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Diss Factsheets

Administrative data

Description of key information

- not sensitising; OECD TG 406, in vivo, Guinea pig maximisation test, guinea pig (no GLP, RL2); read-across: C8-18 and C18 unsatd. AAPB
- not sensitising; OECD TG 406, in vivo, Guinea pig maximisation test, guinea pig (GLP, RL1); read-across: Formamidopropylbetaine

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1980-09 to 1980-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
No data on positive control.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Principles of method if other than guideline:
Guinea pig maximization test as described by B. Magnusson and A.M. Kligman (1970), the later released OECD Guideline 406 (1981) is based to a great extent on this publication.
GLP compliance:
no
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A valid GPMT conducted comparable to guideline with acceptable restrictions is available, which is reliable with restrictions and adequate for classification and labelling purposes. Potency estimation is not mandatory when existing guideline and GLP conforming data are available, which were conducted before the new annex of the REACH Regulation entered into force. Moreover, no indication for skin sensitisation was observed in this study, thus, no dose response information is needed. For this reason and for reasons of animal welfare no additional LLNA was conducted.
Species:
guinea pig
Strain:
other: Pirbright white
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 4 - 6 weeks
- Weight at study initiation: 400 +/- 50 g
- Housing: suspended cages
- Diet: ad libitum, Guinea-pig diet (Ssniff G)
- Water: ad libitum, tap water
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 5 % RH
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
Induction:
Intradermal injection:0.5 % v/v,
Topical application after 7 days: epicutaneous 60 % v/v
Day(s)/duration:
1 week after injections; topical application 48 h
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Challenge: 10 % v/v
Day(s)/duration:
2 weeks after the induction; 24 h exposure
No. of animals per dose:
15 test animals,
5 control animals
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 times
- Exposure period: 0 and 1 week after injections topical application for 48 h
- Test groups: 15
- Control group: 5
- Site: a 4 x 6 cm area of dorsal skin on the scapular region was clipped free of hair.

First stage:: 3 pairs of intradermal injections were made simultaneously. A volume of 0.1 ml was injected into both infection sites (left and right site)
- Concentrations:
Test group:
1. 0.1 ml of Freund´s complete adjuvant 50 : 50 with water for injection
2. 0.1 ml of 0.5 % v/v test item in sterile isotonic saline
3. 0.1 ml of 0.5 % v/v test item in a 50 : 50 mixture of isotonic saline and Freund´s complete adjuvant
Control group:
1. 0.1 ml of Freund´s complete adjuvant 50 : 50 with water for injection
2. 0.1 ml of sterile isotonic saline
3. 0.1 ml of Freund´s complete adjuvant 50 : 50 with isotonic saline
Control animals: during the induction period control animals were treated similarly to the test animals but the test substance was omitted form intradermal injections and topical applications.

Second stage: 7 days after the first induction the same area was clipped and shaved free of hair. A 2 x 4 cm patch of filter paper was saturated with 60 % v/v test item in distilled water. Patch was placed on the skin and covered by a impermeable plastic adhesive tape ("Dermicel"). Tape
was secured by a elastic adhesive bandage ("Elastoplast"). Occulsive dressing was left in place for 48 h.

B. CHALLENGE EXPOSURE
- No. of exposures: 1 time
- Day(s) of challenge: 2 weeks after the induction
- Exposure period: 24 h
- Test groups:
2 x 2 cm filter paper was saturated with the test sample in a similar as used for second stage induction. Occlusive dressing for 24 h.
- Control group: The 5 animals of the control group were similarly challenged
- Site: hair was removed by clipping and then shaving a 5 x 5 cm area on the left flank.
- Concentrations: 10 % v/v test item in distilled water
- Evaluation (hr after challenge): 24, 48, 72 h after patch removal

Scoring:
Erythema and eschar formation:
No erythema 0
Slight erythema (barely perceptible): 1
Well-defined erythema 2
Moderate erythema 3
Severe erythema (beet. redness) to slight eschar formation (injuries in depth) 4

Oedema formation:
No oedema 0
Slight oedema (barely perceptible): 1
Well-defined oedema (edges of area well-defined by definite raising) 2
Moderate oedema (raised app. 1 mm 3
Severe oedema (raised more than 1 mm and extending beyond the area 4
of exposure



Challenge controls:
5
Positive control substance(s):
not specified
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10 %
No. with + reactions:
2
Total no. in group:
15
Clinical observations:
erythema score 1 (Slight erythema (barely perceptible))
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
15
Remarks on result:
no indication of skin sensitisation
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
15
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
5
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
5

Induction:

Intradermal injections containing Freund´s complete adjuvant: elicited well-defined dermal irritation

Intradermal injection of 0.5 % v/v test item in all test animals: temporary slight dermal irritation

Second stage (topical application) of 60 % v/v test item:slight dermal reactions

Challenge (10 % v/v Test item):

Test animals:

After 24 h: slight erythema (1): 2 animals

Control animals: no reactions

All animals (test and control animals) showed slight bandage reactions

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
In this guinea-pig maximization test, performed in 20 albino guinea-pigs, Coco AAPB (30 % a.i.) did not produce evidence of delayed contract hypersensitivity.
Executive summary:

In a dermal sensitization study with Coco AAPB (commercial product) 20 male Pirbright white guinea pig were tested using the MAXIMIZATION TEST described by b. Magnusson and A.M. Kligman (1970). The test method is similar to OECD Guideline 406 (Skin Sensitisation).

The analytical purity of the test item is not stated in study report, according to producer information test material has 30 % a.i., impurities relevant to sensitisation and referred to a.i. are 8.3 % Alkylamidopropylamine and 33 -50 ppm DMPA (3-dimethylaminopropylamine).

At the first scoring, 24 h after challenge, 2/15 showed a skin reaction score 1 (slight erythema (barely perceptible)). The animals were free of erythema and edema at the second (48 h) and the third (72 h) scoring. None of the five negative animals showed up to and including the third scoring a positive skin reaction to challenge treatment.

In this study, Coco AAPB (30 % a.i.) is not a dermal sensitizer.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
February and March 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
No data on positive control.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
12. May 1981
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A valid GPMT conducted comparable to guideline with acceptable restrictions is available, which is reliable with restrictions and adequate for classification and labelling purposes. Potency estimation is not mandatory when existing guideline and GLP conforming data are available, which were conducted before the new annex of the REACH Regulation entered into force. Moreover, no indication for skin sensitisation was observed in this study, thus, no dose response information is needed. For this reason and for reasons of animal welfare no additional LLNA was conducted.
Species:
guinea pig
Strain:
other: Pirbright white, bor: DHPW (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: no data
- Weight at study initiation: 258 - 348 g
- Housing: collective housing up to a maximum of 5 animals per cage (Macrolon type IV)
- Diet: ad libitum, standard laboratory guinea pig diet Ssniff-G
- Water: ad libitum, drinking water as for human consumption
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 +/- 2
- Humidity (%): 50 - 85
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): artificial lighting (120 lux), 7.00 a.m. - 7.00 p.m.
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
Induction:
- intradermal: appropriate concentration (concentration not further specified)
- epicutaneous: undiluted test substance as delivered (30 % a.i.). Because the test article was non-irritating at all tested concentrations, the clipped area was pretreated with 10 % sodium lauryl sulfate (SLS) in petrolatum to create a local irritation.
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Challenge: 30 % a.i., undiluted test substance as delivered
Adequacy of challenge:
other: undiluted test substance as delivered
No. of animals per dose:
20 test animals (10 female, 10 male)
20 control animals (10 female, 10 male)
Details on study design:
RANGE FINDING TESTS:
- Intradermal Injection:
The test article was diluted with equal volumes of aqua ad inject. and Freund's complete adjuvant (FCA; Sigma, Deisenhofen, Germany) to give a final concentration of 5 %.
Two animals were employed for each concentration tested, skin reactions were recorded 48 h and 6 days after treatment.

- Dermal application:
The liquid was used at the highest concentration which did not produce excessive inflammation (30% a.i., undiluted). A closed patch exposure was effected by means of an occlusive bandage. Since this maximum concentration proved irritating, lower concentrations were tested, two animals were employed for each concentration tested and skin reactions were recorded 48 h post applicationem.

MAIN STUDY
A. INDUCTION EXPOSURE
- First stage -an area of 4 x 6 cm over the shoulders was clipped short with electric clippers and cleaned with 70 % (v/v) ethanol. Three pairs of intradermal injections were then made symmetrically in two rows on either side of the spine:
-- Test group:
1. 0.1 ml FCA alone (diluted 1 : 2 in water)
2. 0.1 ml test article alone (in the appropriate concentration)
3. 0.1 ml test article emulsified in FCA (in the appropriate concentration)
-- Control group:
1. 0.1 ml FCA alone (diluted 1 : 2 in water)
2. 0.1 ml aqua ad inject (undiluted)
3. 0.1 ml aqua ad inject (diluted 1 : 2 with FCA)

-- Second stage - 7 days after the intradermal injections, the same area was clipped and cleaned again. Because the test article was non-irritating at all tested concentrations, the clipped area was pretreated with 10 % sodium lauryl sulfate (SLS) in petrolatum. 24 hrs later, the test article was spread in a thick layer [to saturation] over a 2 x 4 cm patch (gauze). The latter was firmly secured over the previous injection sites by an occlusive dressing for 48 h.
Control animals received a patch loaded with the vehicle alone.

B. CHALLENGE EXPOSURE
Both control and test animals were subjected to a challenge exposure 14 days after the second stage of induction. The challenge test was performed on a 5 x 5 cm clipped and shaved area on each flank. The maximal non-irritating concentration of the test article (30 % a.i. undiluted) was applied to the left flank and the vehicle to the right using the patch technique described. In each case the duration of exposure was 24 h under an occlusive
dressing. If necessary, the skin was cleaned with 70 % ethanol 21 h after removal of the patch. 24 and 48 h after patch removal, responses were evaluated on a numerical scale according to Draize.
Positive control substance(s):
not specified
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
30 % a.i.
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
30 % a.i.
No. with + reactions:
4
Total no. in group:
20
Clinical observations:
skin reactions score 1 (scattered mild redness)
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
30 % a.i.
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
30 % a.i.
No. with + reactions:
5
Total no. in group:
20
Clinical observations:
skin reactions score 1 (scattered mild redness)

Pilot study (range finding):

- Intradermal:

After 48 h: No specific findings were observed at 1, 0.5 and 0.1 % (1 females and 1 male animal).

5 %: Black discoloration (6 mm diameter) of the injection site (1 male)

After 6 days: No specific findings were observed at 0.5 and 0.1 % (1 female and 1 male animals).

1 %: Immoderate erythema (8 mm diameter) (1 female, 1 male)

- Dermal:

After 48 h: no reactions after dermal application and at a concentration of 30 % a.i. (undiluted testsubstance as delivered)

Main study:

Test group: At 48 h 4/20 test animals showed skin reactions score 1 (scattered mild redness).

Control group: At 48 h 5/20 test animals showed skin reactions score 1.

The sensitization rate at 24 h and 48 h: 0

Interpretation of results:
GHS criteria not met
Conclusions:
As incidence and severity of reactions seen in the test group were not higher than responses seen in the negative control group, the calculated sensitisation rate was 0. The test substance Coco AAPB is not a dermal sensitizer in this study.
Executive summary:

In a dermal sensitization study with Coco AAPB (30 % a.i.) Pirbright white guinea pig (10 male, 10 female) were tested using the Maximisation Test method according to OECD Guideline 406, May 12, 1981.

The analytical purity of the test item is not stated in study report, according to producer information test material is Coco AAPB and has 30 % a.i. , impurities relevant to sensitisation and referred to a.i. are max 1.7% Alkylamidopropylamine and 33 ppm DMPA (3-dimethylaminopropylamine).

Responses to the challenge procedure were evaluated 24 and 48 h after the end of the exposure period.

There were no skin reactions at the first reading. At the second reading 4/20 test animals and 5/20 control animals showed skin reactions score 1 (scattered mild redness).

As Incidence and severity of reactions seen in the test group were not higher than responses seen in the negative control group, the calculated sensitisation rate was 0.

In this study, Coco AAPB is not a dermal sensitizer.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15. Nov. 2004 - 17. Dec. 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A valid GPMT conducted according to guideline is available, which is reliable without restrictions and adequate for classification and labelling purposes. Potency estimation is not mandatory when existing guideline and GLP conforming data are available, which were conducted before the new annex of the REACH Regulation entered into force. Moreover, no indication for skin sensitisation was observed in this study, thus, no dose response information is needed. For this reason and for reasons of animal welfare no additional LLNA was conducted.
Species:
guinea pig
Strain:
other: Pirbright White, BOR DHPW
Sex:
female
Details on test animals and environmental conditions:
Species: guinea pig
Breed: Pirbright White, BOR DHPW
Breeder: Harlan Winkelmann, Borchen, Germany
Date of receipt: 2004-11-03
Sex: female
Number: 20 (test group 10, control group 5, pilot experiment 5)
Body weights: 313.3 g - 415.6 g
Age: - 4 weeks on delivery
Identification: dye marks and cage numbers
Diet: Altromin International, Lage, Germany, 3022 - 1420
Water: Drinking water for the animals consisted of normal tap water from municipal sources: Stadtische Werke Krefeld AG, Abt. 2 TGW, 47804 Krefeld, Germany. The composition of the water is regularly determined. The data are retained in the archive. The water was supplied to the animals ad libitum via drinking bottles with rubber stoppers and steel pipes.
Bedding Material: Lignocel BK 10/20, Rettenmaier, Jagstzell, Germany
Husbandry: The animals were housed with 2 or 3 animals in Makrolon®-cages No.4. A non-barrier system with air conditioning was used. The air conditioning had the following nominal values: Temperature: 22°C ± 3°C, Humidity: 30 % - 70 %, Air exchange: 8 times/hour. Climate control was run automatically. The lightening was in a 12-hour light/dark-cycle. Temperature and humidity were recorded continuously using a thermohygrometer, Lambrecht GmbH, Gbttingen, Germany. In cause of a technical default of the thermohygrometer the climate could not be recorded on the 2nd, 3rd, 4th and 5th of December 2004. The humidity was lower then 30 % from the 6th to 13th December 2004. These deviations are not supposed to have any significant effect on the experimental result.
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Concentration / amount:
Pilot experiment:
Intradermal: 0.1 mL of 5.0 %, 3.5 %, 2.0 % and 0.5 % (w/w) dilution in saline (0.9 % NaCI solution).
Dermal: soaked patch with 100 %, 75 %, 50 % and 25 % (w/w) of the test substance in saline.

Main study:
Intradermal: 0.1 mL of 3.5 % (w/w) solution of the test substance in saline.
Dermal: soaked patch with the undiluted test substance.
Day(s)/duration:
Dermal induction (48 h exposure) 7 days after induction by intradermal injection
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
Pilot experiment: duhring chamber with 50 and 30% (w/w) of the test substance in saline.
Main study: duhring chamber with 30% (w/w) solution of the test substance in saline.
Day(s)/duration:
day 21, 24 h exposure
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
main study: test group 10 animals, control group 5 animals.
Details on study design:
RANGE FINDING TESTS: Pilot Experiment:
The purpose of the pilot experiment was to determine which concentration of the test substance 1. led to slight irritation after intradermal applicatio (determination of the maximum compatible dose), 2. led to slight irritation after dermal application and 3. can just be applied dermally without leading to skin irritation (subirritative dose).

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and dermal)
- Exposure period: Dermal induction (for 48 h) 7 days after induction by Intradermal injection
- Test groups: 1 with 10 animals
- Control group: 1 with 5 animals
- Frequency of applications: once
- Concentrations: Intradermal: 0.1 mL of 3.5 % (w/w) solution of the test substance in saline. Dermal: soaked patch with the undiluted test substance. Challenge: duhring chamber with 30% (w/w) solution of the test substance in saline.

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: at day 21 of the application period
- Exposure period: 24 hours
- Test groups: 1 with 10 animals
- Control group: 1 with 5 animals
- Site: left flank
- Concentrations: 30 % (w/w) dilution of the test substance in saline
- Evaluation (hr after challenge): 24 h and 48 h after end of challenge exposure
Positive control substance(s):
yes
Remarks:
The corresponding validation experiment produced a sensitisation rate of 70 % with the reference substance 2-mercaptobenzothiazole showing the sensitivity of the test system.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
30%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
30%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
30%
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
30%
No. with + reactions:
0
Total no. in group:
5

Challenge results

 

Animal number

Code

Sex

24 hours

48 hours

Erythema

Edema

Erythema

Edema

Test Group

 

 

 

 

 

 

1

lv

female

0

0

0

0

2

Rv

female

0

0

0

0

3

Lh

female

0

0

0

0

4

Rh

female

0

0

0

0

5

2xv

female

0

0

0

0

6

Lv

female

0

0

0

0

7

rv

female

0

0

0

0

8

Lh

female

0

0

0

0

9

Rh

female

0

0

0

0

10

2xv

female

0

0

0

0

Control Group

 

 

 

 

 

 

1

Lv

female

0

0

0

0

2

Rv

female

0

0

0

0

3

Lh

female

0

0

0

0

4

Rh

female

0

0

0

0

5

2xv

female

0

0

0

0

 

Interpretation of results:
GHS criteria not met
Conclusions:
The observed sensitisation rate of 0% indicates that the test substance Formamidopropyldimethylbetaine is a non-sensitising compound in this test system.
Executive summary:

For labelling and classification purposes the test substance Formamidopropyldimethylbetaine was tested regarding its skin sensitisation potential. The test was performed according to the stringent protocol of Magnusson and Kligman, in which adjuvant is used to elicit an immunologic reaction. The challenge application was executed with Duhring chambers to aim for better, i.e. more exaggerated, exposure conditions. The test protocol is in compliance with the OECD Guideline for the Testing of Chemicals No. 406 Skin Sensitisation (1992). The maximum compatible doses for the intradermal and dermal application as well as the subirritative dose for the challenge were determined in a pilot experiment. In the main experiment ten (10) female guinea pigs of the strain "Pirbright White" were treated intradermally with 0.1 mL of a 3.5 % (w/w) dilution of the test substance in saline. For the dermal induction the undiluted test substance was used. Intradermal application of the 3.5 % (w/w) dilution of the test substance in saline caused slight to moderate erythema and edema formation and the topical application of the undiluted test substance caused slight erythema and edema formation. In the challenge application Duhring chambers with a 30 % (w/w) dilution of the test substance in saline were used, a test concentration which was definitely non-irritating. Besides crust formation at the injection sides after intradermal application with FCA no other systemic toxic symptoms or toxic reactions were observed at any time during the study. Body weight development of the animals was positive and within normal ranges. In the challenge no visible changes on the skin (no erythema and no edema) were observed at any time point; i.e. there was no positive challenge result in the test and the control group animals. Thus, the sensitisation rate was 0 %. Therefore, the test substance Formamidopropyldimethylbetaine has to be regarded as non-sensitising under the applied test conditions when exposed to the skin of experimental animals.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

No experimental data on the sensitisation potential of C8-10 Alkylamidopropyl betaine are available. However, reliable Guinea pig maximisation tests conducted with the closely related source substances Formamidopropylbetaine as well as C8-18 and C18 unsatd. AAPB are reported. A justification for read-across is given below.

In a dermal sensitisation study according to OECD Guideline 406 (1992) with Formamidopropylbetaine female young adult Pirbright White guinea pigs were tested using the Maximization test method.

The maximum compatible doses for the intradermal and dermal application as well as the subirritative dose for the challenge were determined in a pilot experiment. In the main experiment the animals were treated intradermally with 0.1 mL of a 3.5 % (w/w) dilution of the test substance in saline. For the dermal induction the undiluted test substance was used. Intradermal application of the 3.5 % (w/w) dilution of the test substance in saline caused slight to moderate erythema and edema formation and the topical application of the undiluted test substance caused slight erythema and edema formation. In the challenge application Duhring chambers with a 30 % (w/w) dilution of the test substance in saline were used, a test concentration which was definitely non-irritating.

Besides crust formation at the injection sides after intradermal application with FCA no other systemic toxic symptoms or toxic reactions were observed at any time during the study. Body weight development of the animals was positive and within normal ranges. In the challenge no visible changes on the skin (no erythema and no edema) were observed at any time point; i.e. there was no positive challenge result in the test and the control group animals. Thus, the sensitisation rate was 0 %. Therefore, Formamidopropylbetaine has to be regarded as non-sensitising under the applied test conditions when exposed to the skin of experimental animals.

In a dermal sensitisation study with C8-18 and C18 unsatd. AAPB (30 % a.i.) Pirbright white guinea pig (10 male, 10 female) were tested using the Maximisation Test method according to OECD Guideline 406, May 12, 1981.

The analytical purity of the test item is not stated in study report, according to the manufacturer's information the test material is C8-18 and C18 unsatd. AAPB and has 30 % a.i. , impurities relevant to sensitisation and referred to a.i. are max 1.7% Alkylamidopropylamine and 33 ppm DMAPA (3-dimethylaminopropylamine).

Responses to the challenge procedure were evaluated 24 and 48 h after the end of the exposure period.

There were no skin reactions at the first reading. At the second reading 4/20 test animals and 5/20 control animals showed skin reactions score 1 (scattered mild redness). As incidence and severity of reactions seen in the test group were not higher than responses seen in the negative control group, the calculated sensitisation rate was 0.

In this study, C8-18 and C18 unsatd. AAPB is not a dermal sensitiser.

In a dermal sensitisation study with C8-18 and C18 unsatd. AAPB (commercial product) 20 male Pirbright white guinea pig were tested using the MAXIMIZATION TEST described by b. Magnusson and A.M. Kligman (1970). The test method is similar to OECD Guideline 406 (Skin Sensitisation).

The analytical purity of the test item is not stated in study report, according to producer information test material has 30 % a.i., impurities relevant to sensitisation and referred to a.i. are 8.3 % Alkylamidopropylamine and 33 -50 ppm DMAPA (3-dimethylaminopropylamine).

At the first scoring, 24 h after challenge, 2/15 showed a skin reaction score 1 (slight erythema (barely perceptible)). The animals were free of erythema and edema at the second (48 h) and the third (72 h) scoring. None of the five negative animals showed up to and including the third scoring a positive skin reaction to challenge treatment.

In this study, C8-18 and C18 unsatd. AAPB (30 % a.i.) is not a dermal sensitiser.

Conclusion

Overall, the sensitising potential of the source substances was low. As fatty acids independently from their chain length and degree on unsaturation are generally considered to be non-sensitisers, a variability in the fatty acid moiety is not expected to have any influence on the sensitising potential of the substances.

Based on interpolation of the results from the available studies on sensitisation conducted with the closely related source substances C8-18 and C18 unsatd. AAPB and Formamidopropylbetaine, the target substance C8-10 Alkylamidopropyl betaine is considered to be not sensitising.

There is no information available for respiratory sensitisation. Therefore, there is a data gap in this respect. However, the data gap cannot be fulfilled with experimental data, since there is no internationally accepted animal model for respiratory sensitisation. In case human data for respiratory sensitisation emerges, this will be taken into account.

 

Justification for read-across

For details on substance identity and detailed toxicological profiles, please refer also to the general justification for read-across given at the beginning of the CSR and attached as pdf document to IUCLID section 13.

This read-across approach is justified based on structural similarities. The target and source substances contain the same functional groups. Thus a common mode of action can be assumed.

The only deviation within this group of substances is a variety in their carbon chain length, which is not assumed to have a relevant impact on sensitisation potential as demonstrated by the available data on the source substances on the extremes of the chain length distribution.

 

a. Structural similarity and functional groups

The target substance C8-10 Alkylamidopropyl betaine is a UVCB substance manufactured from fatty acids (C8 and C10) and N, N-dimethylpropylenediamine (DMAPA) and further reacted with sodium monochloroacetate.

The source substances C8-18 AAPB and C8-18 and C18 unsatd. AAPB are UVCB substances manufactured from natural fatty acids or oils and N, N-dimethylpropylenediamine (DMAPA) and further reacted with sodium monochloroacetate. As their origin is from natural sources, the used fatty acids may have a mixed slightly varying composition with an even numbered chain length from C8 to C18. Unsaturated C18 amounts may be included.

The source substance Formamidopropylbetaine is a monoconstituent substance manufactured from formic acid and N, N-dimethylpropylenediamine (DMAPA) and further reacted with sodium monochloroacetate.

 

b. Differences

Differences in chemical and other intrinsic properties of the target and source substances could potentially arise from the following facts:

-Different amounts of different carbon chain lengths (carbon chain length distribution):

Higher amounts of higher chain lengths and corresponding lower amounts of lower chain length lead to a rising average lipophilicity as can be seen from the increasing log Kow from Formamidopropylbetaine (log Kow: -3.3), C8-10 Alkylamidopropyl betaine (log Kow: 2.2), C12 AAPB (log Kow: 3.54), C8-18 AAPB (log Kow:4.23).

 

- Different amounts of unsaturated fatty ester moieties:

The source substance C8-18 and C18 unsatd. AAPB contains considerable amounts of unsaturated C18 chains, which represents a worst case with respect to some toxicological endpoints, mainly local effects (e.g. irritation, sensitisation) and reactivity.

 

The provided structural similarities and impurity profiles support the proposed read-across hypothesis with high confidence.

 

Comparison of sensitisation data

Endpoints

Source substance

Target substance

Source substance

 

C8-18 and C18 unsatd. AAPB

C8-10 Alkylamidopropyl betaine

Formamidopropylbetaine

Sensitisation,

Animal data

WoE_RA_GPMT_Skin sensitisation: 61789_40-0_8.3_REWO_1990_OECD 406

 

OECD TG 406, in vivo, Guinea pig maximisation test, guinea pig

 

not sensitising

 

Reliability: 2 (reliable with restrictions), GLP

No data, read-across

WoE_RA_LTOE 16280 Skin sensitization

 

OECD TG 406, in vivo, Guinea pig maximisation test, guinea pig

 

not sensitising

 

Reliability: 1 (reliable without restrictions), GLP

WoE_RA_(highest level of impurities)_Skin sensitisation: 61789-40-0_8.3_THG_1980a

similar to OECD TG 406, in vivo, Guinea pig maximisation test, guinea pig

 

not sensitising

 

Reliability: 2 (reliable with restrictions), no GLP

Supporting data on genotoxicity

Sup_RA_Genetic toxicity in vitro: 147170-44-3_8.4.1_Zschimmer_1996_EEC 92_69


EU Method B.13/14,gene mutation: bacterial reverse mutation assay (e.g. Ames test),
S. typhimurium TA 1535, TA 100, TA 1537, TA 1538 and TA 98

Metabolic activation: with and without

cytotoxicity: first evidence of toxicity at 10000 µg/plate with and without S9;

vehicle controls valid: yes; negative controls valid: yes; positive controls valid: yes

 

Genotoxicity: negative

 

Reliability: 2 (reliable with restrictions), no GLP

Key_Genetic toxicity in vitro: 73772-45-9 / 73772-46-0_8.4.1_Evonik_2014_OECD471

 

OECD TG 471, gene mutation: bacterial reverse mutation assay (e.g. Ames test),
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation: with and without

cytotoxicity: only in strain TA 100 at 5000 μg/plate;

vehicle controls valid: yes; negative controls valid: yes; positive controls valid: yes

 

Genotoxicity: negative

 

Reliability: 1 (reliable without restrictions), GLP

sup_RA_Genetic toxicity in vitro: 120128-90-7_8.4.1_Evonik_2005_OECD471

 

OECD TG 471, gene mutation: bacterial reverse mutation assay (e.g. Ames test),
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation: with and without

cytotoxicity: no;

vehicle controls valid: yes; negative controls valid: yes; positive controls valid: yes

 

Genotoxicity: negative

 

Reliability: 1 (reliable without restrictions), GLP

 

No experimental data on sensitisation are available for target substance C8-10 Alkylamidopropyl betaine.

The source substances C8-18 and C18 unsatd. AAPB and Formamidopropylbetaine were both not sensitising in a Guinea pig maximisation test.

Apart from structural similarities, further support is given by the negative outcome of all three substances in the bacterial reverse mutation assay hinting at low reactivity/electrophilicity as both endpoints, skin sensitisation and genotoxicity, are characterised by covalent binding as a rate determining step.

 

Quality of the experimental data of the analogues:

The available data are adequate and sufficiently reliable to justify the read-across approach.

The studies used in this weight-of-evidence approach were conducted according to (or comparable to) OECD Guideline 406 (Guinea pig maximisation test) and are reliable (RL1) or reliable with restrictions (RL2).

The test materials used in the respective studies represent the source substance as described in the hypothesis in terms of substance identity and minor constituents. A study with the test item containing the minor constituent DMAPA (3-dimethylaminopropylamine) in concentrations between 33-50 ppm and thus representing the upper level of the specifications, has been selected as one of the key studies.

Overall, the study results are adequate for the purpose of classification and labelling and risk assessment.

Conclusion

Based on structural similarities of the target and source substances as presented above and in more detail in the general justification for read across, it can be concluded that the available data from the source substances C8-18 and C18 unsatd. AAPB and Formamidopropylbetaine are also valid for the target substance C8-10 Alkylamidopropyl betaine.

As fatty acids independently from their chain length and degree on unsaturation are generally considered to be non-sensitisers, a variability in the fatty acid moiety is not expected to have any influence on the sensitising activity of the substances.

C8-18 and C18 unsatd. AAPB is a worst case model substance, as it contains short chain fatty acid moieties as well as unsaturated moieties, fatty acid amounts which might have some increasing influence on the irritating potential. Moreover, one maximisation test on this test substance with relatively high amounts of the potentially sensitising minor constituents alkylamidopropylamine and DMAPA is also included in this weight of evidence approach and serves as worst case with respect to potentially sensitising minor constituents.

Certain endpoints such as skin sensitisation and genotoxicity are characterised by covalent binding as a rate determining step or MIE (molecular initiating event). The consistency across endpoints - both, the source substances and target substance were not genotoxic in the bacterial reverse mutation assay - also helps to increase confidence in the read-across approach especially when MIEs are common for example, skin sensitisation and genotoxicity are underpinned by electrophilicity. 

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on reliable, adequate and relevant data, C8-10 Alkylamidopropyl betaine does not need to be classified for skin sensitisation according to regulation (EC) 1272/2008.