Registration Dossier

Administrative data

Description of key information

The substance and/or a structural analogue is irritating to skin and severely irritating to eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
skin corrosion: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Recently conducted guideline study to GLP.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
GLP compliance:
yes (incl. certificate)
Species:
other: reconstructed human epidermis
Strain:
not specified
Details on test animals and environmental conditions:
The purpose of this test is to evaluate the corrosivity potential of the test item using the EPISKIN™ in vitro Reconstructed Human Epidermis (RHE) Model. The EPISKIN™ model is able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied, and is also able to distinguish between known R35 (UN packing group I) and R34 (UN packing group II & III) test items.
Type of coverage:
open
Preparation of test site:
other: none: reconstructed human epidermis
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
50 µL
Duration of treatment / exposure:
3, 60 and 240 minutes
Details on study design:
The procedure followed is based on the recommended EpiSkin™ Skin Corrosivity Test protocol INVITTOX N° 118(5) The test item is applied topically to the stratum corneum surface, at the air interface, so that undiluted and/or end use dilutions can be tested
directly. The test is based on the experience that corrosive chemicals are cytotoxic after a short term exposure to the EPISKIN™ model. Corrosive chemicals are able to penetrate the stratum corneum and are sufficiently cytotoxic to cause cell death in the underlying cell layers. Toxicity is determined by the metabolic conversion of the vital dye MTT to formazan by viable cells in the test item treated cultures relative to the negative control.
Irritation / corrosion parameter:
other: percentage relative viability
Run / experiment:
240 minutes
Value:
ca. 106.7
Remarks on result:
other: MTT reduction in the test item treated issues relative to negative control tissues
Irritation / corrosion parameter:
other: percentage relative viability
Run / experiment:
60 minutes
Value:
ca. 125.5
Remarks on result:
other: MTT reduction in the test item treated tissues relative to negative control tissues
Irritation / corrosion parameter:
other: percentage relative viability
Run / experiment:
3 minutes
Value:
ca. 132.7
Remarks on result:
other: MTT reduction in the test item treated tissues relative to negative control tissues

Table 1 - Mean 0D540 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

 Item  Exposure Period  Mean OD540 of duplicate tissues Relative mean viability (%) 
 Negative Control Item 240 minutes  0.208  100* 
 Positive Control Item 240 minutes  0.037  17.8 
 Test Item       240 minutes  0.222  106.7
60 minutes  0.261  125.5 
3 minutes  0.276  132.7 
* The mean viability of the negative control tissues is set at 100%
Interpretation of results:
other: not corrosive
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The substance was non-corrosive to the skin.
Executive summary:

The corrosivity potential of CAS# 10595 -49 -0 was evaluated using the EPISKIN™ in vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes. This method was designed to be compatible with OECD Guideline 431 and Method 8.40 of (EC) No. 440/2008.

At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-Ioading. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 540 nm (OD540).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results: The relative mean viability of the test item treated tissues was:

240 minutes exposure: 106.7 %

60 minutes exposure: 125.5 %

3 minutes exposure: 132.7 %

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion: The test item was considered to be Non-Corrosive to the skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Recently conducted guideline study to GLP
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes
Species:
other: EPISKIN reconstructed human epidermis
Strain:
not specified
Details on test animals and environmental conditions:
The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

EPISKINTM MODEL KIT
Supplier : SkinEthic Laboratories, Nice, France
Date received : 05 June 2012
Type of coverage:
open
Preparation of test site:
other: none: reconstructed human epidermis
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
As supplied: 40% solution in water
Duration of treatment / exposure:
15 minutes
Observation period:
42 hours
Number of animals:
triplicate tissues were treated with the test item
Details on study design:
The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treatedtissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hour post-exposure incubation period is also determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.
Irritation / corrosion parameter:
other: other: percentage mean viability
Value:
40.3
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 min. Remarks: MTT reduction in the test item treated tissues relative to negative control tissues.

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Mean OD540 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item
 Item  OD540 oftissues  Mean OD540of triplicatetissues  ± SD ofOD540  Relativeindividualtissueviability (%)  Relativemeanviability (%)  ± SD ofRelativemeanviability (%)
 Negative      Control Item  0.733  0.856        0.107        85.6  100*        12.5      
 0.928  108.4
 0.906  105.8
 Positive      Control Item  0.032  0.036        0.005        3.7  4.2        0.6      
 0.036  4.2
 0.041  4.8
 Test Item        0.326  0.345        0.030        38.1  40.3        3.5      
 0.329  38.4
 0.379  44.3

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

Interpretation of results:
irritating
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The substance was irritating to skin in an in vitro assay.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro eye irritation study does not need to be conducted because adequate data from an in vivo eye irritation study are available
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to the guideline study with acceptable restrictions.
Qualifier:
according to
Guideline:
other: USA Interagency Regulatory Liaison Group (IRLG, January 1981)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
GLP compliance:
not specified
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: H. Fortkamp, 4540 Lengerich, Germany
- Weight at study initiation: 2.20 - 2.35 kg
- Age: 11-17 weeks
- Housing: single caging, metal cages
- Diet: ad libitum, Rabbit Diet, Ssniff Versuchstier Diäten GmbH, 4770 Soest/Westfalen, Germany
- Water: ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 40-70
- Air changes (per hr): 10 per hour
- Photoperiod: 12 hours daily
Vehicle:
unchanged (no vehicle)
Controls:
other: Each animal served as its one control. The test article was introduced into the conjunctival sac of one eye, the untreated eye serving as a control
Amount / concentration applied:
0.1 ml of the test material was instilled into the conjunctival sac of the left eye while the right eye served as control.
Duration of treatment / exposure:
not rinsed
Observation period (in vivo):
Readings of ocular reactions were made 1-2 h, 24 h, 48 h, 72 h, 4 days, 7 days after treatment.
Number of animals or in vitro replicates:
6
Details on study design:
PREPARATION OF ANIMALS
- 24 h before treatment eyes were examined for corneal lesions after application of one drop of fluorescein-sodium solution (2%)

APPLICATION OF TEST SUBSTANCE
- 0.1 mL test substance was instilled into the conjunctival sac of the left eye. The lids were then held together for 1-2 seconds. The untreated right eye served as control. Because one of test animals showed a moderate pain reaction (score 3; see table 1), all remaining rabbits received a local anaesthetic in both eyes shortly before the application of the test substance.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): the treated eye was not rinsed after treatment

SCORING SYSTEM (modified Draize system): the ocular reactions were assessed using the following numerical scoring system (see table 2)
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
#1, #3, #5, #6
Time point:
other: 24 h, 48 h, 72 h
Score:
1
Max. score:
4
Reversibility:
not reversible
Remarks:
within 7 days
Remarks on result:
other: Pannus
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
#2, #4
Time point:
other: 24 h, 48 h, 72 h
Score:
0
Max. score:
4
Reversibility:
other: delayed opacity formation after 7 days
Remarks on result:
other: Pannus
Irritation parameter:
iris score
Basis:
mean
Remarks:
#1, #2, #3, #4, #5, #6
Time point:
other: 24 h, 48 h, 72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
#2, #3, #4, #5, #6
Time point:
other: 24 h, 48 h, 72 h
Score:
2
Max. score:
3
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
#1
Time point:
other: 24 h, 48 h, 72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
#1, #2, #3, #4, #5, #6
Time point:
other: 24 h, 48 h, 72 h
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
#1
Time point:
other: 24 h, 48 h, 72 h
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritant / corrosive response data:
The most remarkable reaction to the instillation of the test substance was a not discernible iris through cornea opacity in one animal on day 7 after the application. The investigation of the remaining animals showed Pannus formations also on day 7 after the application. As the post exposure observation period was less then 21 days reversibility of effects could not be determined.
Other effects:
no data

Table 3: Irritant/corrosive response for each animal at each observation time

Score at time point / Reversibility

Cornea opacity

Cornea area

Iris

Conjunctivae

Redness

Conjunctivae

Chemosis

Conjunctivae

discharge

Max. score: 4

Max. score: 4

Max. score: 2

Max. score: 3

Max. score: 4

Max. score: 3

1-2 hours

1/0/1/0/1/1

2/0/2/0/2/2

1/1/1/1/1/1

2/2/2/2/2/2

2/2/2/2/2/2

1/1/1/1/1/1

1 day

1/0/1/0/1/1

2/0/2/0/2/2

1/1/1/1/1/1

2/2/2/2/2/2

2/2/2/2/2/2

2/2/2/2/2/2

2 days

1/0/1/0/1/1

2/0/2/0/2/2

1/1/1/1/1/1

2/2/2/2/2/2

2/2/2/2/2/2

2/2/2/2/2/2

3 days

1/0/1/0/1/1

2/0/2/0/2/2

1/1/1/1/1/1

2/2/2/2/2/2

2/2/2/2/2/2

2/2/2/2/2/2

4 days

1/0/1/0/1/1

2/0/2/0/2/2

1/1/1/1/1/1

1/2/2/1/2/2

1/2/2/1/2/2

1/2/2/1/2/2

7 days

1/2/2/1/3/4

4P/4P/4P/4P/4P/4P

0/0/0/0/0/*

0/1/1/1/2/2

0/1/1/1/2/2

0/1/1/0/1/2

Average 24h, 48h, 72h

1/0/1/0/1/1

2/0/2/0/2/2

1/1/1/1/1/1

2/2/2/2/2/2

2/2/2/2/2/2

2/2/2/2/2/2

Reversibility

n./n./n./n./n./n.

n./n./n./n./n./n.

c./c./c./c./c./*

c./n.c./n.c./n.c./n./n.

c./n.c./n.c./n.c./n./n.

c./n.c./n.c./c./n.c./n.

 

P = Pannus,

* Evaluation is not possible because of corneal opacity

c. = completely reversible

n.c. = not completely reversible

n.= not reversible

Reactions observed at 2 hours reading after the application

- Clear redness and chemosis of the conjunctiva (score 2)

- Light ocular secretion production (score 1)

- Isolated small diffuse areas in more as a quarter of a cornea (score 1) observed in 4/6 rabbits.

 

Reactions observed within 1 – 7 days after the application

- Clear redness and chemosis of the conjunctiva (score 2), decreased in 4 animals by the end of the study.

- Moderate pericorneal Hyperaemia of iris (score 1), decreased within the 7 days after the treatment

- Isolated small diffuse areas in more as a quarter of a cornea (score 1) observed in 4/6 rabbits turned into the corneal opacity in all animals by the end of the study.

- Conjunctivae discharge with moistening of the lids and hairs just adjacent to the lids (score 2), decreased in 5/6 of the rabbits by the end of the study.

Table 4: Assessment of results (mean values)

Days after treatment

Mean values

Conjunctivae (max. 20)

Iris (max. 10)

Cornea (max. 80)

Mean total score (max. 110)

0 (2 h)

10

5

6.7

21.7

1

12

5

6.7

23.7

2

12

5

6.7

23.7

3

12

5

6.7

23.7

4

10

5

6.7

21.7

7

6.3

0*

43.3

49.6

*Evaluation is not possible because of corneal opacity

 

Interpretation of results:
Category 1 (irreversible effects on the eye)
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test material caused serious damage to eyes in this study
Executive summary:

A primary eye irritation study, performed according to the test plan P 4/152, 3-rd revision, refers to the recommended guidelines of the USA Interagency Regulatory Liaison Group (IRLG, January, 1981). Study performance is comparable to the guideline study.

0.1 ml of the substance (48 % a.i) undiluted were instilled into the conjunctival sac of one eye of 6 young female adult New Zealand White rabbits. Eyes were not washed. Animals then were observed for 7 days. Irritation was scored by the method of Draize.

Calculated mean scores following grading at 24, 48 and 72 hours after installation of the test material for effects on the cornea opacity are 1 for 4 of 6 animals and 0 for 2 of 6 animals. Mean scores for effects on the conjunctiva (redness) and chemosis were 2 for all animals; the effects decreased in 3 of 6 animals were reversible in one animal within 7 days. Mean scores for effects on the iris were 1 for all animals and were fully reversible within 7 days.

 

As the most remarkable reaction to the instillation of the test substance was cornea opacity (score 4) in one of 6 animals and pannus in all animals at day 7 after application. As the post exposure observation period was less then 21 days reversibility of effects could not be determined.

 

In this study, the substance (48 % a.i) induced serious damage to eyes.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: The study was conducted prior to OECD guideline and GLP. It is only reported in very limited detail.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
Only 10 mg was instilled into the eye
GLP compliance:
no
Species:
rabbit
Strain:
other: Albino
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
10 mg
Duration of treatment / exposure:
Not applicable
Observation period (in vivo):
7 days
Number of animals or in vitro replicates:
5 males
Remarks on result:
other: The eye scores were not reported in the study report.
Irritant / corrosive response data:
The eye scores were not reported in the study report.
Other effects:
The substance produced moderate to severe conjunctivitis and chemosis within 24 hours in all animals. Mild corneal opacity and severe pannus developed at 1 week in 3/5 animals. Mild iritis appeared at 4 hours in 3/5 animals however gradually dissapeared during the week.
Interpretation of results:
irritating
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The substance gave rise to significant irritatation to the rabbit eye after dosing of 10mg.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Based on two reliable in vitro studies, a structural analogue (3-(dodecanoylamino)-N,N,N-trimethylpropan-1-aminium methyl sulfate; 10595-49-0) has been shown to be irritating to skin but not corrosive. Given the in vitro positive response for skin irritation, there is no scientific justification for proceeding with an in vivo skin irritation study.

In a reliable in vivo eye irritation study the substance was tested as a 48% solution and gave a severe eye response. In a second unreliable study on a structurally related substance (3-(dodecanoylamino)-N,N,N-trimethylpropan-1-aminium methyl sulfate; 10595-49-0), dosing of only 10mg gave rise to significant irritation.


Justification for selection of skin irritation / corrosion endpoint:
This is a Klimisch 1 study (or Klimisch 2 allowing for read-across) which demonstrates skin irritation in vitro.

Justification for selection of eye irritation endpoint:
This study is Klimisch 2 whereas the other available study is Klimisch 3.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: corrosive

Justification for classification or non-classification

Given the observed in vitro skin responses (irritant but not corrosive) the substance is classified as Skin irritant, Category 2.

Given the severe response observed in an in vivo eye irritation study, the substance will be classified as an Eye Irritant, Category 1.