Registration Dossier

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Administrative data

Description of key information

Both a primary dermal irritation study in rabbits (20% formulation) and human patch test with registered

substance (purity not specified) indicated that there was no irritation potential. Read across substance

CAS 37294-49-8 (Butanedioic acid, sulfo-, 1,4-diisodecyl ester, sodium salt ) containing 50% active

ingredient was demonstrated not to be a skin irritant based on primary irritation score of 0.375/4 and

reversible findings within 72h. Further weight of evidence was provided by read across substance

CAS 90268-36-3 (Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts) containing >95%

active ingredient which was also predicted to be non-corrosive and non-irritant to skin based on the

three-dimensional EST 1000 human skin model: viability values were 78.8% and 74.9% in the corrosivity

test and 98.3% in the irritation test (all >50% threshold values), therefore it can be considered to be non-irritant

to human skin. Nevertheless a subgroup classification CLP category 2 was proposed for skin irritation, with

application of concentration limit of 20% for the registered substance for non-classification.
For eye irritation, supporting studies for the registered substance, including a rabbit study (5% formulation)

and an in vitro study (unspecified concentration) already indicated irritation. However from a key study with

read across substance CAS 37294-49-8 (Butanedioic acid, sulfo-, 1,4-diisodecyl ester, sodium salt)

containing 50% active ingredient, mean 24 -48h scores were 20.6/80 for cornea, 1.9/10 for iris and 4.1/20

for conjuctiva irritation,, with 6/6 eyes positive for corneal opacity and 3/6 with iritis. As observations were

only performed up to 72 hours, reversibility could not be scored, and worst case classification CLP class 1

was proposed, with application for the registered substance of concentration limits of 20% or less for CLP

category 2 classification, and 5% or less for non-classification.

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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
See attached read-across justification
Reason / purpose for cross-reference:
read-across source
Irritation parameter:
other: primary dermal irritation score
Basis:
mean
Time point:
other: 24h, 72h
Score:
0.375
Max. score:
4
Reversibility:
not specified
Remarks on result:
other: intact & abraded
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
not specified
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
not specified
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
not specified
Irritation parameter:
erythema score
Basis:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
not specified
Irritation parameter:
erythema score
Basis:
animal #5
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
not specified
Irritation parameter:
erythema score
Basis:
animal #6
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
not specified
Irritant / corrosive response data:
See table

Table 1. Rabbit skin irritation after 0.5mL of [Trade name] under covered patch (FHSA procedure)

Time,

Hours

Reaction

Rabbit Number, Value

Mean Value

1

2

3

4

5

6

 

ERYTHEMA

 

 

 

 

 

 

 

24

Intact

0

0

0

0

2

0

0.33

72

Intact

0

0

0

0

0

0

0.0

24

Abraded

0

2

0

0

2

2

1.0

72

Abraded

0

1

0

0

0

0

0.17

 

EDEMA

 

 

 

 

 

 

 

24

Intact

0

0

0

0

0

0

0.0

72

Intact

0

0

0

0

0

0

0.0

24

Abraded

0

0

0

0

0

0

0.0

72

Abraded

0

0

0

0

0

0

0.0

Interpretation of results:
GHS criteria not met
Conclusions:
The read-across test item containing +-50% active ingredient was not an irritant to the rabbit skin.
Executive summary:

6 Male albino rabbits underwent a covered application to the clipped intact and abraded skin of 0.5 mL read-across test item containing +-50% active ingredient. Scoring according to Draize et al. (1944) demonstrated a primary irritation score of 0.375, based on erythema which was reversible wihtin 72 hours. It was concluded that the read-across test item was not irritating to the rabbit skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
See attached read-across justification
Reason / purpose for cross-reference:
read-across source
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
test group
Value:
98.3
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
positive control group
Value:
1.2
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: Not provided.
- Direct-MTT reduction: Not applicable.
- Colour interference with MTT: Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Each batch of the epidermal model used meets defined production release criteria, set by the supplier, among which those for viability and for barrier function are the most relevant (MTT, 2 hours Triton X-100: target > 50%). The barrier properties of the tissues were verified by the supplier.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. For each assay using valid batches, tissues treated with the negative control exhibit OD reflecting the quality of the tissue that followed all shipment and receipt steps and all the irritation protocol process. The OD values of controls should not be below historical established lower boundaries. SD of 0.067 of the control is within the one sided 95% tolerance interval of standard deviations calculated from historical data.
- Acceptance criteria met for positive control: Yes. Tissues treated with the positive control, i.e.5% aqueous SDS, should reflect the sensitivity retained by tissues and their ability to respond to an irritant substance in the conditions of each individual assay (e.g. viability ≤ 50% for the validated method). Mean tissue viability of positive control was 1.2%.
- Acceptance criteria met for variability between replicate measurements: Standard deviations should be within the one sided 95% tolerance interval of standard deviations calculated from historical data. The 95% tolerance interval of standard deviations of historical control data of the year 2012 was calculated as 0.024 - 0.067 (n = 17). In this study there is a slight deviation from the criteria as given in the guideline as the SD of 0.133 of the test item is outside this range, while the SD of 0.067 of the control is within this range. The study director regards this deviation as negligible due to the fact that the test item revealed a clear negative effect and, hence, the deviation does not affect the validility of the study.
- Range of historical values if different from the ones specified in the test guideline:

The cell viability was measured by determining theoptical density (OD) at a wavelength of 540 nm. An exposure time of 20 minutes was employed.

The test item, Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts,was applied to the model skin surface. Water for injection was used as the negative control.5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item.

The mean viability of the cells exposed to the test item was 98.3% of the mean negative control value. The OD540values were well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of >50% for a 20 -minute exposure.

The test item was considered to be non-cytotoxic and predicted to be not irritant to skin.

The viability of cells treated with the positive reference item, 5% SDS, was 1.2% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the present test conditions the read-across test item, applied at an exposure time of 20 minutes, was non-cytotoxic and not irritant to skin in an experiment with an artificial three-dimensional model of human skin.
Executive summary:

The purpose of this study was to determine cytotoxic properties to skin cells, which might lead to irritation by the read-across test item to human skin, in an experiment with an artificial three-dimensional model of human skin. The EST-1000 model was employed. The cell viability was measured by determining the optical density (OD) at a wavelength of 540 nm. An exposure time of 20 minutes was employed. The read-across test item was applied to the model skin surface. Water for injection was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item. The mean viability of the cells exposed to the test item was 98.3% of the mean negative control value. The OD540 values were well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of >50% for a 20-minute exposure. The read-across test item was considered to be non-cytotoxic and predicted to be not irritant to skin.
The viability of cells treated with the positive reference item, 5% SDS, was 1.2% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.

Endpoint:
skin corrosion: in vitro / ex vivo
Remarks:
in vitro
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
See attached read-across justification
Reason / purpose for cross-reference:
read-across source
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
test item 3 minutes
Value:
78.8
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
test item 1 hour
Value:
74.9
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
positive control 3 minutes
Value:
0.5
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
positive control 1 hour
Value:
< 0.1
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: Not provided.
- Direct-MTT reduction: Not applicable.
- Colour interference with MTT: Not applicable.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Each batch of the epidermal model used meets defined production release criteria, set by the supplier, among which those for viability and for barrier function are the most relevant (MTT, 2 hours Triton X-100: target > 50%). The barrier properties of the tissues were verified by the supplier.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The magnitude of viability was quantified by using MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue ). In this case the optical density (OD) of the extracted (solubilised) dye from the negative control tissue was at least 20-fold greater than the OD of the extraction solvent alone. The negative control tissue has been shown to be stable in culture (provide similar viability measurements) for the duration of the test exposure period.
- Acceptance criteria met for positive control: Yes. The mean viability of cells treated with the positive reference item 8 N KOH were 0.5% (3-minute incubation) and <0.1% (1-hour incubation) of the negative controls and were below the cut-off values. Hence, 8 N KOH caused pronounced corrosion in this skin model and is predicted to be corrosive to human skin.
- Acceptance criteria met for variability between replicate measurements: The tissue employed has been shown to demonstrate reproducibility over time between laboratories.
- Range of historical values if different from the ones specified in the test guideline:

The test item, Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts,was applied to the skin surface. Dulbecco’s phosphate buffered saline (D-PBS) was used as the negative control.8 N KOH was used as the positive reference item.Two exposure times of 3 minutes or 1 hour were employed.

In comparison to the negative controls, the mean viability of cells exposed to the test item was 78.8% after a 3-minute exposure period and 74.9% after a 1-hour exposure.The OD540values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or <15% for a 3-minute or 1-hour treatment, respectively.Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

The mean viability of cells treated with the positive reference item 8 N KOH were 0.5% (3-minute incubation) and <0.1% (1-hour incubation) of the negative controls and were below the cut-off values. Hence,8 N KOH caused pronounced corrosion in this skin model and is predicted to be corrosive to human skin.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the present test conditions the read-across test itemapplied at two exposure times of 3 minutes or 1 hour was predicted non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin.
Executive summary:

The purpose of this study was to determine cytotoxic properties to skin cells which might lead to corrosion by a read-across test item to human skin, in an experiment with an artificial three-dimensional model of human skin. The EST-1000 model was employed. The read-across test item was applied to the skin surface. Dulbecco’s phosphate buffered saline (D-PBS)was used as the negative control. 8 N KOH was used as the positive reference item.Two exposure times of 3 minutes or 1 hour were employed.

In comparison to the negative controls, the mean viability of cells exposed to the read-across test item was 78.8% after a 3-minute exposure period and 74.9% after a 1-hour exposure. The OD540 values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or<15% for a 3-minute or 1-hour treatment, respectively. Therefore, the read-across test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin. The mean viability of cells treated with the positive reference item 8 N KOH was 0.5% (3-minute incubation) and <0.1% (1-hour incubation) of the negative controls and below the cut-off values. Hence, 8 N KOH caused pronounced corrosion in this skin model and is predicted to be corrosive to human skin.

Under the present test conditions the read-across test item containing >95% active ingredient, tested at two exposure times of 3 minutes or 1 hour, was non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached read-across justification
Reason / purpose for cross-reference:
read-across source
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: 24-72h
Score:
20.6
Max. score:
80
Reversibility:
not fully reversible within: 72h
Irritation parameter:
iris score
Basis:
mean
Time point:
other: 24-72h
Score:
1.9
Max. score:
10
Reversibility:
not fully reversible within: 72h
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: 24-72h
Score:
4.1
Max. score:
20
Reversibility:
not fully reversible within: 72h
Other effects:
6 of 6 eyes were positive for corneal opacity; 3 of 6 had irritis.

Table 1. Rabbit eye irritation after application of 0.1mL of [Trade name] (FHSA procedure)

Time,

Hours

Reaction

Rabbit Number, Value

Mean Value

1

2

3

4

5

6

24

CORNEA

IRIS

CONJUNCTIVAE

20

0

4

10

5

2

30

0

4

40

5

6

15

0

4

40

5

6

25.8

2.5

4.3

48

CORNEA

IRIS

CONJUNCTIVAE

5

0

4

10

0

2

20

0

4

30

5

6

5

0

4

40

5

6

18.3

1.7

4.3

72

CORNEA

IRIS

CONJUNCTIVAE

5

0

4

5

0

2

5

0

2

30

5

6

5

0

4

40

5

6

15.0

1.6

3.7

Interpretation of results:
other: irritating
Remarks:
FHSA definition
Conclusions:
The read-across test item containing +-50% active ingredient was irritating to the rabbit eye (an irritant by FHSA definition). 6 of 6 eyes were positive for corneal opacity; 3 of 6 had iritis.
Executive summary:

6 Male albino rabbits underwent an eye irritation test with 0.1 mL read-across test item containing +-50% active ingredient. According to FHSA procedure. Scoring according to Draize et al. (1944) demonstrated mean 24 -48h score of 20.6/80 for cornea, 1.9/10 for iris and 4.1/20 for conjuctiva irritation, therefore the read-across substance was concluded to be irritating to the rabbit eye (by FHSA definition), with 6 of 6 eyes positive for corneal opacity and 3 of 6 with iritis.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation was assessed based on supporting data from registered substance and weight-of-evidence

information from read-across:

A separate read across justification document for the mono-ester sulfosuccinates is provided in Section 13.

- In a supporting primary dermal irritation study with the registered substance, six New Zealand White

rabbits were dermally exposed to 0.5 mL of test item (20% act.ingr. in water) (Consultox Laboratories Ltd,

1969a). Test sites were covered with an occlusive dressing for 24 hours. Animals were then observed for

72 hours. Irritation was scored by the method of Draize test. There was no irritation at all throughout the test.

- In a supporting human test with registered substance, 20 test persons (19 females and 1 male) between

33 and 56 years of age (that had not used cosmetics during 3 days before the test), were exposed on the

forearms for 24 hours to the test item under gauze occlusion, followed by methylene blue exposure to

measure tensio-active effect (Biefeldt and Schrader, 1991). The test formulation showed a strong

roughening effect. The test item in this test turned out to do only slightly better than sodium lauryl sulphate.

The test method is not a regulatory standard method for skin irritation.

- Weight of evidence from read across data were available from category member, CAS No. 37294-49-8

(Disodium C-isodecyl sulphonatosuccinate), for which an in vivo study was done in male albino rabbits

under covered application to the clipped intact and abraded skin of 0.5 mL test item containing +- 50%

act. ingr. (Carpenter, 1971b). Scoring according to Draize et al. (1944) demonstrated a primary irritation

score of 0.375, based on erythema which was reversible within 72 hours. It was concluded that the test

item was not irritating to the rabbit skin.

- Additional weight of evidence was generated from read across substance CAS No. 90268-36-3

(Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts):

A first in vitro study was conducted to study corrosive properties by means of a three-dimensional EST-1000

human skin model (Flügge, 2013a). The test item containing >95% active ingredient was applied to the

skin surface. In comparison to the negative controls, the mean viability of cells exposed to the test item

was 78.8% after a 3-minute exposure period and 74.9% after a 1-hour exposure. The values were well

above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items

of <50% or <15% for a 3-minute or 1-hour treatment, respectively. Therefore, the test item was non-corrosive

in this skin model and was predicted to be non-corrosive to human skin.

A second in vitro study was conducted with the same read across substance CAS No. 90268-36-3

(Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts) to determine irritating properties by means

of the three-dimensional EST-1000 model of human skin (Flügge, 2013b). The test item containing >95%

active ingredient was applied to the skin surface for 20 minutes, followed by refreshment of the medium

and a further incubation period of 42 hours. The mean viability of the cells exposed to the test item was

98.3% of the mean negative control value. The value was well above the cut-off percentage cell viability

value that distinguishes irritant from non-irritant test items of >50% for a 20-minute exposure. The test

item was considered to be non-cytotoxic and predicted to be not irritant to skin.

- According to ECHA progress report 2010 (p 32), it is accepted that in vitro methods for skin irritation

represent a full replacement of the in vivo method in a tiered testing strategy and in conjunction with in

vitro skin corrosivity tests, if necessary. A negative result in the human skin model for irritation does not

need to be confirmed by additional testing. Taking into account all the above information, the registered

test substance is considered not to be irritating for skin.

In conclusion, the test substance was predicted to be non-corrosive and non-irritant to human skin and

found non-irritating in in vivo testing.

Nevertheless a subgroup classification CLP category 2 was proposed for skin irritation, with application

of concentration limit of 20% for the registered substance for non-classification.

 

Eye irritation was assessed based on supporting data from registered substance and weigth-of-evidence

information from read-across:

A separate read across justification document for the mono-ester sulfosuccinates is provided in Section 13.

- In a supporting primary eye irritation study the registered test substance (5 % act.ingr. in water) was

instilled into the conjunctival sac of the left eye of 9 New Zealand White rabbits (Consultox Laboratories Ltd,

1969b). The eyes of three animals, respectively were washed 2 sec and 4 sec after substance instillation.

The eye of three animals remained unwashed. Animals were then observed for 7 days. Irritation was

scored by the method of Draize. Irritation was confined to the conjunctiva. Cornea or iris was not involved.

Conjunctival effects of the test item were fully reversible at day 3 in the unwashed eye. In this study, the

test item is mildly irritating to the eye based on ocular reactions at 24, 48, and 72 hours p.a., using the

Draize test (Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics FDA, 1959).

The findings however did not result in the need of classification.

- In a supporting in vitro test on bovine red blood cells, haemoloysis and denaturation were measured

after exposure to the test item, followed by calculation of a mean ocular irritation index (Löffert and Lang,

1992). The test formulation showed an L/D value of 0.8, representing for an irritating effect. The test method

however is not a regulatory standard method for eye irritation.

- Read across data were available from category member, CAS No. 37294-49-8 (Disodium C-isodecyl

sulphonatosuccinate), for which a key in vivo study was done in 6 male albino rabbits given 0.1 mL test

item containing +- 50% act. ingr. according to FHSA procedure (Carpenter, 1971). Scoring according to

Draize et al. (1944) demonstrated mean 24 -48h score of 20.6/80 for cornea, 1.9/10 for iris and 4.1/20 for

conjuctiva irritation, therefore the substance was concluded to be irritating to the rabbit eye (by FHSA

definition), with 6 of 6 eyes positive for corneal opacity and 3 of 6 with iritis. As observations were only

performed up to 72 hours, reversibility could not be scored and worst case classification CLP class 1

was proposed.

- In conclusion, the registered substance can be considered to evoke severe damage to the eye, therefore

CLP class 1 is proposed. However a non-classification threshold to CLP class 2 (eye irritating) can be

applied for a formulation containing 20% active ingredient or less, whereas non-classification can be

applied for formulations of 5% or less.

 

Conclusion

- Based on the available data of registered and read across substances, a subgroup classification

CLP category 2 is proposed, however with concentration limit of 20% or less where the registered

substance can also be accepted to be non irritating for skin.

- Based on the available data of registered and read across substances, the substance needs to be

classified as causing severe damage to the eye. A classification threshold to CLP class 2 (eye irritating)

can be applied for a formulation containing 20% active ingredient or less, whereas for formulations of

5% or less no classification is needed.

- Further information is provided in the read across justification for the Mono-ester sulfosuccinate subgroup. 

 

Justification for classification or non-classification

Based on these results of registered and read across substances, and according to the CLP
(No. 1272/2008 of 16 December 2008), a subgroup classification Category 2 classification is proposed
with signal word 'WARNING' and hazard statement H315 'CAUSES SKIN IRRITATION'. However a
concentration limit of 20% or less can be applied for the registered substance for non-classification.

Based on results of registered and read across substances, the test substance needs to be classified to

under CLP regulation (No. 1272/2008 of 16 December 2008) as Category 1, with signal word 'Danger'
and hazard statement: H318 -Causes serious eye damage. A specific concentration limit can be
applied for formulations containing 5 to 20 % active ingredient, with CLP Category 2, signal word
'Warning' and hazard statement H319 - Causes serious eye irritation. At and below 5 % there is no
classification needed.