyes.

The above in vivo results are further supported by in vitro experimental studies performed on various cell lines to determine the degree of ocular damage caused by Test chemical.

The human corneal epithelial cell (HCE-2) culture line was used to study cellular morphology, cytotoxicity and inflammatory responses after exposure to Test chemical (C16, 0.0002%). Human corneal epithelial cells (HCE-2) were obtained from American Type Culture Collection (ATCC, Manassas, VA).Test chemical(0.0002%), normal cell culture medium served as treatment control. Cell viability was determined by measuring the release of lactate dehydrogenase (LDH) and mitochondrial dehydrogenase activity was evaluated using 3-(4,5 -dimethyldiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. The possible induction of apoptosis was analyzed by measuring the activity of caspase-3, and Cell Counting Kit-8 (CCK-8) was used to evaluate the number of viable cells after the exposure to test compounds. Furthermore, the tendency of the test compounds to produce inflammatory reaction was determined by analyzing the production of pro- inflammatory cytokines IL-6 and IL-8. The mitochondrial dehydrogenase activity of the cells was evaluated using MTT analysis. Test chemical did not show any significant difference when compared to control cells.In order to get a broader view on the cellular viability, Cell Counting Kit-8 (CCK-8) was also used. Color intensity of the WST-8 formazan was proportional to the number of viable cells. The cell number/viability increased with Test chemical after 15 min exposure. In addition to positive cell viability tests, the possible induction of apoptosis was determined by measuring the activity of caspase-3. There was an increasing trend in the caspase-3 activity in all groups but the differences were not significant in any time points evaluated.

For analyzing the tendency of test compounds to induce inflammation in HCE-2 cells, the production of pro-inflammatory cytokines IL-6 and IL-8 from the cell culture medium samples was measured. After 15 min of exposure, the differences in the expression levels of IL-8 were not significant between the control group and Test chemical group.

On the basis of the above findings, Test chemical can be considered to be irritating in the HCE assay.

This in vitro result is supported by the study conducted to evaluate acute corneal epithelial toxicity induced by test chemical using an in vivo corneal TER measurement system. Corneal transepithelial electrical resistance (TER) was measured in living Japanese white rabbits by 2 Ag/AgCl electrodes placed in the anterior aqueous chamber and on the cornea.TER changes were evaluated after a 60 second exposure to 0.1 ml of 0.0025%, 0.005%, and 0.01% test chemical in rabbit corneal epithelial cell growth medium (RCGM) or Hank balanced salt Ca2+ and Mg2+ free solution (HBSS). The corneal exposure to HBSS served as control.The TER of the cornea of normal live rabbits was 821.8 6 36.6 Ohm-cm2 (43 eyes). The results of the TER measurement were expressed as the mean ± standard error of at least 3 experiments. Statistical comparisons were performed by analysis of variance followed by Scheffe test for the TER measurement.

0.0025% test chemical did not induce any significant changes in the TER, exposure to 0.005% test chemical led to decrease of 61.1% in the TER value and exposure to 0.01% test chemical led to decrease of 43.7% in the TER value.

Test chemical exhibited moderate corneal barrier dysfunction. Hence, test chemical can be considered to be irritating to rabbit eyes.

The above results of the in vitro studies are ably supported by the a study performed on the normal rabbit corneal epithelial cells using a WST-1 assay to determine the cytotoxicity of test chemical.

The normal rabbit corneal epithelial (NRCE) cells at the second passage were obtained from Kurabo, Co, Ltd. The NRCE cells were maintained in RCGM, which was supplemented with 5 mg/mL of insulin, 10 ng/mL of epidermal growth factor, 0.5 mg/mL of hydrocortisone, 50 mg/mL of gentamicin, 50 ng/mL of amphotericin B, and 0.4% bovine pituitary extractives reagent. The cells were grown at 37 deg C in a humidified atmosphere with 5% CO2. The stock solutions of WST-1 (5.5 mM) and 1-methoxy PMS (2 mM) were prepared in sterilized phosphate-buffered saline.

Just before each of the experiments, the mixture was prepared by combining 0.9 mL of WST-1 solution and 0.1 mL of 1-methoxy PMS solution. NRCE cells were plated at 3*103cells per well in 96-well microtiter plates (Becton-Dickinson, Franklin, NJ). At 6 days after plating, the growth medium was replaced with 100 mL of the test solutions for 60 seconds. The cells were washed with fresh growth medium, with 100 mL of fresh growth medium and 10 mL of WST-1 mixture solution, and then added to each well. After incubation of the cells for 3 hours at 37 deg C, a microplate reader (Thermo Fisher Scientific, Inc, MA) was used to measure the absorbance at a wavelength of 450 nm, with a reference wavelength of 630 nm.

Test chemical showed a concentration-dependent cytotoxicity as compared with the untreated cells (control).

The mean cell viabilities of 0.002%, 0.005% and 0.01% test chemical were 55%, 40%, 15% respectively. Test chemical was comparatively less cytotoxic compared to other benzalkonium chlorides.

These in vivo and in vitro results are also confirmed by the Draize study performed on rabbits to assess the irritation potential of the structurally similar chemical.

10% solution of the test chemical was instilled in the eyes of 43 rabbits and observed for signs of irritation till 21 days. The reactions observed were scored according to Draize method. Mean scores are calculated for each animal from gradings at 24, 48, and 72 h after instillation of the test chemical and these “severity scores” are then used to determine the classification of the test chemical.

The corneal opacity, iris and conjunctival irritation persisted till day 21 in all the tested rabbits. The Corneal opacity score was 4 between day 1 to day 21. Based on these observations, the test chemical was considered to highly irritating to rabbit eyes, was classified under the category “Category 1”.

Based on the available studies for the target as well its read across chemicals, test chemical can be considered to be irritating to eyes. Since Test chemical was applied to the conjunctival sac as a solution in suitable solvent in very low concentrations, hence there is slight possibility that it can cause damage to the eyes at higher concentrations and when tested in undiluted form. Comparing the above annotations with criteria in CLP regulation, test chemical can be classified under the category “Category 1”.

Justification for classification or non-classification

Available studies for test chemical indicate a strong possibility of causing irritation/corrosion to eyes and skin.

Hence, test chemical can be considered to be irritating to skin and eyes and it can be classified under the category "Category 1" as per CLP regulation.