Amines, di-C12-18-alkylmethyl

Endpoint summary

Description of key information

There is no information of Amines, di-C12-18-alkylmethyl (CAS No.: 68439-75-8; EC No.: 270-418-8) itself but there is information available for 1-Decanamine, N-decyl, N-methyl (CAS No.: 7396-58-9; EC No.: 230-990-1) and Amines, di-C16-18-alkylmethyl (CAS No.: 1227096-04-9; EC No.: 627-132-7) which is structurally similar to Amines, di-C12-18-alkylmethyl.

Conclusion: negative for skin sensitization.

A read-across document has been constructed to validate conclusions drawn from these structurally similar chemicals and is present in Section 13.2 'Other assessment reports'.

 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 10-03-1990 to 11-23-1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

There is no information of Amines, di-C12-18-alkylmethyl (CAS No.: 68439-75-8; EC No.: 270-418-8) itself but there is information available for Amines, di-C16-18-alkylmethyl (CAS No.: 1227096-04-9; EC No.: 627-132-7) which is structurally similar to Amines, di-C12-18-alkylmethyl.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

Reliability scoring based on 1981 guideline for test n°406

Deviations:

yes

Remarks:

: no data reported on the positive control in the report

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Olac Limited, Bicester, Oxfordshire, England
- Age before acclimatation period: 4-6 weeks old
- Weight at study initiation: 330-451 g
- Housing: in stainless steel cages with grid floors, 5 animals of the same sexe in each cage
- Diet : ad libitum (samples of diet were analysed to detect contamination by PCB and chlorinated hydrocarbons)
- Water: ad libitum
- Acclimation period: yes but no more precision

ENVIRONMENTAL CONDITIONS
- Temperature : 18-20 °C
- Humidity: below 40 %
- Air changes : 15 per hour
- Photoperiod (hrs dark / hrs light):12/12 (no source of natural light)

Route:

intradermal and epicutaneous

Vehicle:

paraffin oil

Concentration / amount:

- Induction exposure:
intrademal: 10% w/v Test Substance (TS) in vehicle
occlusive epicutaneous: 10% w/v
- Challenge exposure:
occlusive epicutaneous: 3 % w/v and 0.5 % w/v

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

- Induction exposure:
intrademal: 10% w/v Test Substance (TS) in vehicle
occlusive epicutaneous: 10% w/v
- Challenge exposure:
occlusive epicutaneous: 3 % w/v and 0.5 % w/v

No. of animals per dose:

Control group: 10 animals (5 males and 5 females)
Treated group: 20 animals (10 males and 10 females)

Details on study design:

RANGE FINDING TESTS: conducted to define the concentrations to be tested in the main study.

Intradermal exposure:
24 hours before treatment: dorsal region clipped.
Intradermal administration of 0.1 mL of the test substance (TS) at increasing concentrations.
Evaluation of the potential cutaneous reactions 24 and 48 hours and approximately 168 hours after injection.

Epicutaneous exposure:
24 hours before treatment: dorsal region clipped.
0.4 mL of each concentration applied to a gauze patch of approximately 4 cm2 for 48 hours by occlusive dressing (4 concentrations per animal).
Evaluation of the potential cutaneous reactions 24 and 48 hours after patch removal.


MAIN STUDY

A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal, 1 epicutaneous
- Exposure period: epicutaneous: 48 hours

-> TEST GROUPS:
Intradermal exposure
Three injections of 0.1 mL were injected into each side of the animal as follows:
. Freund's complete adjuvant (FCA) diluted to 50% with distilled water
. TS at 10% w/v in vehicle
. TS at 5% w/v in a 50/50 (v/v) mixture of FCA and distilled water

Epicutaneous exposure
Application of 0.6 mL of TSat 10% w/v in paraffin oil applied to the scapular region and held in place for 48 hours using an occlusive dressing.

-> CONTROL GROUP:
Intradermal exposure
Three injections of 0.1 mL were injected into each side of the animal as follows:
. Freund's complete adjuvant (FCA) diluted to 50% with distilled water
. vehicle
. A mixture 50/50 (v/v) FCA diluted to 50% with distilled water, and vehicle

Epicutaneous exposure
Application of 0.6 mL of vehicle applied to the scapular region and held in place for 48 hours using an ocular dressing.

- Site:
Intradermal exposure
6 injections in the clipped area (4 x 2 cm) in the scapular region

Epicutaneous exposure
4 x 2 cmarea over the scapulae

- Frequency of applications:
One intradermal injection and one epicutaneous application 8 days after on the same site.

- Duration:
Epicutaneous exposure: 48 hours


B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 22
- Exposure period: 24 hours

-> TEST GROUPS:
0.03 mL of the TS at 3% w/v in vehicule to one site and 0.03 mL of the TS at 0.5% w/v in vehicule to a second site on the posterior right flank (occlusiv e epicutaneous application)
0.03 mL of the vehicle on the posterior left flank (occlusive epicutaneous application)

-> CONTROL GROUPS:
Same treatment as test group
- Site: posterior right and left flanks
- Evaluation (hr after challenge): 24 and 48 hours after removal of the dressing

Challenge controls:

Vehicle was applied at the challenge application on the posterior left flank of both test and control groups of animals.

Positive control substance(s):

not specified

Positive control results:

no data.
DNCB - within 6 month validation window on two seperte compounds.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

challenged with Armeen M2HT 3%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: challenged with Armeen M2HT 3%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Challenged with Armeen M2HT 3%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Challenged with Armeen M2HT 3%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Induced with Armeen M2HT and challenged with Armeen M2HT 3%

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

slight erythema

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Induced with Armeen M2HT and challenged with Armeen M2HT 3%. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: slight erythema.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Induced with Armeen M2HT and challenged with Armeen M2HT 3%

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

slight erythema

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Induced with Armeen M2HT and challenged with Armeen M2HT 3%. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: slight erythema.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

challenged with Armeen M2HT 0.5 %

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: challenged with Armeen M2HT 0.5 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

challenged with Armeen M2HT 0.5 %

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: challenged with Armeen M2HT 0.5 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Induced with Armeen M2HT and challenged with Armeen M2HT 0.5 %

No. with + reactions:

3

Total no. in group:

20

Clinical observations:

slight erythema

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Induced with Armeen M2HT and challenged with Armeen M2HT 0.5 %. No with. + reactions: 3.0. Total no. in groups: 20.0. Clinical observations: slight erythema.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Induced with Armeen M2HT and challenged with Armeen M2HT 0.5 %

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

slight erythema

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Induced with Armeen M2HT and challenged with Armeen M2HT 0.5 %. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: slight erythema.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

challenged with paraffin oil

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: challenged with paraffin oil. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

challenged with paraffin oil

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: challenged with paraffin oil. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

induced with Armeen M2HT and challenged with paraffin oil

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: induced with Armeen M2HT and challenged with paraffin oil. No with. + reactions: 0.0. Total no. in groups: 20.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Induced with Armeen M2HT and challenged with paraffin oil

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Induced with Armeen M2HT and challenged with paraffin oil. No with. + reactions: 0.0. Total no. in groups: 20.0.

CHALLENGE PHASE: individual dermal responses after removal of occlusive dressings

Group	Sexe	Animal	Topical application of 0.03 ml 3 %w/v Armeen in paraffin oil		Topical application of 0.03 ml 0.5 %w/v Armeen in paraffin oil		Topical application of 0.03 ml paraffin oil
			24 hours after removal of dressing	48 hours after removal of dressing	24 hours after removal of dressing	48 hours after removal of dressing	24 hours after removal of dressing	48 hours after removal of dressing
Control	M	601	0	0	0	0	0	0
		602	0	0	+-	+-	0	0
		603	0	0	0	0	0	0
		604	0	0	0	0	+-	+-
		605	0	0	0	0	0	0
Control	F	606	0	0	0	0	0	0
		607	0	0	0	0	0	0
		608	0	0	0	0	+-	+-
		609	0	0	0	0	0	0
		610	0	0	0	0	0	0
Treated	M	611	+-	0	0	0	0	0
		612	+-	0	0	0	0	0
		613	0	0	0	+-	0	0
		614	+-	+-	+-	+-	+-	+-
		615	+-	+-	+-	0	+-	0
		616	0	0	0	+-	0	0
		617	+-	0	0	0	+-	0
		618	0	0	0	0	+-	+-
		619	+-	0	+-	0	0	0
		620	0	+-	0	+-	+-	0
Treated	F	621	0	0	0	0	0	0
		622	0	0	0	0	0	0
		623	0	0	0	0	+-	+-
		624	+-	0	0	0	0	0
		625	+-	1	0	1	+-	+-
		626	+-	+-	+-	1	0	0
		627	+-	0	+-	0	+-	+-
		628	1	0	0	+-	0	0
		629	+-	0	0	0	+-	0
		630	0	0	0	1	0	0

0: No response

+-: Barely perceptible erythema

1: Slight erythema

2: Moderate erythema

3: Severe erythema

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

In the treated group, following cutaneous reactions were noted:
-Challenge application of 3 % w/v Armeen M2HT in paraffin oil caused slight erythema in 1 out of 20 animals in the treated group after the 24 and the 48-hour readings and no cutaneous reactions in the control group.
-Challenge application of 0.5 % w/v Armeen M2HT in paraffin oil caused slight erythema in 3 and 1 out of 20 treated animals after the 24 and 48-hour readings respectively. No cutaneous reactions were observed in the control group.
-Challenge application of paraffin oil alone caused caused no significant cutaneous reaction.

Executive summary:

The potential of Armeen M2HT to induce delayed contact hypersensitivity was assessed in guinea pigs according to the OECD guideline 406 and conducted in compliance with the principles of Good Laboratory Practice regulations.

Positive controls were not included in the study though guideline equivalent GPMT studies on Armoblen 600 & NAPAA were conducted with positive controls that were successful 6 months prior to and 3 months after respectively of this test in the same contract laboratory. This is consistent with the OECD guideline need for reliability checks in the place of positive controls. Further information on these studies can be found in section 7.12 "Additional toxicological information".

The induction phase has been realized both by intradermal route on day 1 (Armeen M2HT 10 % w/v in paraffin oil) and by cutaneous route on day 8 (Armeen M2HT 10% w/v in paraffin oil ) in 2 groups of guinea pigs: 5 males and 5 females for control group and 10 males and 10 females for treated group. The challenge phase was realized on day 22 by cutaneous application of Armeen M2HT 3% and Armeen M2HT 0.5% w/v in paraffin oil on two sites on the right flank (vehicle on the left flank). the cutaneous reactions were scored 24 and 48 hours after the challenge phase.

 

After the challenge application with 3 % w/v Armeen 2HT in paraffin oil, a slight erythema was observed in 1/ 20 animals of the treated group at the 24 and 48-hour readings. No cutaneous reactions were recorded in the control group.

 

After the challenge application with 0.5 % w/v Armeen 2HT in paraffin oil, a slight erythema was observed in 3 and 1 out the 20 animals of the treated group at the 24 and 48-hour readings respectively.

 

The persistent cutaneous reactions observed in 4/20 animals of the treated group after the challenge application may be attributable to delayed contact hypersensitivity but as the cutaneous reactions were more confined to the 24 hour examination, they were more consistent with irritation than delayed contact hypersensitivity.

 

Therefore, the cutaneous reactions observed in 4/20 animals (20%) of the treated groups were considered as irritative response and the substance was not considered as a skin sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 2010-06-22 to 2010-08-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Justification for type of information:

There is no information of Amines, di-C12-18-alkylmethyl (CAS No.: 68439-75-8; EC No.: 270-418-8) itself but there is information available for 1-Decanamine, N-decyl, N-methyl (CAS No.: 7396-58-9; EC No.: 230-990-1) which is structurally similar to Amines, di-C12-18-alkylmethyl.

Reason / purpose for cross-reference:

read-across source

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 (incl. QA statement)

Type of study:

guinea pig maximisation test

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Breeder: Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: on the first day of treatment, the animals of the main test were 1-2 months old
- Weight at study initiation: a mean body weight ± standard deviation of 349 ± 17 g for the males and 345 ± 15 g for the females
- Housing: housed individually in polycarbonate cages with stainless steel lid (43 cm x 22 cm x 20 cm). Autoclaved sawdust were placed in each cage
- Diet (e.g. ad libitum): free access to 106 pelleted diet
- Water (e.g. ad libitum): water (filtered with a 0.22 µm filter)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 220+/- 2°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00).

IN-LIFE DATES: From: 22 June 2010 To: 07 August 2010.

Route:

intradermal and epicutaneous

Vehicle:

other: intradermal: corn oil / topical: acetone

Concentration / amount:

Induction phase:
Concentration for intradermal injection: 5% *
Concentration for topical application: 50% *
*: highest to cause mild-to-moderate skin irritation based on preliminary assays

Challenge phase:
Concentration for topical application: 0.1%**
** highest non-irritant concentration based on preliminary assays

Route:

epicutaneous, occlusive

Vehicle:

other: intradermal: corn oil / topical: acetone

Concentration / amount:

Induction phase:
Concentration for intradermal injection: 5% *
Concentration for topical application: 50% *
*: highest to cause mild-to-moderate skin irritation based on preliminary assays

Challenge phase:
Concentration for topical application: 0.1%**
** highest non-irritant concentration based on preliminary assays

No. of animals per dose:

Number and sex:
- preliminary test: 14 animals (6 males and 8 females),
- main test: 30 animals (15 males and 15 females = for vehicle group: 5 males and 5 females + for treated group: 10 males and 10 females)

Details on study design:

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: topical induction lasted 48h
- Site: induction: interscapular region
- Frequency of applications: once intradermal, once topical

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 4 weeks between injection (induction) and challenge
- Exposure period: 24h
- Site: right flank (test item) and left flank (vehicle)
- Evaluation (hr after challenge): 24, 48, 72 after removal of dressing

Positive control substance(s):

yes

Remarks:

mercaptobenzothiazole (within a 6-month interval of the test)

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

Induction: 1% (w/w) on day 1 (intradermal route)
Induction: 20% (w/w) on day 8 (cutaneous route)
Challenge: 20% (w/w) on day 22 (cutaneous route)

No. with + reactions:

9

Total no. in group:

10

Clinical observations:

edema at site of contact in 1 aminal. Average score of 1.4 at 24hrs.

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

Induction: 1% (w/w) on day 1 (intradermal route)
Induction: 20% (w/w) on day 8 (cutaneous route)
Challenge: 20% (w/w) on day 22 (cutaneous route)

No. with + reactions:

9

Total no. in group:

10

Clinical observations:

dryness of skin and/or edema at site of contact in 7 aminals. Average score of 1.55 at 48hrs. evaluation in 1 animal unobtainable due to severty of dryness of skin.

Remarks on result:

positive indication of skin sensitisation

Reading:

other: 1st reading, 24 or 48h (combined) after challenge

Group:

negative control

Dose level:

0.1%

No. with + reactions:

6

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: other: 1st reading, 24 or 48h (combined) after challenge. Group: negative control. Dose level: 0.1%. No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: none.

Reading:

1st reading

Hours after challenge:

72

Group:

negative control

Dose level:

0.1%

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

Dryness of skin, 10/10 animals. Reaction is irritation and not sensitisation.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 0.1%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: Dryness of skin, 10/10 animals. Reaction is irritation and not sensitisation..

Reading:

other: 1st reading, 24 or 48h (combined) after challenge

Group:

test chemical

Dose level:

0.1%

No. with + reactions:

17

Total no. in group:

20

Clinical observations:

Dryness of skin at 48h, 5/20 animals. Reaction is irritation and not sensitisation.

Remarks on result:

other: see Remark

Remarks:

Reading: other: 1st reading, 24 or 48h (combined) after challenge. Group: test group. Dose level: 0.1%. No with. + reactions: 17.0. Total no. in groups: 20.0. Clinical observations: Dryness of skin at 48h, 5/20 animals. Reaction is irritation and not sensitisation..

Reading:

1st reading

Hours after challenge:

72

Group:

test chemical

Dose level:

0.1%

No. with + reactions:

5

Total no. in group:

20

Clinical observations:

Dryness of skin in 15/20 animals. Its severity masked the skin reading in 5/20 animals. Reaction is irritation and not sensitisation.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 72.0. Group: test group. Dose level: 0.1%. No with. + reactions: 5.0. Total no. in groups: 20.0. Clinical observations: Dryness of skin in 15/20 animals. Its severity masked the skin reading in 5/20 animals. Reaction is irritation and not sensitisation..

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Under these experimental conditions, the test itemdid not induce delayed contact hypersensitivity in guinea pigs.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce delayed contact hypersensitivity in guinea pigs according to the maximization method of Magnusson and Kligman and to OECD (No. 406, 17th July 1992) and Commission Regulation (EC) (No. 440/2008, B.6, 30 May 2008) guidelines. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

 

 Thirty guinea-pigs were allocated to 2 groups: a control group of 5 males and 5 females and a treated group of 10 males and 10 females.

 On day 1, three pairs of intradermal injections were performed in the interscapular region of all animals:

.            Freund's complete adjuvant (FCA) diluted to 50% (v/v) with 0.9% NaCl (both groups),

.            test item at the concentration of 5% in corn oil (treated group) or vehicle alone (control group),

.            test item at the concentration of 5% in a mixture FCA/0.9% NaCl (50/50, w/w) (treated group) or vehicle at the concentration of 50% (w/v)

in a mixture FCA/0.9% NaCl (50/50, v/v) (control group).

 

On day 8, the treated group animals received a topical application of the test item at the concentration of 50% (w/w) in acetone to the same test site, which was then covered by an occlusive dressing for 48 hours. The control group animals received an application of the vehicle under the same experimental conditions.

 

On day 29, all animals of both groups were challenged by a cutaneous application of the test item at the concentration of 0.1% (w/w) in acetone to the right flank. The test item was maintained under an occlusive dressing for 24 hours. The vehicle was applied to the left flank under the same experimental conditions.

Cutaneous reactions were evaluated in each animal 24, 48 and 72 hours after removal of the dressing.

 

Each animal was observed at least once a day for mortality and clinical signs during the treatment and observation periods. Body weight was recordedon the day of allocation into the groups, on day 1 and day 25.

On completion of the observation period, the animals were sacrificed then discarded without macroscopic post-mortem examination. Skin samples were taken each flank (challenge application sites) of all animals. No histological examination was performed.

 

No unscheduled mortality and no clinical signs were recorded in any animals. After the challenge application of the test item, a moderate erythema was noted on the right flank (test item application) of 1/10 animals at the 72-hour reading. A discrete erythema was observed on the right flank of 6/10, 5/10 and 0/10 animals of the control group at the 24, 48 and 72-hour readings, respectively. Dryness of the skin was also recorded in 6/10 animals. No cutaneous reactions were noted on the left flank (vehicle application) of the control animals during the whole challenge phase.

In the treated group, a moderate erythema was recorded on the right flank (test item application) of 4/20 and 1/20 animals at the 48 and 72-hour reading. A discrete erythema was noted on the right flank of 13/20, 11/20 and 3/20 animals at the 24, 48 and 72-hour readings, respectively. Dryness of the skin was recorded in 5/20 and 15/20 animals at the 48 and 72-hour readings, respectively. A severe dryness of the skin masked the evaluation of cutaneous reactions in 5/20 animals at the 72-hour reading. No cutaneous reactions were noted on the left flank (vehicle application) of the treated animals.

 

A higher incidence and severity in local reactions was noted on 20% of the animals of the treated group at the 48-hour reading. However, as the cutaneous reactions observed at the 24 and 72-hour readings in the animals of the treated group were of similar incidence and severity when compared to those recorded in the animals of the control group, they were attributed to the irritant properties of the test item but not to delayed contact hypersensitivity.

 

Therefore, under the experimental conditions of this study,the test item did not induce delayed contact hypersensitivity in guinea pigs.

 

Reference 3

Endpoint:

skin sensitisation, other

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Remarks:

Two of the 12 constituents fall outside the applicability domain. This is not seen to effect the results as the two structures out of domain show similar structure to other members within applicability domain and are unlikely to change mechanisms of action within the context of skin sensitization. As a result the prediction is considered reliable without restriction. The OECD Toolbox uses a standardized workflow approved under OECD Guideline No. 497.

Justification for type of information:

1. SOFTWARE
OECD QSAR Toolbox

2. MODEL (incl. version number)
Version: 4.5

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
As the product is a UVCB the boundary composition was utilized as a starting point. The substance was subsequently deaminated into its various constituents. All known constituents were examined via the toolbox for possible links to skin sensitization through the use of Integrated Testing Strategy (ITS) v2.

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
[[Explain how the model fulfils the OECD principles for (Q)SAR model validation. Consider attaching the QMRF and/or QPRF or providing a link]
- Defined endpoint:
Skin sensitization
EC3 from LLNA or Skin sensitization from GPMT assays for defined approaches (SS AW for DASS) Automated Workflow
- Unambiguous algorithm: takes the highest mode value from the 1 nearest neighbours
- Defined domain of applicability: ECHA REACH Database , OECD Toolbox V4.5
- Appropriate measures of goodness-of-fit and robustness and predictivity:
OECD Toolbox has shown to have repeatability, predictivity, and robustness as a model. As read-across is used goodness-of-fit is not applicable in this context. The model undergoes appropriate subcategorization dependent on the molecule being examined for read-across in an automated and standardized way to obtain a read-across source substance the closely represents target in three layers: structural, parametric, mechanistic. Accuracy and robustness of the model for this endpoint approach are covered in detail in OECD GD 497.
- Mechanistic interpretation:
No protein binding alerts were indicated by OASIS.

5. APPLICABILITY DOMAIN
When using ITS v2 the DASS AW predictions and consists of three layers: structural, parametric and mechanistic. In a read-across approach with the toolbox both structural and parametric are not considered as the toolbox has ensured some level of similarity via the training set to ensure suitable analogues. (OECD GD 497).
Those constituents that are not considered in-domain should be evaluated for their results presented here with caution.
- Descriptor domain: Not considered
- Structural domain: Not considered. Organic functional groups (US EPA) was the initial categorization for the read-across.
- Mechanistic domain: Considered
- Similarity with analogues in the training set: there is a high similarity with analogues among all constituent structures that were considered in-domain.
- Other considerations (as appropriate):
Structural similarity for interpretation of analogue substances for the use in the read-across predictions vary per individual constituent and were dependent on the standardized workflow analysis. Mechanistic domain remained similar for all constituents and was based off the endpoint being predicted and the available data within workflow selected databases.

6. ADEQUACY OF THE RESULT
The results predict structural similar endpoints for skin sensitization based off the automated workflow: EC3 from LLNA or Skin sensitization from GPMT assays for defined approaches (SS AW for DASS). This approach is recognized as part of the OECD Guideline No. 497: Guideline on Defined Approaches for Skin Sensitisation. This approach is acceptable to equivalent examination in combination with in vitro methods of select key event relationships in the Skin sensitization AOP to equate conclusions on skin sensitization as an IATA.

Qualifier:

according to guideline

Guideline:

other: Guideline No. 497, Defined Approaches for Skin Sensitisation 2022

Version / remarks:

ITS V2 was utilized for this approach

Outcome of the prediction model:

other: in silico - Negative for protien binding

Interpretation of results:

GHS criteria not met

Conclusions:

Results indicate a Negative response for skin sensitization for Amines, di-C12-18-alkylmethyl (CAS No.: 68439-75-8; EC No.: 270-418-8) after analysis on all knonw constituents of the UVCB.

OECD QSAR Toolbox, EC3 from LLNA or Skin sensitization from GPMT assays for defined approaches (SS AW for DASS) Automated Workflow was performed to examine potential for skin sensitization. 2 of 12 constituents fell outside of the applicability domain for the OECD Toolbox performed read-across.

Executive summary:

Summary:

In this approach the OECD QSAR Toolbox was used to examine the potential for skin sensitization and was performed in accordance with Guideline No. 497, Defined Approaches for Skin Sensitization, Integrated Testing Strategy (ITS) V2. EC3 from LLNA or Skin sensitization from GPMT assays for defined approaches (SS AW for DASS) Automated Workflow was used as a predefined approach which is contained within the OECD Toolbox v4.5. 

 

As the primary Amines, di-C12-18-alkylmethyl (CAS No.: 68439-75-8; EC No.: 270-418-8) is a UVCB it was decided before implementation of the read-across to apply ITS V2 to all known constituents to maintain transparency of results. There are 12 known constituents in the currently registered substance under REACH.

 

Results of read-across prediction and DASS AW domain:

CCCCCCCCCCCCCCN(C)CCCCCCCCCCCC : in domain – negative for skin sensitization
CCCCCCCCCCCCCCCCN(C)CCCCCCCCCCCCCCCC : in domain – negative for skin sensitization
CCCCCCCCCCCCN(C)C : in domain – negative for skin sensitization
CCCCCCCCCCCCN(CCCCCCCCCCCC)CCCCCCCCCCCC : out of domain – negative for skin sensitization
CCCCCCCCCCCCCCN(CCCCCCCCCCCC)CCCCCCCCCCCC : out of domain – negative for skin sensitization
CCCCCCCCCCCCN(C)CCCCCCCC : in domain – negative for skin sensitization
CCCCCCCCCCCCCCN(C)CCCCCCCCCCCCCC : in domain – negative for skin sensitization
CCCCCCCCCCCCCCCCCCN(C)CCCCCCCCCCCCCCCC : in domain – negative for skin sensitization
CCCCCCCCCCCCCCCCCCN(C)CCCCCCCCCCCC : in domain – negative for skin sensitization
CCCCCCCCCCCCN(C)CCCCCCCCCCCC : in domain – negative for skin sensitization
CCCCCCCCCCN(C)CCCCCCCC : in domain – negative for skin sensitization
CCCCCCCCCCCCN(C)CCCCCCCCCC : in domain – negative for skin sensitization

 

Identifier: Guideline No. 497, Defined Approaches for Skin Sensitization, Integrated Testing Strategy (ITS) V2

 

Date: 2022-11-03

 

Proprietary aspects: N/A, OECD Toolbox is Co-owned by ECHA and OECD.

 

Endpoint:Skin Sensitization

 

Species Modeled: Guinea Pig

 

Definition of the purpose and regulatory relevance: 

Integrated Testing Strategy (ITS) V2 is a recognized optional part of defined approaches to skin sensitization in the aim of identification and classification of skin sensitizers. It is important in helping to identify Key Events that can led to skin sensitization in humans through in silico techniques.

Rationale underlying the construction of the defined approach:

Guideline No. 497 was followed for the framework of the ITS and its role in defined approach to skin sensitization.

Description of the individual information sources used:

ECHA Skin sensitization Database was used as an initail qualifier for the predictions of read-across substances to the target substance.

Data interpretation procedure applied (DIP): The ITS was originally developed to use three information sources (DPRA, h-CLAT, and an in silico tool (Derek Nexus or OECD QSAR Toolbox)). Conclusions about classification can only be made with results from all three or in some select cases two. 

OECD Toolbox uses standardized workflows to determine the ITS outcome. The Read-across must go through both subcategorization of both structural and mechanistic as well as domain checks for a result to be validated. 

 

Chemicals used to develop and test the DIP:The initial validation of the DIP was performed by the OECD using both LLNA and human data on skin senstization which sensitivity and specificity were evaluated. See guideline No. 497 for details. Proficency has been demonstrated for the defined approach to ITS V1 and V2 in accordance with teh procedure outlined in Guideline No. 497.

 

Limitations in the application of the defined approach: For classification the defined approach for skin sensitization must follow the steps putlined in the DIP. OECD Toolbox is and the ITS V2 are limited by the databases utilizied in making the prediction which confines read-across to those chemicals confined within the database. 

 

Predictive capacity of the defined approach:

70% correct classification overall
ITSv2 vs. Human reference data: Statistics based on the UN GHS 1A/1B sub-categorisation
Performance (total N=57), NC (N=9), 1B (N=30), 1A (N=18)
Correct classification (%), 44%, 80%, 67%
Underpredicted (%,) NA, 10% (NC), 0% (NC); 33% (1B)
Overpredicted (%), 56% (1B); 0% (1A), 10% (1A), NA

Hazard identification performance of the ITSv2 DA in comparison to Human
reference data (N=62)

Accuracy (%) 87%
Sensitivity (%) 94%
Specificity (%) 44%
Balanced Accuracy (%) 69%

Consideration of uncertainties associated with the application of the defined approach:

As two of the twelve constituents fall outside applicability domains it is important to consider there are some unknowns with regard to the UVCBs potential for skin sensitization. Expert judgementa pplied to these constituents outside of domain leds to the conclusion that there is an unlikely possibility that they will have diffrent mechanistic behavior compared to that of the constituents within the domain.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

One study is recorded for this endpoint and was chosen as a key study. The delayed contact hypersensitivity of sodium hypophosphite was assessed using the Maximization method of Magnusson and Kligman. This study was conducted in compliance with the principles of Good Laboratory Practices and performed according to the OECD guideline 406.

 

While this study lacks a positive control similar GPMT studies on other substances were conducted around the time (<6 months) this study took place in a renowned contract laboratory. This provide adequate consistency and reliability check for the laboratory that conducted the study.

 

The induction phase has been realized both by intradermal route on day 1 (Test item at 10 % w/v in paraffin oil) and by cutaneous route on day 8 (Test item at 10% w/v in paraffin oil ) in 2 groups of guinea pigs: 5 males and 5 females for control group and 10 males and 10 females for treated group. The challenge phase was realized on day 22 by cutaneous application of Test item at 3% and 0.5% w/v in paraffin oil on two sites on the right flank (vehicle on the left flank). the cutaneous reactions were scored 24 and 48 hours after the challenge phase.

 

After the challenge application with the test item at 3 % in paraffin oil, a slight erythema was observed in 1/ 20 animals of the treated group at the 24 and 48-hour readings. No cutaneous reactions were recorded in the control group.

 

After the challenge application with the test item at 0.5 % w/v in paraffin oil, a slight erythema was observed in 3 and 1 out the 20 animals of the treated group at the 24 and 48-hour readings respectively.

 

The persistent cutaneous reactions observed in 4/20 animals of the treated group after the challenge application may be attributable to delayed contact hypersensitivity but as the cutaneous reactions were more confined to the 24 hour examination, they were more consistent with irritation than delayed contact hypersensitivity.

 

Therefore, the low incidence of cutaneous reactions observed in treated animals (20%) were considered as irritative response and the substance was not considered as a skin sensitiser.

 

Migrated from Short description of key information:

The potential of the substance to induce delayed contact hypersensitivity was investigated using the Maximization method of Magnusson and Kligman(OECD 406, GLP) . The test item was found to be non-sensitising.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Migrated from Short description of key information:

There is no reported case of respiratory sensitisation in humans.

Justification for classification or non-classification

Skin sensitisation:

In accordance with column 2 of REACH annex VII point 8.3, the murine Local Lymph Node Assay (LLNA) (OECD guideline 429) is the first-choice method for in vivo testing of skin sensitisation. However a reliable guinea-pig maximization test was already available (Rees, 1991).

According to the results of the guinea-pig maximization test and the criteria laid down in EU regulation (EC) n°1272/2008 (CLP) and EU Directive 67/548/EEC, the substance is not classified for skin sensitisation.