1,6-Hexanediamine, N1,N1,N6,N6-tetramethyl-, propoxylated (>1 < 4,5 mol PO)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-Jul-2020 - 02-Feb-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

substance is highly irritating, therefore out of applicability domain of LLNA

Test material

Test material information

Constituent 1

Reference substance name:

1,6-Hexanediamine, N1,N1,N6,N6-tetramethyl-, propoxylated (>1 < 4,5 mol PO)

EC Number:

605-146-4

Cas Number:

158451-78-6

Molecular formula:

C16H38N2O2

IUPAC Name:

1,6-Hexanediamine, N1,N1,N6,N6-tetramethyl-, propoxylated (>1 < 4,5 mol PO)

Test material form:

liquid

Details on test material:

Purity: 13,5% a.s., 19% a.i. (contains a.s. plus 2,3% 1,6-Hexa… isomere plus 3,2% Propandiol) see substance characterisation No. 20L00045 for details
CAS No.: 158451-78-6
BASF substance No.: 14/0384-3
Batch: 21245033
Purity/ content: identity confirmed / water 81.0 g/100 g

Specific details on test material used for the study:

Name of Test Item: 1,6-Hexanediamine, N1,N1,N6,N6-tetramethyl-, propoxylated
CAS No.: 158451-78-6
BASF substance No.: 14/0384-3
Batch: 21245033
Purity/ content: identity confirmed / water 81.0 g/100 g; an analytical characterization is given (No. 20L00045)
Purity: 13,5% a.s., 19% a.i.

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

not specified

Details on test animals and environmental conditions:

In total, ten adult SPF albino guinea pigs of the stock Dunkin Hartley (Envigo RMS GmbH) were utilized in four pre-tests whereas each 2 animals were allocated to two intradermal pre-tests and each 3 animals in two dermal pre-tests. For the two main tests, in total 30 animals were used with a body weight between 300 g and 394 g at the beginning of the tests.
The guinea pigs were randomly allocated into two groups of 2 to 3 animals each for two consecutive main tests. In order to ensure secure identification, the animal with the highest number per cage was marked with red ink on its forehead whereas the second highest was marked likewise with green colour.
An acclimatization period of at least five days was allowed.
The study took place in rooms provided with filtered air at a temperature of 203 °C, relative humidity being at least 30 % and preferably not exceed 70 % and air changes of 10 times/hour. The room was illuminated to give a 24-hour cycle of 12 hours light and 12 hours darkness whereas light was on from 6 a.m. to 6 p.m.
The guinea pigs were housed in Macrolone® cages (approx. 2280 cm²), two or three animals to a cage. The cages were cleaned and the bedding was changed 3 times a week. Fur¬ther-more, specific enrichment material like hay bricks (Granovit AG, CH-4303 Kaiser¬augst, Switzerland) as well as red polycarbonate houses (Plexx, The Netherlands) were pro¬vided to the animals.
“JELUXYL HW 300/500” (Altromin, D-32791 Lage, Lippe) served as bedding material. Analyses are performed regularly and certificates of analysis are retained as well as archived
The guinea pigs had free access to a pelleted diet "Altromin 3123" (Altromin, D-32791 Lage, Lippe). Analyses are performed regularly and certificates of analysis are retained as well as archived.
The guinea pigs had free access to bottles with domestic quality drinking water being en¬riched with vitamin C and acidified with hydrochloric acid to pH 2.5 in order to prevent micro¬bial growth. The drinking water is analysed regularly and certificates of analysis are retained as well as archived.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

40 % w/w (corresponding to 5.48 % a. s. or 7.6 % total a. i., respectively)

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Induction 2

Route:

intradermal

Vehicle:

water

Concentration / amount:

0.69 %w/w (corresponding to 0.09 % a. s. or 0.13 % total a. i., respectively)

Day(s)/duration:

2d

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

Challenge 1

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

15 %w/w (corresponding to 2.03 % a. s. or 2.85 % total a. i., respectively)

Day(s)/duration:

2d

Adequacy of challenge:

other: 2nd main study 1st challenge due to irritating results of 1st main study 1st challenge

Challenge 2

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

15 %w/w (corresponding to 2.03 % a. s. or 2.85 % total a. i., respectively)

Day(s)/duration:

1d

Adequacy of challenge:

other: 2nd main study 1st challenge due to irritating results of 1st main study 1st challenge

Challenge 3

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

15 %w/w (corresponding to 2.03 % a. s. or 2.85 % total a. i., respectively)

Day(s)/duration:

2d

Adequacy of challenge:

other: Due to observed ambiguous challenge results during 1st main test a reduction of TI concentration to 15 %w/w

Challenge 4

No.:

#1

Route:

epicutaneous, occlusive

Concentration / amount:

30 % w/w (corresponding to 4.05 % a. s. or 5.7 % total a. i., respectively)

Day(s)/duration:

1d

Adequacy of challenge:

other: ambiguous challenge results during 1st main test indicating a probable irritant effect of the applied 30 %w/w concentration of the test item

No. of animals per dose:

10

Details on study design:

RANGE FINDING TESTS: yes

Two intradermal pre-tests were accomplished with each one test concentration in 2 guinea pigs as following described. Generally, the intradermal pre-test served for concentration deter-mination of the test item to be applied in the main test. Therefore, a concentration for intradermal induction has to be determined which is not causing skin necrosis 24 and 48 hours post application.

Two dermal pre-tests were accomplished with each 3 guinea pigs as following described. Generally, the 2x 3 animals were treated with adjuvant (FCA) 3 to 4 weeks before conduction of the respective dermal pre-test. Therefore, animals were shaved in the scapular region and pairwise injected in two rows of 4 intradermal injections of 0.1 mL of adjuvant. The dermal pre-test served for concentration determination of the test item to be applied in the main test. For that, the minimal irritating concentration for dermal induction and the maximal non-irritating concentration for challenge have to be determined.

Based on the results of pre-tests, the following concentrations of the test item were chosen in agreement with the sponsor initially for the 1st main test:
 Intradermal induction of
 test group with the 0.69 %w/w (corresponding to 0.09 % a. s. or 0.13 % total a. i., respectively) test item
 control group with the vehicle water
 Dermal induction of
 test group with the 40 %w/w (corresponding to 5.4 % a. s. or 7.6 % total a. i., respectively) test item
 control group with the vehicle water
 Challenge treatment of test group and control group
 with the 30 %w/w (corresponding to 4.05 % a. s. or 5.7 % total a. i., respectively) test item
 challenge application of the vehicle was waived because the vehicle was water only
Due to observed ambiguous challenge results during 1st main test indicating a probable irri¬tant effect of the applied 30 %w/w concentration of the test item, a reduction of TI concen¬tra¬tion to 15 %w/w (corresponding to 2.025 % a. s. or 2.85 % total a. i., respectively) was agreed with the sponsor for both the re-challenge of the 1st main test on one hand and for the challenge of the 2nd main test on the other hand.


MAIN STUDY Two Main Studies where conducted

A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal, 1 dermal
- Exposure period: intradermal injection and on day 7 afterward dermal induction
- Test groups: 2
- Control group: 1
- Frequency of applications: one injection and one 48h dermal patch application
- Duration: one injection and one 48h dermal patch application
- Concentrations:
Injection - 0.69 %w/w (corresponding to 0.09 % a. s. or 0.13 % total a. i., respectively) test item in the vehicle water
dermal induction - patch with 0.5 mL of the 40 %w/w (corresponding to 5.4 % a. s. or 7.6 % total a. i., respectively) test item in the vehicle water

B. CHALLENGE EXPOSURE Two Main Studies where conducted
- No. of exposures: On day 21, challenge treatment was performed
- Day(s) of challenge: challenge day 21, rechallenge day 28
- Exposure period: 24h
- Test groups: 2
- Control group: 2
- Site: challenge caudal right flank , rechallenge cranial left flank
- Concentrations: The difference between the challenge treatments in the 1st and 2nd main tests was the test item concentration which was applied. Whereas the test item was prepared with concentration of 30 %w/w (corresponding to 4.05 % a. s. or 5.7 % total a. i., respectively) for the 1st main test, the TI preparation for the 2nd main test and rechallenge had a reduced concentration of only 15 %w/w (corresponding to 2.025 % a. s. or 2.85 % total a. i., respectively).
- Evaluation (hr after challenge): 24h and 48h

Challenge controls:

yes

Positive control substance(s):

no

Remarks:

Verification of test method The sensitivity and reliability of the experimental procedure and test systems were verified. The last positive control study (Lab. No. 04239) with the reference material α-Hexylcinnam-aldehyde, technical grade, 85 % was

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

15 %w/w (corresponding to 2.03 % a. s. or 2.85 % total a. i., respectively)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no erythema

Results 2

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

15 %w/w (corresponding to 2.025 % a. s. or 2.85 % total a. i., respectively)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no erythema

Results 3

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

15 %w/w (corresponding to 2.03 % a. s. or 2.85 % total a. i., respectively)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no erythema

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

15 %w/w (corresponding to 2.03 % a. s. or 2.85 % total a. i., respectively)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no erythema

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

30 %w/w (corresponding to 4.05 % a. s. or 5.7 % total a. i., respectively)

No. with + reactions:

5

Total no. in group:

5

Clinical observations:

erythema 4 animals grade 1, 1 animal grade 2, 0 animals grade 3

Results 6

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

30 %w/w (corresponding to 4.05 % a. s. or 5.7 % total a. i., respectively)

No. with + reactions:

4

Total no. in group:

5

Clinical observations:

erythema 3 animals grade 1, 1 animal grade 2, 0 animals grade 3

Results 7

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

30 %w/w (corresponding to 4.05 % a. s. or 5.7 % total a. i., respectively)

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

erythema 3 animals grade 1, 0 animal grade 2, 2 animals grade 3

Results 8

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

30 %w/w (corresponding to 4.05 % a. s. or 5.7 % total a. i., respectively)

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

erythema 4 animals grade 1, 1 animal grade 2, 3 animals grade 3

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the Guinea Pig Maximization Test (GPMT) described in this Final Report, the test item 1,6-Hexanediamine, N1,N1,N6,N6-tetramethyl-, propoxylated of batch: 21245033 has no skin sensitizing potential.

Executive summary:

The skin sensitization potential of the test item 1,6-Hexanediamine, N1,N1,N6,N6-tetra­methyl-, propoxylated was examined based on the methods of the OECD guideline 406 (1992), council regulation (EC) No 440/2008 part B.6. (2008)and U.S. EPA guideline OPPTS 870.2600 (2003)in a modified manner. For the study, the Guinea Pig Maximization test (GPMT) of MAGNUSSON and KLIGMAN (1970)¹ was applied (BASF SE 2021).

Altogether, two main tests were accomplished with 30 guinea pigs being organized in two control groups (1 & 2) of each 5 animals and two test groups (1 & 2) of each 10 animals. The test concen­trations for the main tests were determined according to the results of preliminary investi­ga­tions (each two dermal and intra­der­mal pre-tests).

The main tests started with a 2-week induction period in which the animals of the test group were induced with the test item whereas animals of the control group were induced with the vehicle water (distilled or for injections).

In the first induction, the 0.69 %w/w (corresponding to 0.09 % a. s. or 0.131 % total a. i., respectively) test item in the vehicle water and in an adjuvant solution [1:1-mixture of Freund’s Complete Adjuvant (FCA) and water for injections (WFI)] was intradermal injected in pairs into the scapular area of the animals of the test groups. Animals of the control groups were pairwise injected only with the vehicle or a 50 %w/v concen­tration of the vehicle in the adjuvant solution in an intradermal manner. Furthermore, all ani­mals got a third pair of intradermal injections consisting of the adjuvant solution as a 1:1-mixture of FCA and WFI.

One week after intradermal induction, the 40 %w/w (corresponding to 5.4 % a. s. or 7.6 % total a. i., respectively) test item was applied under occlusive conditions percutaneous on the skin of the scapular area of the animals of the test groups for 48 hours whereas in the control groups, dermal induction was accomplished with the vehicle water only.

Following intradermal and dermal induction in the 1^st main test, all 15 animals of the control and test group displayed graduated skin reactions in form of erythema (grade 1 to 3) or no reactions (grade 0) on the different injection sites. Injections sites involving the adjuvant appeared necrotic and open/bloody on the day before the dermal induction and at the end of dermal induction application. The skin reactions during the 2^nd main test following intradermal and dermal induction were similar to those from the 1^st main test and ranged between grades 0 and 3 for ery­the­ma on the different injection sites. Injections sites involving the adjuvant appeared necrotic, scabbed as well as open/bloody on the day before the dermal induction and at the end of the dermal induction application.

Three weeks after the first induction, animals of the control and test groups in both main tests were subjected to challenge treatments comprising a 24-hour occlusive application of the test item onto skin of the caudal right flank. However, the challenge treatment was performed with the 30 %w/w (corresponding to 4.05 % a. s. or 5.7 % total a. i., respectively) test item during the 1^st main test and with the 15 %w/w (corresponding to 2.025 % a. s. or 2.85 % total a. i., respectively) test item during the 2^nd main study. Simultaneous, occlusive appli­ca­tion of the vehicle onto the skin of the caudal left flank was waived in both main tests, because the vehicle was water only. Skin reac­tions were always assessed 24 and 48 hours after patch removal.

In the 1^st main test, 7 of the 10 animals of the test group (70 %, 7/10) responded with skin reactions to the challenge treatment ranging between grades 1 and 3. Also 4 of 5 control group animals (80 %, 4/5) displayed a discrete or patchy erythema (grade 1), whereas a moderate and confluent erythema (grade 2) was observed in 1 of 5 control group animals (20 %, 1/5). Since no control group animal displayed intense erythema and swelling, only this maximum grade-3-response which was observed in 2 of 10 test group animals (20 %, 2/10) both 24 and 48 hours after the challenge treatment with the 30 %w/w (corresponding to 4.05 % a. s. or 5.7 % total a. i., respectively) test item may be considered as positive reac­tion. However, this incidence rate of 20 % potentially positive reactions in the test group 1 was below the sensitizer threshold value of 30 %. 

In accordance to agreement with the sponsor, a re-challenge with halved test item con­cen­tration should verify the outcomes of the 1^st main test described above which supplied indications for an irritation potential of the chosen test item concentration of 30 %w/w although none of the three pre-test animals responded with skin reactions to the treatment with the 30 %w/w test item during the 2^nd dermal pre-test.

One week after the challenge in the 1^st main test, the same animals of the control and test group were subjected to a re-challenge treatment comprising a 24-hour occlusive application of the 15 %w/w (corresponding to 2.025 % a. s. or 2.85 % total a. i., respectively) test item onto the skin of the cranial left flank. Simultaneous, occlusive appli­ca­tion of the vehicle onto the skin of the cranial right flank was waived again, because the vehicle was water only. Skin reac­tions were assessed 24 and 48 hours after patch removal. Due to simultaneous observations concerning the skin areas on the caudal right flank, which were treated with the 30 %w/w test item one week before, these skin reactions were also recorded 8 and 9 days after termination of challenge application during the 1^st main test. During the re-challenge in the 1^st main test, none of the 10 animals of the test group 1 (0 %, 0/10) and none of the 5 animals of the control group 1 (0 %, 0/5) responded with skin reactions to the re-challenge treatment with the 15 %w/w (corresponding to 2.025 % a. s. or 2.85 % total a. i., respectively) test item. However, a majority of control and test group animals displayed open skin defects (with varying sizes of about 0.5 cm² to 2 cm²), eschar or scales on the caudal right flanks one week after the challenge treatment on these skin sites. These observations confirmed the indications of an irritant or even corrosive effect of the 30 %w/w test item concentration used for the challenge during in the 1^st main test (contrary to the pre-test results).

Moreover, sudden death of one test group animal was noticed about 24 hours after re-challenge patch removal. However, conducted necropsy of this deceased animal No. 14 revealed doubtlessly a ruptured aneurysm of the abdominal aorta and internal bleeding as causes of death.

In a 2^nd main test with 15 additional animals the non-sensitizing outcome of the 1^st main test should be confirmed under consideration of gained knowledge that 30 %w/w was not the maximal non-irritating concentration of the test item.

In the 2^nd main test, none of the 10 animals of the test group 2 (0 %, 0/10) and none of the 5 animals of the control group 2 (0 %, 0/5) responded with any skin reaction to the challenge treatment with the 15 %w/w (corresponding to 2.025 % a. s. or 2.85 % total a. i., respectively) test item neither 24 nor 48 hours post application.

Performance of a re-challenge was not necessary due to this unambiguous outcome of the challenge in the 2^nd main test.

The absence of positive skin reactions in the two test groups of 1^st and 2^nd main test after re-challenge or challenge treatment with the non-irritating test item concentration of 15 %w/w (corresponding to 2.025 % a. s. or 2.85 % total a. i., respectively) indicated no sensitizing effect of the test item.