Registration Dossier

Administrative data

Description of key information

- Skin irritation: not irritating; OECD TG 439 / EU method B.46; RL1; GLP: relative mean tissue viability after 15 minutes treatment, 42 h observation: 98%. 
- Eye irritation: Category 1 (irreversible effects on the eye) at 30% a.i.; OECD TG 405; RL2; GLP

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-12-15 to 2016-01-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted July 28, 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
recommended by guideline
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEMEpiSkin™ Kit Lot No.: 16-EKIN-002, SkinEthic Laboratories (69007 Lyon, France)EpiSkin™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.The tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS on 12 January 2016. On day of experiment EpiSkin™ tissues were transferred to 12-well plates with maintenance medium and the preincubation phase of the EpiSkin™ tissues started.REMOVAL OF TEST SUBSTANCEAfter the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium.MTT ASSAY:The MTT concentrate was prepared freshly. A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.After the treatment procedure was completed for all tissues the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted 69 hours and 15 min without shaking in the refrigerator.Per each tissue sample 2 × 200 μL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader with 570 ± 1 nm filter. Mean values were calculated from the 2 wells per tissue sample.PREDICTION MODEL / DECISION CRITERIA A test substance is considered irritant in the skin irritation test if:The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.A test substance is considered non-irritant in the in vitro skin irritation test if:The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit): 5 μL of deionised water were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 10 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.NEGATIVE CONTROL- Amount(s) applied (volume or weight): 10 µL deionised waterPOSITIVE CONTROL- Amount(s) applied (volume or weight): 10 μL 5% SDS
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
triplicates
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
test substance
Value:
98
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
32.7% tissue viability
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour. Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour. In a further pre-test the test item was not a MTT reducer

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
- Range of historical values if different from the ones specified in the test guideline:
Positive Control range of viability: 1.7% - 35.4% (19.3% ± 10.0%; n = 232)
Negative Control range of viability (OD): 0.607 – 1.521 (1.004 ± 0.219; n = 232)

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour. Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Group

Absorbance

570 nm

Tissue

1

Absorbance

570 nm

Tissue

2

Absorbance

570 nm

Tissue

3

Mean

Absorbance

of

3

Tissues

Relative

Standard

Deviation

[%]

Rel. Absorbance

[% of

Negative

Control]

Negative

Control

0.872

0.981

0.915

0.922

 

6.0

100.0

Positive

Control

0.307

0.313

0.286

0.302

4.7

32.7

Test item

0.941

0.794

0.976

0.904

10.7

98.0

 

Interpretation of results:
GHS criteria not met
Conclusions:
C16 Alkylamidopropyltrimethylammonium Chloride (100% a.i.) was not irritating in the in vitro skin irritation test under the experimental conditions described in this report.
Executive summary:

In a dermal irritation study performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (adopted July 28, 2015), C16 Alkylamidopropyltrimethylammonium Chloride (100% a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 15 minutes in triplicates. 5 μL of deionised water were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 10 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.

After 15 minutes exposure at room temperature, the tissues were washed with phosphate buffered saline to remove any residual test material. Subsequently the tissue constructs were incubated for 42 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (5% SDS) and negative (deionised water) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 15 minutes treatment with C16 Alkylamidopropyltrimethylammonium Chloride compared to the negative control tissues was 98%. Since the mean relative tissue viability for the test substance was above 50%, C16 Alkylamidopropyltrimethylammonium Chloride is identified to be not irritating.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
February 24, 1987
Deviations:
yes
Remarks:
observation time only 7 d, thus, reversibility cannot finally be judged / test substance was used as 30% solution
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS- Age at study initiation: young adult- Weight at study initiation: Males: 2.175-2.500 kg; Females: 2.250-2.650 kg- Housing: individually in suspended wire bottom stainless steel cages- Diet (e.g. ad libitum): PM1 Rabbit Diet #5321; presented in measured amounts- Water (e.g. ad libitum): municipal water supply, ad libitum- Acclimation period: 5 dENVIRONMENTAL CONDITIONS- Temperature (°C): 72 ± 5°F (= 22°C)- Humidity (%): 30 - 80- Air changes (per hr): 10 - 12- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
other: The untreated left eyes served as controls.
Amount / concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit): 0.1 mL
Duration of treatment / exposure:
3 animals: treated eyes were washed after 30 s, 6 animals: treated eyes were washed after 24 h
Observation period (in vivo):
7 d (the study was terminated on Day 7)
Number of animals or in vitro replicates:
9 (3 washed after 30 s, 6 washed after 24 h)
Details on study design:
REMOVAL OF TEST SUBSTANCE- Washing (if done): Three of the treated eyes (“washed eyes”) were each washed with room temperature deionized water for one minute beginning 30 seconds after treatment. All treated eyes were washed with room temperature deionized water for one minute immediately after recording the 24-hour observation.SCORING SYSTEM: Kay & Calandra ScaleTOOL USED TO ASSESS SCORE: white room lighting (daylight-type fluorescent tubes) + additional source of white light / fluorescein
Irritation parameter:
cornea opacity score
Basis:
animal: #1, #2, #3
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
not reversible
Remarks:
within 7 d
Remarks on result:
positive indication of irritation
Remarks:
eyes washed after 24 h; 24 h unable to score due to severity of chemosis
Irritation parameter:
cornea opacity score
Basis:
animal #4
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not reversible
Remarks:
within 7 d
Remarks on result:
positive indication of irritation
Remarks:
eyes washed after 24 h;24 h unable to score due to severity of chemosis
Irritation parameter:
cornea opacity score
Basis:
animal #5
Time point:
24/48/72 h
Score:
1.5
Max. score:
4
Reversibility:
not reversible
Remarks:
within 7 d
Remarks on result:
positive indication of irritation
Remarks:
eyes washed after 24 h; 24 h unable to score due to severity of chemosis
Irritation parameter:
cornea opacity score
Basis:
animal #6
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not reversible
Remarks:
within 7 d
Remarks on result:
positive indication of irritation
Remarks:
eyes washed after 24 h; 24 h unable to score due to severity of chemosis
Irritation parameter:
iris score
Basis:
animal: #1, #2, #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
other: eyes washed after 24 h; 24 h unable to score due to severity of chemosis
Irritation parameter:
iris score
Basis:
animal: #4
Time point:
24/48/72 h
Score:
0.5
Max. score:
2
Reversibility:
fully reversible within: 4 d
Remarks on result:
other: eyes washed after 24 h; 24 h unable to score due to severity of chemosis; constricted pipils still present at day 7
Irritation parameter:
iris score
Basis:
animal #5
Time point:
24/48/72 h
Score:
0.5
Max. score:
2
Reversibility:
not fully reversible within: 7 d
Remarks on result:
other: eyes washed after 24 h; 24 h unable to score due to severity of chemosis
Irritation parameter:
iris score
Basis:
animal #6
Time point:
24/48/72 h
Score:
0.5
Max. score:
2
Reversibility:
fully reversible within: 4 d
Remarks on result:
other: eyes washed after 24 h; 24 h unable to score due to severity of chemosis
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
not reversible
Remarks:
within 7 d
Remarks on result:
positive indication of irritation
Remarks:
eyes washed after 24 h
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2.7
Max. score:
3
Reversibility:
not reversible
Remarks:
within 7 d
Remarks on result:
positive indication of irritation
Remarks:
eyes washed after 24 h
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
not reversible
Remarks:
within 7 d
Remarks on result:
positive indication of irritation
Remarks:
eyes washed after 24 h
Irritation parameter:
conjunctivae score
Basis:
animal: #4, #5, #6
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
not reversible
Remarks:
within 7 d
Remarks on result:
positive indication of irritation
Remarks:
eyes washed after 24 h
Irritation parameter:
chemosis score
Basis:
animal: #1, #2, #4, #5, #6
Time point:
24/48/72 h
Score:
2.3
Max. score:
4
Reversibility:
not reversible
Remarks:
within 7 d
Remarks on result:
positive indication of irritation
Remarks:
eyes washed after 24 h
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2.7
Max. score:
4
Reversibility:
not reversible
Remarks:
within 7 d
Remarks on result:
positive indication of irritation
Remarks:
eyes washed after 24 h
Irritant / corrosive response data:
Washing after 30 s: Fluorescein staining was observed in all three eyes at 24 hours after treatment and persisted in two eyes through Day 7 after treatment. Corneal opacity was not possible to score after 1 h in 2/3 animals due to severity of chemosis. A slight dulling of normal luster (graded as 0.5) was present in all 3 animals from 24 h after treatment to day 4 or 7, respectively, after treatment. In 1/3 animals corneal opacity, grade1 was observed on day 7. Iris scores were 1 in all three animals 1 h and 24 h after treatment, and zero thereafter. Constricted pupils were observed in 2/3 animals up to day 4. Conjunctial redness grade 2 was present in all three animals after 1 h, advancing to grade 3 in 2/3 animals after 24 h. Grade 1-2 conjunctival redness was observed after 48 h, 72 h and after 4 d, and was still present in 2/3 animals at the end of the observation period of 7 d.Grade 3 chemosis was observed in 3/3 animals at the 1 h observation, grade 2 chemosis in 3/3 animals at the 24 h observation, grade 1-2 chemosis was observed after 48 h, 72 h and after 4 d, and was still present in 2/3 animals at the end of the observation period of 7 d.Grade 3 discharge was present in 3/3 animals after 1 h, receding to grade 2 after 2 h and grade 1 thereafter. Slight discharge (grade 1) was still present on day 7 in 1/3 animals.Washing after 24 h: Fluorescein staining was observed in all six eyes at 48 hours after treatment and persisted in all six eyes through Day 7 after treatment.Corneal opacity and iris were not possible to score after 1 and 24 h in all 6 animals due to severity of chemosis. Corneal opacity, grade 1 (4/6 animals) and 2 (2/6 animals) was observed after 48 h. From day 3 to 7, corneal opacity grade 2 was present in 3/6 animals and grade 1 in 3/6 animals. Iris scores were 0 in all six animals 48 h after treatment, 1 at the 72 h observation in 3/6 animals and was still present (grade 1) in 1/6 animals after 7 d. Constricted pupils were observed in 5/6 animals at the 48 h observation, in 4/6 animals at the 72 h observation, in 3/6 animals on day 4 and was still present in 2/6 animals on day 7.Conjunctial redness grade 2 was present in all three animals after 1 h. Grade 2-3 conjunctival redness was present at the 24, 48, 72 and 4 d observations. Grade 2 conjunctival redness was still observed after 7 d in all six animals. Grade 3 chemosis was present in all six animals at the 1 h and 24 h observations, and in 1/6 animals at the 48 h observation. Grade 2 chemosis was present in 5/6 animals at the 48 h observation, and in all six animals at the 72 h and 4 d observation. Grade 1 (1/6 animals) – 2 (5/6 animals) chemosis was still present in all six animals at the end of the observation period of 7 d.Grade 1 – 3 discharge was observed throughout the study period and was still present at the end of the observation time in 5/6 animals.

Irritant/corrosive response data for each animal at each observation time up to removal of each animal from the test

Washed after 30 s

Score at time point / Reversibility

Cornea

Iris

Conjunctivae

Chemosis

Discharge

 

Max. score: 4

Max. score: 2

Max. score: 3

Max. score: 4

Max. score: 3

60 min

+/f/f

1/1/1

2/2/2

3/3/3

3/3/3

24 h

 +/+/+

1/1/1

2/3/3

2/2/2

2/2/2

48 h

 +/+/+

0c/0c0c/

1/1/2

1/2/1

1/1/1

72 h

 +/+/+

0c/0c/0

1/2/2

1/2/1

1/1/1

4 d

+/+/+

0c/0c/0

1/2/2

1/2/1

0/1/1

7 d

+/1e/0

0/0/0

1/2/0

1/2/0

0/1/0

Average 24h, 48h, 72h

0.5/0.5/0.5

0.3/0.3/0.3 

1.3/2/2.3 

1.3/2/1.3

1.3/1.3/1.3

 

Washed after 24 h

Score at time point / Reversibility

Cornea

Iris

Conjunctivae

Chemosis

Discharge

 

Max. score: 4

Max.score: 2

Max. score: 3

Max. score: 4

Max. score: 3

60 min

f/f/f/f/f/f

f/f/f/f/f/f

2/2/2/2/2/2

3/3/3/3/3/3

3/3/2/3/3/3

24 h

f/f/f/f/f/f

 f/f/f/f/f/f

3/3/2/3/3/3

3/3/3/3/3/3

2/3/2/3/3/3

48 h

1/1/1/2/1/2

 0/0c/0c/0c/0c/0c

3/3/2/3/3/3

2/2/3/2/2/2

2/2/2/2/2/2

72 h

1/1/1/2/2/2

 0/0/0c/1c/1c/1c

3/2/2/3/3/3

2/2/2/2/2/2

2/2/2/2/2/2

4 d

1/1/1/2/2/2

0/0/0c/0c/0c/0

3/2/2/3/2/3

2/2/2/2/2/2

2/1/1/2/2/2

7 d

1/1/1/2/2/2

0/0/0/0c/1c/0

2/2/2/2/2/2

2/2/1/2/2/2

2/1/0/2/2/2

Average 24h, 48h, 72h

1/1/1/2/1.5/2

0/0/0/0.5/0.5/0.5 

3/2.7/2/3/3/3 

2.3/2.3/2.7/2.3/2.3/2.3

2/2.3/2/2.3/2.3/2.3

 

+Slight dulling of normal luster, scored as 0.5

f) unable to score due to severity of chemosis

c) constricted pupils

e) apparent invasion of cornea by blood vessels

 

Fluorescein staining: 

Eyes washed after 30 s

Eyes washed after 24 h

 

24 h

D/D/C

f/f/f/f/f/f

48 h

D/C/C

D/D/D/D/D/D

72 h

D/C/B

D/D/C/D/D/D

4 d

C/B/B

C/C/B/C/C/C

7 d

B/B/0

C/B/A/C/C/B

 

 

Area of cornea involved

No fluorescein staining: 0

One quarter (or less), but not zero: A

Greater than one quarter, but less than half: B

Greater than half, but less than three quarters: C

Greater than three quarters, up to whole area: D

 

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
In this study, C16 Alkylamidopropyltrimethylammonium Chloride has irreversible effects on the eyes (Category 1) based on the effects being not reversible at the end of the observation period of 7 d.
Executive summary:

In a primary eye irritation study according to OECD guideline 405 (February 24, 1987) 0.1 mL of C16 Alkylamidopropyltrimethylammonium Chloride (30% a.i.) was instilled into the conjunctival sac of the right eye of 9 young adult New Zealand White rabbits. The untreated left eyes served as controls. In 3 animals, the treated eyes were rinsedwith room temperature deionized water for one minute beginning 30 seconds after treatment. In the remaining 6 animals, the treated eyes were rinsedwith room temperature deionized water for one minute immediately after recording the 24-hour observation.

Animals were then observed for 7 days.  Irritation was scored by the method of Kay and Calandra.

 

Washing after 30 s:

Fluorescein staining was observed in all three eyes at 24 hours after treatment and persisted in two eyes through Day 7 after treatment.

Corneal opacity was not possible to score after 1 h in 2/3 animals due toseverity of chemosis.A slight dulling of normal luster (graded as 0.5) was present in all 3 animals from 24 h after treatment to day 4 or 7, respectively, after treatment. In 1/3 animals corneal opacity, grade1was observed on day 7.

Iris scores were 1 in all three animals 1 h and 24 h after treatment, and zero thereafter. Constricted pupils were observed in 2/3 animals up to day 4.

Conjunctial redness grade 2 was present in all three animals after 1 h, advancing to grade 3 in 2/3 animals after 24 h. Grade 1-2 conjunctival redness was observed after 48 h, 72 h and after 4 d, and was still present in 2/3 animals at the end of the observation period of 7 d.

Grade 3 chemosis was observed in 3/3 animals at the 1 h observation, grade 2 chemosis in 3/3 animals at the 24 h observation, grade 1-2 chemosis was observed after 48 h, 72 h and after 4 d, and was still present in 2/3 animals at the end of the observation period of 7 d.

Grade 3 discharge was present in 3/3 animals after 1 h, receding to grade 2 after 2 h and grade 1 thereafter. Slight discharge (grade 1) was still present on day 7 in 1/3 animals.

Score at time point / Reversibility

Cornea

Iris

Conjunctivae

Chemosis

Discharge

 

Max. score: 4

Max. score: 2

Max. score: 3

Max. score: 4

Max. score: 3

60 min

+/f/f

1/1/1

2/2/2

3/3/3

3/3/3

24 h

 +/+/+

1/1/1

2/3/3

2/2/2

2/2/2

48 h

 +/+/+

0c/0c0c/

1/1/2

1/2/1

1/1/1

72 h

 +/+/+

0c/0c/0

1/2/2

1/2/1

1/1/1

4 d

+/+/+

0c/0c/0

1/2/2

1/2/1

0/1/1

7 d

+/1e/0

0/0/0

1/2/0

1/2/0

0/1/0

Average 24h, 48h, 72h

0.5/0.5/0.5

0.3/0.3/0.3 

1.3/2/2.3 

1.3/2/1.3

1.3/1.3/1.3

 

Washing after 24 h:

Fluorescein staining was observed in all six eyes at 48 hours after treatment and persisted in all six eyes through Day 7 after treatment.

Corneal opacity and iris were not possible to score after 1 and 24 h in all 6 animals due toseverity of chemosis.

Corneal opacity, grade 1 (4/6 animals) and 2 (2/6 animals) was observed after 48 h. From day 3 to 7, corneal opacity grade 2 was present in 3/6 animals and grade 1 in 3/6 animals.

Iris scores were 0 in all six animals 48 h after treatment, 1 at the 72 h observation in 3/6 animals and was still present (grade 1) in 1/6 animals after 7 d. Constricted pupils were observed in 5/6 animals at the 48 h observation, in 4/6 animals at the 72 h observation, in 3/6 animals on day 4 and was still present in 2/6 animals on day 7.

Conjunctial redness grade 2 was present in all three animals after 1 h. Grade 2-3 conjunctival redness was present at the 24, 48, 72 and 4 d observations. Grade 2 conjunctival redness was still observed after 7 d in all six animals.

Grade 3 chemosis was present in all six animals at the 1 h and 24 h observations, and in 1/6 animals at the 48 h observation.

Grade 2 chemosis was present in 5/6 animals at the 48 h observation, and in all six animals at the 72 h and 4 d observation. Grade 1 (1/6 animals) – 2 (5/6 animals) chemosis was still present in all six animals at the end of the observation period of 7 d.

Grade 1 – 3 discharge was observed throughout the study period and was still present at the end of the observation time in 5/6 animals.

 

Score at time point / Reversibility

Cornea

Iris

Conjunctivae

Chemosis

Discharge

 

Max. score: 4

Max.score: 2

Max. score: 3

Max. score: 4

Max. score: 3

60 min

f/f/f/f/f/f

f/f/f/f/f/f

2/2/2/2/2/2

3/3/3/3/3/3

3/3/2/3/3/3

24 h

f/f/f/f/f/f

 f/f/f/f/f/f

3/3/2/3/3/3

3/3/3/3/3/3

2/3/2/3/3/3

48 h

1/1/1/2/1/2

 0/0c/0c/0c/0c/0c

3/3/2/3/3/3

2/2/3/2/2/2

2/2/2/2/2/2

72 h

1/1/1/2/2/2

 0/0/0c/1c/1c/1c

3/2/2/3/3/3

2/2/2/2/2/2

2/2/2/2/2/2

4 d

1/1/1/2/2/2

0/0/0c/0c/0c/0

3/2/2/3/2/3

2/2/2/2/2/2

2/1/1/2/2/2

7 d

1/1/1/2/2/2

0/0/0/0c/1c/0

2/2/2/2/2/2

2/2/1/2/2/2

2/1/0/2/2/2

Average 24h, 48h, 72h

1/1/1/2/1.5/2

0/0/0/0.5/0.5/0.5 

3/2.7/2/3/3/3 

2.3/2.3/2.7/2.3/2.3/2.3

2/2.3/2/2.3/2.3/2.3

 

Reversibility cannot finally be judged as thestudy was terminated on day 7.

Thus, C16 Alkylamidopropyltrimethylammonium Chloride (30% a.i.), produced irreversible effects to the eye in this study and thus has to be classified as Category 1 (irreversible effects on the eye).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

In a dermal irritation study performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation) (adopted July 28, 2015), C16 Alkylamidopropyltrimethylammonium Chloride (100% a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 15 minutes in triplicates. 5 μL of deionised water were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 10 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.

After 15 minutes exposure at room temperature, the tissues were washed with phosphate buffered saline to remove any residual test material. Subsequently the tissue constructs were incubated for 42 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive (5% SDS) and negative (deionised water) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The relative mean tissue viability obtained after 15 minutes treatment with C16 Alkylamidopropyltrimethylammonium Chloride compared to the negative control tissues was 98%. Since the mean relative tissue viability for the test substance was above 50%, C16 Alkylamidopropyltrimethylammonium Chloride is identified to be not irritating.

 

Data on the source substance are provided to justify the read-across approach for other endpoints:

The source substance Stearic acid 3-(dimethylaminopropyl)amide was not irritating to skin in an in vitro dermal irritation study according to OECD guideline 439. The source substance Cetrimoniumchloride is classified as ...

 

Eye irritation

In a primary eye irritation study according to OECD guideline 405 (February 24, 1987) 0.1 mL of C16 Alkylamidopropyltrimethylammonium Chloride (30% a.i.) was instilled into the conjunctival sac of the right eye of 9 young adult New Zealand White rabbits. The untreated left eyes served as controls. In 3 animals, the treated eyes were rinsedwith room temperature deionized water for one minute beginning 30 seconds after treatment. In the remaining 6 animals, the treated eyes were rinsedwith room temperature deionized water for one minute immediately after recording the 24-hour observation. Animals were then observed for 7 days.  Irritation was scored by the method of Kay and Calandra.

 

Washing after 30 s:

Fluorescein staining was observed in all three eyes at 24 hours after treatment and persisted in two eyes through Day 7 after treatment.

Corneal opacity was not possible to score after 1 h in 2/3 animals due toseverity of chemosis.A slight dulling of normal luster (graded as 0.5) was present in all 3 animals from 24 h after treatment to day 4 or 7, respectively, after treatment. In 1/3 animals corneal opacity, grade1was observed on day 7.

Iris scores were 1 in all three animals 1 h and 24 h after treatment, and zero thereafter. Constricted pupils were observed in 2/3 animals up to day 4.

Conjunctial redness grade 2 was present in all three animals after 1 h, advancing to grade 3 in 2/3 animals after 24 h. Grade 1-2 conjunctival redness was observed after 48 h, 72 h and after 4 d, and was still present in 2/3 animals at the end of the observation period of 7 d.

Grade 3 chemosis was observed in 3/3 animals at the 1 h observation, grade 2 chemosis in 3/3 animals at the 24 h observation, grade 1-2 chemosis was observed after 48 h, 72 h and after 4 d, and was still present in 2/3 animals at the end of the observation period of 7 d.

Grade 3 discharge was present in 3/3 animals after 1 h, receding to grade 2 after 2 h and grade 1 thereafter. Slight discharge (grade 1) was still present on day 7 in 1/3 animals.

 

Washing after 24 h:

Fluorescein staining was observed in all six eyes at 48 hours after treatment and persisted in all six eyes through Day 7 after treatment.

Corneal opacity and iris were not possible to score after 1 and 24 h in all 6 animals due toseverity of chemosis.

Corneal opacity, grade 1 (4/6 animals) and 2 (2/6 animals) was observed after 48 h. From day 3 to 7, corneal opacity grade 2 was present in 3/6 animals and grade 1 in 3/6 animals.

Iris scores were 0 in all six animals 48 h after treatment, 1 at the 72 h observation in 3/6 animals and was still present (grade 1) in 1/6 animals after 7 d. Constricted pupils were observed in 5/6 animals at the 48 h observation, in 4/6 animals at the 72 h observation, in 3/6 animals on day 4 and was still present in 2/6 animals on day 7.

Conjunctial redness grade 2 was present in all three animals after 1 h. Grade 2-3 conjunctival redness was present at the 24, 48, 72 and 4 d observations. Grade 2 conjunctival redness was still observed after 7 d in all six animals.

Grade 3 chemosis was present in all six animals at the 1 h and 24 h observations, and in 1/6 animals at the 48 h observation.

Grade 2 chemosis was present in 5/6 animals at the 48 h observation, and in all six animals at the 72 h and 4 d observation. Grade 1 (1/6 animals) – 2 (5/6 animals) chemosis was still present in all six animals at the end of the observation period of 7 d.

Grade 1 – 3 discharge was observed throughout the study period and was still present at the end of the observation time in 5/6 animals.

Reversibility cannot finally be judged as the study was terminated on day 7.

Thus, C16 Alkylamidopropyltrimethylammonium Chloride (30% a.i.), produced irreversible effects to the eye in this study and thus has to be classified as Category 1 (irreversible effects on the eye).

 

Data on the source substances are provided to justify the read-across approach for other endpoints:

Both source substances Stearic acid 3-(dimethylaminopropyl)amide and Cetrimonium chloride are also classified as Category 1 (irreversible effects on the eye).

 

Respiratory irritation

No data on the respiratory irritation of C16 Alkylamidopropyltrimethylammonium Chloride are available.

 

There are no data gaps for the endpoint irritation/corrosion. No human information is available for this endpoint. However, there is no reason to believe that these results would not be applicable to humans.


Justification for classification or non-classification

Skin irritation

Based on reliable, adequate and relevant data, C16 Alkylamidopropyltrimethylammonium Chloride does not need to be classified for skin irritation according to regulation (EC) 1272/2008.

 

Eye irritation

Based on reliable, adequate and relevant data, C16 Alkylamidopropyltrimethylammonium Chloride has to be classified as Category 1, irreversible effects on the eye according to CLP, EU GHS (Regulation (EC) No 1272/2008) and is assigned the hazard statement H318 and the signal word “Danger”.