Butanedioic acid, 2-sulfo-, mono(C16-18 and C18-unsatd. alkyl) esters, ammonium sodium salts

Endpoint summary

Description of key information

 

A key in vivo skin irritation study showed that the test item containing 40% active ingredient was irritating on rabbit skin, showing mean 24 -72h scores of 2.22/4 for erythema and 2.44/4 for edema. Although slight erythema was still observed after 21 days, a supporting study with similar formulation (40% purity) showed similar findings (mean 24-72h scores of 2.67/4 and 2.67/4, respectively) with complete reversibility within 14 days. The test item was considered to be irritating to skin (CLP category 2).
A key in vivo eye irritation study showed that the test item containing 40% active ingredient was irritating in rabbit eye, showing mean 24 -72h scores of 1/4 for cornea, 0.25/2 for iris, 2.33/3 for conjunctiva redness and 0.75/4 for chemosis. The test item was considered to be irritating to eye (CLP category 2). This was confirmed in a supporting in vivo and in vitro study for eye irritation.  Subgroup classification however was proposed to be CLP category 1.

 

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:

key study

Study period:

May 4-June 24, 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to GLP and valid test methods, therefore the study was considered relevant, adequate and reliable for classification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

Off. J. Europ. Commun. L 251, 27, 106-108 (1984)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

other: Kleinrussen, CHbb:HM

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Thomae GmbH, Biberach/D.
- Age at study initiation: Approximately 17 months
- Weight at study initiation: Approximately 2630 g
- Housing: Individual accomodation
- Diet (e.g. ad libitum): Ssniff rabbit diet K4, Ssniff GmbH, Soest/D Batch: 550492/09, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: At least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Approximately 20-25°C
- Humidity (%):Approximately 45-70%
- Air changes (per hr): At least 8 per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1992-05-04 To:1992-05-24

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

Amount/concentration applied: 0.5 g
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g
- Concentration (if solution): 40%

Duration of treatment / exposure:

4 hours

Observation period:

21 days (22 days for first experimental animal)

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: flank
- % coverage: Not provided
- Type of wrap if used: The semi-occlusive exposure was performed by means of a 6 cm2 adhesive air- and vapor permeable gauze-patch which was fixed by a hypoallergenic adhesive gauze-bandage.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 4 hours

SCORING SYSTEM:
Erythema and eschar formation
No erythema 0
Very slight, barely perceptible erythema (-slight-) 1
Well-defined erythema (-weak-) 2
Moderate to severe erythema (-moderate-) 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) (-severe-) 4

Edema formation
No edema 0
Very slight, barely perceptible edema (-slight-) 1
Slight edema, edges of area well defined (-weak-) 2
Moderate edema, raised approx. 1mm (-moderate-) 3
Severe edema, raised more than 1mm and extending beyond the area of exposure (-severe-) 4

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

>= 2

Max. score:

4

Reversibility:

not fully reversible within: 21-22 days

Remarks on result:

other: mean score= 2.22

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

not fully reversible within: 22 days

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 21 days

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

>= 2

Max. score:

4

Remarks on result:

other: mean score = 2.44

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.67

Max. score:

4

Reversibility:

fully reversible within: 22 days

Remarks on result:

other: eschar on day 7 & 14

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2.33

Max. score:

4

Reversibility:

fully reversible within: 21 days

Remarks on result:

other: brownish discolouration on day 3; eschar on day 7 & 14

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2.33

Max. score:

4

Reversibility:

fully reversible within: 21 dyas

Remarks on result:

other: brownish discolouration up to 3 days; eschar on day 7 & 14

Table 1. Summary of the symptoms

dermal reactions	mean value (24/48/72h)	Number of animals
erythema	≥2	3
edema	≥2	3

 

Table 2. Evaluation of the skin reaction

Animal no./flank	Hours after exposure
	1		24		48		72
	er	ed	er	ed	er	ed	er	ed
1251/r	2	1	2	2	3	3	3	3
1256/l	1	1	2	2	2	2	2	2 b
1573/l	1	1	2	3 b	2	2 b	2	2 b

 

Animal no./flank	Days after exposure
	7		14		21/22^*	er	ed
1251/r	ᴓ	es	ᴓ	es	ᴓ	1	0
1256/l	ᴓ	es	ᴓ	es	ᴓ	0	0
1573/l	ᴓ	es	ᴓ	es	+	0	0

 

r = right flank                   ed = edema                         + = reversible
l = left flank                      er = erythema                    ᴓ = irreversible
* animal 1251                   es = eschar b = brownish discolouration

 

Interpretation of results:

Category 2 (irritant)

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item proved to be “moderately irritating” on the skin according to the conditions applied.

Executive summary:

The test item, containing 40% active ingredient, was tested for acute dermal irritation on the dorsal shaved skin of the rabbit in a dose of 0.5 g. The contact time under semi-occlusive conditions lasted 4 hours. According to the guidelines in a preliminary study 1 animal was treated first because of the risk of severe skin injury. Moderate effects of the skin were observed. After 4 weeks 2 other experimental animals were treated. In all animals moderate erythema and edema were observed during the first 72 hours after exposure. Mean 24 -72h scores were 2.22/4 for erythema and 2.44/4 for edema. Two experimental animals showed a brownish discolouration additionally. The skin of all animals showed eschar on day seven and day 14 after exposure. The irritation effects (slight erythema) of 2 experimental animals persisted longer than 21 days. The observed effects of the other experimental animal reversed completely within 21 days.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22-7-1988 to 19-8-1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted non-GLP, however quality inspections were performed and the study was performed according to valid study methods.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

other: Kleinrussen, Chbb:HM/Fa

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source:Fa. Thomae, Biberach, Germany
- Age at study initiation: Adult (males)
- Weight at study initiation: Average 2482.5 g
- Housing: Individually in rabbit batteries (Fa. Heinkel, 7343 Kuchen/Germany)
- Diet (e.g. ad libitum): Altromin-Haltungsdiät 2023 (Fa. Altromin GmbH/Lage/Germany) ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: At least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 20-21°C
- Humidity (%): ca. 45-55%
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated left eyes served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g undiluted test item

Duration of treatment / exposure:

single application, lids were held close briefly

Observation period (in vivo):

10 days after application (Time points: 1, 6, 24, 48 and 72 hours and 7 and 10 days)

Number of animals or in vitro replicates:

4 (males)

Details on study design:

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

SCORING SYSTEM: Draize

TOOL USED TO ASSESS SCORE: fluorescein (0.5% fluorescein-sodium-solution )

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0.25

Max. score:

2

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

2.33

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0.75

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritant / corrosive response data:

-All animals showed a slight corneal opacity after 1 to 6 hours after application, which disappeared 7 to 10 days after application.
-One animal had an iris reaction at 24, 48 and 72 hours after application.
-Conjunctival redness was observed in a mild to severe degree and reached its maximum between 24 and 72 hours after application.
-Edema in a mild degree subsided completely in one animal after 24 hours, in the other animals after 7 to 10 days.
-Strong exudation was observed in all animals.
-Conjunctival reactions disappeared completely after 7 to 10 days in all animals.
Corneal damage was observed in all animals 24 hours after application and was confirmed with the help of the fluorescein method. Complete reversibility 7 to 10 days after application was also confirmed with the fluorescein method.

Table 1. Corneal reactions after application of undiluted Ke 833 B40 ne in continuous contact

Animal no.	Hours after exposure
	1h		6h		24h		48h		72h
	A	B	A	B	A	B	A	B	A	B
924	1	1	1	1	1	2	1	1	1	1
802	1	1	1	3	1	3	1	2	1	2
803	0	0	1	1	1	2	1	1	1	1
804	1	1	1	2	1	2	1	2	1	2
Mean A	0.75		1.00		1.00		1.00		1.00
MeanB	0.75		1.75		2.25		1.50		1.50

 

Animal no.	Days after exposure
	7d		10d
	A	B	A	B
924	0	0	0	0
802	1	1	0	0
803	0	0	0	0
804	1	1	0	0
Mean A	0.50		0
MeanB	0.50		0

A= opacity
B= area of opacity

Table 2. Corneal reactions in % area of damaged cornea after application of undiluted Ke 833 B40 ne as determined by fluorescein method

Animal no.	24 h before application	Days after application
		1d	7d	10d
924	0	50	0	-
802	0	50	-	0
803	0	50	-	0
804	0	50	-	0
Mean	0	50.0	0	0

 

Table 3. Iris reactions after application of undiluted Ke 833 B40 ne in continuous contact

Animal no.	Hours after exposure					Days after exposure
	1h	6h	24h	48h	72h	7d	10d
	A	A	A	A	A	A	A
924	0	0	0	0	1	0	0
802	0	0	0	0	1	0	0
803	0	0	0	0	1	0	0
804	0	0	1	1	1	0	0
Mean A	0	0	0.25	0.25	0.25	0	0

 

Table 4. Conjunctivae reactions after application of undiluted Ke 833 B40 ne in continuous
contact

Animal no.	Hours after Treatment
	1h			6h			24h			48h			72h
	A	B	C	A	B	C	A	B	C	A	B	C	A	B	C
924	1	1	3	1	1	3	1	0	2	1	0	1	1	0	0
802	1	1	3	1	1	3	3	1	3	3	1	2	3	1	3
803	1	1	3	1	1	3	3	1	3	3	1	3	2	1	1
804	1	1	3	1	1	3	2	1	3	3	1	1	3	1	2
Mean A	1.00			1.00			2.25			2.50			2.25
Mean B	1.00			1.00			0.75			0.75			0.75
Mean C	3.00			3.00			2.75			1.75			1.50

 

Animal no.	Days after Treatment
	7d			10d
	A	B	C	A	B	C
924	0	0	0	0	0	0
802	2	0	2	0	0	0
803	0	0	0	0	0	0
804	1	0	1	0	0	0
Mean A	0.75			0
Mean B	0			0
Mean C	0.75			0

A= Redness
B= Chemosis
C= Exudation

Interpretation of results:

irritating

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item containing 40% active ingredient was irritating for the rabbit eyes. .

Executive summary:

The test item containing 40% active ingredient was tested for primary eye irritation on rabbits by a single application of 0.1 g of the undiluted test item and permanent contact. The reactions on the cornea were slight and disappeared totally within 7 to 10 days. One animal had an irrital reaction between 24 and 72 hours after the application. Mean 24 -72h scores were 1/4 for cornea, 0.25/2 for iris, 2.33/3 for conjunctiva redness and 0.75/4 for chemosis. The conjunctival reactions were moderate to severe and disappeared completely within 7 to 10 days after the application in all animals.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

- A key in vivo study for acute dermal irritation with test item containing 40% active ingredient, was performed on the dorsal shaved skin of the rabbit in a dose of 0.5 g (Pittermann, 1992a). The contact time under semi-occlusive conditions lasted 4 hours. In all animals moderate erythema and edema (mean scores >2) were observed during the first 72 hours after exposure; mean 24 -72h scores were 2.22/4 for erythema and 2.44/4 for edema. Two experimental animals showed a brownish discolouration additionally. The skin of all animals showed eschar on day seven and day 14 after exposure.The irritation effects (slight erythema) of 2 experimental animals persisted longer than 21 days. The observed effects of the other experimental animal reversed completely within 21 days.

- A supporting in vivo study for primary skin irritation was performed under occlusive conditions on the shaved back skin of 4 rabbits (Kästner, 1988a). After a contact time of 4 hours with test item containing 40% active ingredient, moderate skin reactions were observed, with mean scores (24, 48, 72 hours) of 2.67 for erythema and 1.92 for edema. Eschar formation was observed in 3 out of 4 rabbits up to day 7. The reactions disappeared totally within 14 days.

- In conclusion: although slight erythema was still observed at 21 days in the key study, the findings of the supporting study demonstrated full reversibility of a similar formulation within 14 days, therefore the findings were considered to be reversible. A CLP category 2 is proposed for skin irritataion (subgroup classification).

Eye irritation

- A key in vivo study for primary eye irritation was conducted with test item containing 40% active ingredient on rabbits by a single application of 0.1 g in the conjuntival sac of one eye (Kästner, 1988b). The reactions on the cornea were slight and disappeared totally within 7 to 10 days. One animal had an irrital reaction between 24 and 72 hours after the application. Mean 24 -72h scores were 1/4 for cornea, 0.25/2 for iris, 2.33/3 for conjunctiva redness and 0.75/4 for chemosis. The conjunctival reactions were moderate to severe and disappeared completely within 7 to 10 days after the application in all animals.

- A support in vivo study for acute eye irritation with test item containing 40% active ingredient was performed in rabbits (Pittermann, 1992b). A single instillation dose of 0.1 mL was placed in the conjunctival sac of one eye. Mild to moderate effects of the eye were observed in a first animal, after which 2 other experimental animals were treated. The observed conjunctival reactions (erythema, edema and lacrimation) of 2 experimental animals were mild to moderate. Weak corneal opacities were observed additionally. The other experimental animal showed mild conjunctival irritation. Mean 24-72h scores were 0.67/4 for cornea, 0.11/2 for iris, 1.56/3 for conjunctiva redness, 1.14/4 for chemosis and 0.78/4 for lacrimation. All reactions disappeared completely within 14 days. The test item containing 40% active ingredient proved to be moderately irritating to the eye according to the conditions applied.

- A supporting in vitro study was conducted with structural analogue to determine the eye irritancy potential of a 60% dilution of test item containing 36.9% active ingredient (or netto 22.1% active ingredient) by means of the chorioallantoic membrane of hens' eggs (HET-CAM) (Steiling, 1991). The potential for irritation was significantly higher than the irritation potential of the positive reference substance (Texapon 5% active ingredient in water). According to the test results it is assumed that the 60% dilution (22.1% act. ingr.) can cause severe eye irritation. In the acute eye irritation in vivo (Draize-Test) a “One-animal-test” was indicated.

- In conclusion, the substance was considered to be irritating to the eye (CLP category 2A upt to 40% tested concentration), however for undiluted substance a higher classification CLP category 1 is proposed (subgroup classification).

Conclusion

- The registered substance was concluded to be irritant to human skin; a CLP category 2 classification is proposed (subgroup classification).

- For the eye, CLP category class 2 was found, however worst case classification CLP class 1 is proposed (subgroup classification). A concentration threshold to CLP class 2 (eye irritating) can be applied for a formulation containing 40% active ingredient or less.

- This is also supported by other data from the mono-ester group (see separate read-across justification attached to Section 13).

Justification for selection of skin irritation / corrosion endpoint:
Key study

Justification for selection of eye irritation endpoint:
Key study

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: irritating

Justification for classification or non-classification

The substance is classified according to the EC Directive (No.93/21/EEC) as 'IRRITATING TO SKIN' and to be signed with “Xi” and the risk-phrase R 38. According to CLP regulation (No. 1272/2008 of 16 December 2008), Category 2 classification is proposed with signal word 'WARNING' and hazard statement H315 'CAUSES SKIN IRRITATION'.'

The substance is classified according the EU labelling regulations Commision Directive 93/21/EEC as 'RISK OF SERIOUS DAMAGE TO THE EYES' and to be signed with “Xi” and the risk-phrase R 41. According to CLP regulation (No. 1272/2008 of 16 December 2008), Category 1 classification is proposed with signal word 'DANGER' and hazard statement H318 'CAUSES SERIOUS EYE DAMAGE'. A classification threshold to CLP class 2 (eye irritating) can be applied for a formulation containing 40% active ingredient or less.