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Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Aug 16 to 14 Oct 16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
The substance has been found to be surface active. The OECD guideline states that surface active substances have been found to cause false positives in the LLNA study; various comparative studies of the local lymph node assay, guinea pig maximization test, and in vitro methods confirm this to be the case. Furthermore, lymphocyte proliferation has also been shown to be influenced by several factors including vehicle selection. OECD recommended vehicles include: acetone/olive oil (AOO: 4 : 1 v/v), dimethylformamide, methyl ethyl ketone, propylene glycol, dimethyl sulphoxide, and dimethyl sulfoxide (DMSO). Several of these vehicles including AOO, DMSO, and propylene glycol have been shown to augment the LLNA response of certain chemicals. For example, AOO has been shown to give highly variable results when used as a vehicle in the LLNA. In addition, research suggests that olive oil itself may cause contact allergy in certain circumstances.. Therefore, it cannot be excluded that a non-specific stimulation of the lymph nodes occurred, not related to skin sensitization, and thus providing a false positive in this test. Based on these results and evaluation and according to the recommendations made in the test guidelines (including all amendments), MLA-3202 may possibly be regarded as skin sensitizer. However, a false positive outcome cannot be excluded, and further investigation work for this endpoint was required. Selection of the subsequent Buehler test was undertaken in order to further evaluate this endpoint, and the results confirm the conclusion that the LLNA provided a false positive result.

Test material

Constituent 1
Chemical structure
Reference substance name:
Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol
EC Number:
701-003-6
Cas Number:
1454803-04-3
Molecular formula:
C20H39NO3 to C26H51NO3
IUPAC Name:
Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol
Test material form:
liquid
Details on test material:
Identification: MLA-3202
Appearance: Clear amber-red liquid
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until 17 February 2019 (expiry date)
Purity/composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: Amides, tallow, N,N-bis(2-hydroxypropyl)
CAS Number: 1454803-04-3
Test item handling No specific handling conditions required
Specific details on test material used for the study:
No further details specified

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Experimental AnimalsSpecies & Strain: Guinea Pig; Hartley-AlbinoJustification of Species: The guinea pig is conventionally used in skin sensitization studies to provide information on which human hazard can be judged, and is preferred by regulatory agencies.Source: Charles River, Hdq: Wilmington, MAQuantity & Sex: 2/sex (Range-fmding); 15/sex (Defmitive); females nulliparous & non-pregnantAcclimation Period: At least 5 daysDate Born/Date Received: 17 & 22 Jun 16 / 22 Aug 16Animal Identification: Ear punch & cage cardWeights When Tested: 388-563 gAnimal HusbandryCage Type: Stainless steel, suspended, wire bottom with plastic liner & beddingHousing: 1 - 5 per cage (males separate from females); enrichment providedEnvironmental ControlsSet to Maintain:20 ± 3 °C target temperature; 12-hr light/12-hr dark cycle; 30- 70% target humidity10+ air changes per hourActual Temp/Rel. Humidity: 16- 25°C / 50- 79%Food: LabDiet® 5025 Guinea Pig Diet; available ad libitumWater: Municipal water supply analyzed by TCEQ Water Utilities Division; available ad libitum from water bowlsAnimal husbandry and housing at STILLMEADOW, Inc. comply with standards outlined in "Guide for the Care and Use of Laboratory Animals" (NRC Publ.). No contaminants were expected to have been present in feed or water that would have interfered with or affected study results.

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
0.4 mL
Day(s)/duration:
Once weekly for three weeks
Adequacy of induction:
not specified
Challenge
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
0.4 mL
Day(s)/duration:
48 hours
Adequacy of challenge:
not specified
No. of animals per dose:
Two male and two female albino guinea pigs were selected for irritation range-finding.Naive control group (5/sex).Test group (10/sex).
Details on study design:
Irritation Range-Finding Two male and two female albino guinea pigs were selected for irritation range-finding to determine both maximum dose producing no more than moderate irritation, and maximum non-irritating dose.Concentrations tested in the range-finder were 100% (undiluted), and 75%, 50% and 25% v/v dilutions in DI water, with each animal receiving 0.4 mL of each concentration at different test sites.Preparation of AnimalsMales and females were selected for each of two treatment groups, designated as Naive control group (5/sex) and Test group (10/sex). On the day prior to each treatment, animals were prepared by clipping the back of the trunk free of hair to expose a sufficiently sized test area on each animal. Individual body weights were recorded on Days 0 and 31.Test Substance AdministrationBased on range-finding results, the dose administered was an application of 0.4 mL of undiluted test substance.For each induction treatment, Test group animals were treated by introducing test substance beneath -5 x 5 cm surgical gauze patch. Each gauze patch was placed laterally from the midline of the back on the left front quadrant of exposure area, held by a strip of clear polyethylene film placed over the patch and secured with non-irritating tape. At the end of the 6-hour exposure period, wrappings and patches were removed, and animals returned to their cages. Test animals were treated once weekly for three weeks (Days 1, 8 and 15) with 0.4 mL of undiluted test substance, using the same treatment regimen and test site location for all three inductions. Naive group animals remained untreated during induction phase of the study.Challenge TreatmentAfter a two-week rest period, all animals (both groups) were each challenged on Day 29 at a virgin test site with an application of 0.4 mL of undiluted test substance. The dose was applied in a manner identical to induction treatments, except the test site was placed laterally on the right rear quadrant of exposure area.Observations and Scoring MethodObservations for skin reactions at each test site were made -24 hours after each unwrap. In addition, observations for skin reactions were made -48 hours after both the first induction and the challenge unwraps.The scoring scale for grading skin reactions is presented as follows.Erythema ScoreNo reaction 0Very faint, usually non-confluent 0.5Faint, usually confluent 1Moderate 2Strong, with or without edema 3An average score for each time period was obtained by adding all scores for each time period and dividing by number of test sites scored for that time period. The test substance is considered a sensitizer if mean irritation scores, total number of animals with scores, and/or total number of scores for the virgin test site in the Test group after challenge treatment are appreciably greater than those for the Naive challenge group.
Challenge controls:
Not specified
Positive control substance(s):
yes
Remarks:
Alpha-Hexylcirmamaldehyde, >95%, CAS# 101-86-0,

Results and discussion

Positive control results:
The mean score of 0.8 for the Test group after challenge treatment, when compared with the Naive control group mean score of 0.0, confmned sensitivity of guinea pigs to the positive control material.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0.4 mL
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Non specified
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0.4 mL
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Non specified

Any other information on results incl. tables

Range-finder

Animal Number

Body Wt (g)

Observation Time After Treatment

24 Hour Score*

48 Hour Score*

Front Site

Rear Site

Front Site

Rear Site

31-M

415

0

0

0

0

0

0

0

0

32-M

420

0

0

0

0

0

0

0

0

33-F

405

0

0

0

0

0

0

0

0

34-F

398

0

0

0

0

0

0

0

0

* - Observations made for erythema; M – Male; F – Female

Level Selected for Induction: 100% as received

Level Selected for Challenge: 100 % as received

 

Skin Reaction Scores

Animal No.

Hours After Day of Treatment

Induction Treatments LF

 

Challenge RR

Day 1

Day 8

Day 15

Day 29

24 hr

48 hr

24 hr

48 hr

24 hr

48 hr

Naïve Control

41-M

42-M

43-M

44-M

45-M

 

46-F

47-F

48-F

49-F

50-F

 

 

Test

51-M

52-M

53-M

54-M

55-M

 

56-F

57-F

58-F

59-F

60-F

 

61-M

62-M

63-M

64-M

65-M

 

66-F

67-F

68-F

69-F

70-F

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0

0

0

0

0

 

0

0

0

0

0

 

0

0

0

0

0

 

0

0

0

0

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0

0

0

0

0

 

0

0

0

0

0

 

0

0

0

0

0

 

0

0

0

0

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0

0

0

0

0

 

0

0

0

0

0

 

0

0

0

0

0

 

0

0

0

0

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0

0

0

0

0

 

0

0

0

0

0

 

0

0

0

0

0

 

0

0

0

0

0

 

0

0

0

0

0

 

0

0

0

0

0

 

 

 

0

0

0

0

0

 

0

0

0

0

0

 

0

0

0

0

0

 

0

0

0

0

0

 

0

0

0

0

0

 

0

0

0

0

0

 

 

 

0

0

0

0

0

 

0

0

0

0

0

 

0

0

0

0

0

 

0

0

0

0

0

 

M – Male; F – Female

LF – Left Front test site; RR – Right Rear test site

Note: Observations were made for erythema

 

Body Weights

Animal Number

Day of Study

Animal Number

Day of Study

Day 0

Day 31

Day 0

Day 31

Naïve Control

41-M

516

669

46-F

407

522

42-M

502

667

47-F

388

508

43-M

449

646

48-F

406

558

44-M

481

680

49-F

421

528

45-M

479

643

50-F

469

577

Test

51-M

549

793

61-M

498

560

52-M

494

707

62-M

466

584

53-M

476

756

63-M

474

579

54-M

464

641

64-M

506

631

55-M

563

873

65-M

505

557

56-F

449

555

66-F

435

696

57-F

440

564

67-F

451

715

58-F

434

526

68-F

464

781

59-F

443

569

69-F

453

712

60-F

429

547

70-F

442

718

M – Male; F – Female

Note: Weights are in grams

 

Positive Control Skin Reaction Scores

Animal No.

Hours After Day of Treatment

Induction Treatment LF

 

Challenge RR

Day 1

Day 8

Day 15

Day 29

24 hr

48 hr

24 hr

24 hr

24 hr

48 hr

Naïve Control

41-M

42-M

43-M

44-M

45-M

 

46-F

47-F

48-F

49-F

50-F

 

 

Test

51-M

52-M

53-M

54-M

55-M

 

56-F

57-F

58-F

59-F

60-F

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0

0

0

0

0

 

0

0

0

0

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0

0

0

0

0

 

0

0

0

0

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0

0

0

0

0

 

0

0

0

0

0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.5

0

0

0.5

0

 

0.5

0

0

0.5

1

 

0

0

0

0

0

 

0

0

0

0

0

 

 

 

2

0.5

0.5

1

1

 

0.5

1

1

0.5

1

 

0

0

0

0

0

 

0

0

0

0

0

 

 

 

1

0

0.5

0.5

1

 

0.5

0.5

1

0

1

 

Male – Male; F – Female; LF – Left Front test site; RR – Right Rear test site

Note: Observations were made for erythema

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance produced no reaction in either Test animals or Naive control animals after the challenge treatment. Therefore, MLA-3202, Batch RC-1045, CAS 1454803-04-3 is not a sensitizer in guinea pigs.
Executive summary:

A skin sensitization study was conducted on 15 male and 15 female short-haired albino guinea pigs to determine if test substance MLA-3202, Batch RC-1045, CAS 1454803-04-3 produced a sensitizing reaction.

 

The study was conducted in accordance with US EPA OCSPP 870.2600 and OECD 406 guidelines.

 

Males and females were assigned to each of two groups, designated Naive control (5/sex) and Test (10/sex).

 

Naive control group animals remained untreated during induction phase of the study. Test group animals were treated with 0.4 mL of undiluted test substance (selected from range-finding). Test animals were treated once weekly for three weeks, for a total of three inductions. After a two-week rest period, all animals (both groups) were challenged at a virgin test site with an application of 0.4 mL of undiluted test substance.

 

The test substance produced no reaction in either Test animals or Naive control animals after the challenge treatment. Therefore, MLA-3202, Batch RC-1045, CAS 1454803-04-3 is not a sensitizer in guinea pigs.