3,6-dioxaoctamethylenediamine

Endpoint summary

Description of key information

two in vivo skin sensitizing study was performed in 1985 (First study - Klimisch 1) and the second one in 1988 (Second study - Klimisch 1). The method used was similar or equivalent to the OECD Guideline 406 (Skin Sensitisation).

The test was performed to assess the capacity of the test item to cause delayed hypersensitivity in guinea pigs under the tested conditions.

Based upon the observations made in both of the delayed contact hypersensitivity study in guinea pigs, the test item, induced at 25%(first study) and 5% (second study) and challenged at 10% (first study) and 5% (second study) resulted in delayed contact hypersensitivity in gunea pigs in the first study and in the second study .

As data from a reliable in vivo test is conducted before the enforcement of Commission Regulation (EU) 2016/1688 of 20 September 2016 amending Annex VII to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) as regards skin sensitisation are available.

Therefore no in vitro test has to be performed and therefore in vitro testing are waived.

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:

experimental study

Adequacy of study:

key study

Study period:

Janury 21 1988 through February 20, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

The study have been performed before the REACh legislation enters into force.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor; 5601-49-1

STABILITY
- Stability under test conditions: There was no apparent change in the physical state of the test article during administration

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass.
- Method and justofication for randomization: Treatement groups were housed by vertical cage positioning. Randomization was carried out by use of a random number table. One week before treatment commenced, all animals were weighrd, ranked according to body wight and assigned to treatment groups using a table of random numbers so that each treatemtn group ad a similar distribution according to the body weight. Animals beyond the extermes of the body weight range were not assigned to treatment groups. There were no statistically significant differences in mean body weights between any two groups of the same sex after allocation to the treatment groups.

- System of identification: Cages merked with animal number and dose level. Guinea pigs were ear tagged.


- Age at study initiation: no data
- Weight at study initiation: 300-500 grams
- Housing: Guine pigs were housed 2 per vage in 1/2 wire mesh cages. Size in accordance with "Guide for the care and use of Laboratory animals" of the Institut of Laboratory Ressources, National Research Council.
- Diet (e.g. ad libitum): yes, Wayne Guinea Pig Diet, ad libitum, food was checked daily and added or replaced as needed. Feeders were designed to reduce soiling, briding and cattering.
- Food analysis: There were no contaminants that were reasonably expected to be present in the dietary material known to be capable of interfering with the purpose or conduct of the study.
- Water (e.g. ad libitum): fresh tap water was available ad libitum, Water is monitored for contaminants at periodic intervals according to standard Operating Procedure-
- Acclimation period: 5 days
- Sanitanization: Wate material removed aily. Cages and feeders sanitized every two weeks.
- Rationale for test system: This test will readily detect moderate to strong skin sensitizers and thus act as an early warning against the use of such materials for human testing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C +/-3°C
- Humidity (%): 30-70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12hours lght and 12 hours dark

Route:

epicutaneous, semiocclusive

Vehicle:

other: Ethanol

Concentration / amount:

25% /0.4 ml

Day(s)/duration:

1/wk for 3 wks/6 h

Adequacy of induction:

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

No.:

#1

Route:

epicutaneous, semiocclusive

Vehicle:

other: 80% Ethanol

Remarks:

Naive animals

Concentration / amount:

10%/ 0.4 ml

Day(s)/duration:

Day 14/6 h

Adequacy of challenge:

not specified

No.:

#2

Route:

epicutaneous, semiocclusive

Vehicle:

other: 80% Ethanol/test animals

Concentration / amount:

10%/ 0.4 ml

Day(s)/duration:

Day 14/6 h

Adequacy of challenge:

not specified

No.:

#3

Route:

epicutaneous, semiocclusive

Vehicle:

other: 80% Ethanol/ Vehicle animals

Concentration / amount:

10%/0.4 ml

Day(s)/duration:

Day 14/6 h

Adequacy of challenge:

not specified

No.:

#4

Route:

epicutaneous, semiocclusive

Vehicle:

other: 80% Ethanol/Positive control

Concentration / amount:

10%/0.4 ml

Day(s)/duration:

Day 14/6 h

Adequacy of challenge:

not specified

No. of animals per dose:

Test article-20 animals
Positive control-6 animals
Negative control-10 animals
Naive challenge-4 animals
Dose range-4 animals

Details on study design:

RANGE FINDING TESTS: Prior to intiation of the study, the irritation potential was determined. Four unexposed animals (2 males and 2 females) were exposed to four concentrations of the test material by the technique described in site preparation treatment. In this test, both sides of the animal were shaved and exposed to four concentrations of the material. For grading of the response the procedure described in table 1 for primary challenge was used, except that only 24 hour grade were obtained. The dose chosen for indiction was 25%.


SITE PREPARATION AND TREATMENT:
The left shoulder of each animal was clipped free of hair with an Oster Model A2 clipper twenty-four hours prior to the 1st, 2nd and 3rd application of the test material. The shaved area was approximately 5x10 cm, i.e. 10% of the body surface.
The test material was applied to 20 guinea pigs (10 males and 10 femelas). The material was applied beneath a 37X40 mm Parke-Davis Readi-Bandage, (Park-Davis and Company, Greenwood, South Carolina) and covered with dental dam. The patch and dam were held in place with clips attached to the sides of the guinea pig restrainer.
The patches were allowed to remain in place for six hours, after which the rubber dams and patches were removed. The treated sites were examined after each dosing day and scored at 24 and 48 hours according to table I. This procedure was performed was performed once a week for three weeks, a total of three six-hour inductions. The initial body weight of the animals was recorded. A concurrent positive control group consisting of six guinea pigs (3 males and 3 females) was treated with (0.3%) 1-chloro-2,4-dinitrobenzene in 80% ethanol (80:20 ethanol/distilled water). An additional group of ten guinea pigs (5 males and 5 females) was treated with vehicle alone (Ethanol 80%).

MAIN STUDY
A. INDUCTION EXPOSURE
Frequency and duration of administration:
- three (3) inductions: test item 25% (experimental group)
- three (3) inductions: DNCB 0.3% (positive control animals)
- three (3) inductions: Ethanol 80% (vehicle control animals)


Fourteen days after the last induction sensitizing exposure, the animal were challenged im the same manner on a naive site on the left side. Four naive pigs (2 males and 2 females) were induced with the test material at a 10 % concentration, on challenge day in the same manner. Local and systemic effects were observed. Twenty four hours after the challenge, all animals were depilated with Neet Cream Hair Remover (Whitehall Laboratories, Inc., New-York, 10017). The depilatory was placed on the test sites and surrounding areas and left for no more than 30 minutes. A minimun of two hours afetr depilation test sites were graded. the grading was repeated 24 hours later (48 hours)
B. CHALLENGE EXPOSURE
- One (1) challenge: Test item 10% (naive animals)
- One (1) challenge: Test item 10% (experimental animals)
- One (1) challenge: DNCB 0.3% (positive control animals)
- One (1) challenge: Ethanol 80% (vehicle control animals)

Challenge controls:

- One (1) challenge: DNCB 0.3% (positive control animals)
- One (1) challenge: Ethanol 80% (vehicle control animals)

Positive control substance(s):

yes

Remarks:

0.3% 1-chloro-2,4-dinitrobenzene (DNCB)

Positive control results:

A positive response was elicited in the animals receiveing the positive control article, 1-Chloro-2,4 dinitrobenzene (DNCB)

Group:

positive control

Remarks on result:

positive indication of skin sensitisation

Group:

negative control

Remarks on result:

no indication of skin sensitisation

Hours after challenge:

24

Group:

test chemical

Remarks on result:

positive indication of skin sensitisation

Hours after challenge:

48

Group:

test chemical

Remarks on result:

positive indication of skin sensitisation

Results:

A positive response was elicited in the animals receiving the positive control article, 1 -chloro-2,4 dinitrobenzene (DNCB).

No positive responses were observed in the vehicle control animals.

Positive responses were observed in the experimental group at 24 and 48 hours after treatment.

One positive response was observed in the naive animals receiving the test item at 10% concentration at 48 hours after treatment. Since a grade of one (1) was seen on the naive animals, then the reaction of the test article group animals that exceed the most severe naive reactions are considered to be positive scores.

Delayed contact hypersensitivity in Guinea Pigs Incidence and severity of responses at challenge.

	Challenge
	Naive site
	24 hours		48 hours
Test Article	Incidence	Severity	Incidence	Severity
Test item (10%)	9/20	1.6	8/20	1.3
Naive animals ( test item (10%)	0/4	0	1/4*	0.3
DNCB (0.3%)	6/6	3.0	6/6	2.5
ethanol (80%)	0/10	0.2	0/10	0.1

*Since a grade of one (1) was seen on the naive animals, then the reactions of the test article group animals that exeed the most severe naive reactions are considered to be positive scores.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test article was found to cause delayed hypersensitivity in guinea pigs under the tested conditions. Therefore the substance is classified as cateogry 1B according to CLP Regulation

Executive summary:

In a preliminary dose-range-finding study to detrmine the highest non-irritating dose, four animals (2males and 2 females) were exposed to four different concentrations of the test item.

Based upon the results of the dose-range-finding study and in discussion with the sponsor, the dose chosen for induction was 25% and the dose chosen for challenge was 10%.

In order to determine if the test article is capable of causing delayed contact hypersensitivity, the test item was dermally applied to twenty guinea pigs (ten males and ten females) for a total of three six hour insult periods at a 25% concentration.

Another group of six guinea pigs (three males and three females) was treated with 1 -chloro-2,4, dinitrobenzene at a 0.3% concentration for a total of three six-hour insult periods. An additional group of ten guinea pigs (five males and five females) was treated with vehicle for a total of three six-hour insult periods.

Fourteen days afetr the last induction period, all animals were challenged at a naive site. A positive response was elicited in the animals receieving the positive control article, 1 -chloro-2,4 dinitrobenzene (DNCB). Positive responses were observed in the experimental group at 24 and 48 hours afetr treatment. No positive responses were observed in the vehicle control animals. One positive response was observed in the naive animals receiveing the test item at a 10% concentration at 48 hours after treatment. Since a grade of one (1) was seen on the naive animals, then the reactions of the test article group animals that exceed the most severe naive reactions are considered to be positive scores.

Based upon the observations made in the delayed contact hxpersensitivity study in guinea pigs, the test item, induced at 25% and challenged at 10% resulted in delayed contact hypersensitivity in gunea pigs.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

other justification

Justification for data waiving:

other:

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2, 1985 - November 1, 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

The study have been performed before the REACh legislation enters into force.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor; 5601-92-20

STABILITY
- Stability under test conditions: There was no apparent change in the physical state of the test article during administration

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass.
- Method and justofication for randomization: Selection of guinea pigs based upon body weight, sex and apparent good health
- System of identification: Cages merked with animal number and dose level. Guinea pigs were ear tagged.


- Age at study initiation: no data
- Weight at study initiation: 300-500 grams
- Housing: Guine pigs were housed 2 per vage in 1/2 wire mesh cages. Size in accordance with "Guide for the care and use of Laboratory animals" of the Institut of Laboratory Ressources, National Research Council.
- Diet (e.g. ad libitum): yes, Wayne Guinea Pig Diet, ad libitum, food was checked daily and added or replaced as needed. Feeders were designed to reduce soiling, briding and cattering.
- Food: Wayne Guinea Pig Dietr, ad libitum, food was checked daily and added or replaced as needed. Feedres were designed to reduce soiling, bridging and scattering.
- Food analysis: There were no contaminants that were reasonably expected to be present in the dietary material known to be capable of interfering with the purpose or conduct of the study.
- Water (e.g. ad libitum): fresh tap water was available ad libitum, Water is monitored for contaminants at periodic intervals according to standard Operating Procedure PH-018.
- Acclimation period: 5 days
- Sanitanization: Wate material removed daily. Cages and feeders sanitized every two weeks.
- Rationale for test system: This test will readily detect moderate to strong skin sensitizers and thus act as an early warning against the use of such materials for human testing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/-3°C
- Humidity (%): 30-70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12hours lght and 12 hours dark

Route:

epicutaneous, semiocclusive

Vehicle:

other: Ethanol 80%

Concentration / amount:

5% /0.4 ml

Day(s)/duration:

1/wk for 3 wks/6 h

Adequacy of induction:

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

No.:

#1

Route:

epicutaneous, semiocclusive

Vehicle:

other: 80% Ethanol

Remarks:

Naive animals

Concentration / amount:

10%/ 0.4 ml

Day(s)/duration:

Day 14/6 h

Adequacy of challenge:

not specified

No.:

#2

Route:

epicutaneous, semiocclusive

Vehicle:

other: 80% Ethanol/test animals

Concentration / amount:

10%/ 0.4 ml

Day(s)/duration:

Day 14/6 h

Adequacy of challenge:

not specified

No.:

#3

Route:

epicutaneous, semiocclusive

Vehicle:

other: 80% Ethanol/ Vehicle animals

Concentration / amount:

10%/0.4 ml

Day(s)/duration:

Day 14/6 h

Adequacy of challenge:

not specified

No.:

#4

Route:

epicutaneous, semiocclusive

Vehicle:

other: 80% Ethanol/Positive control

Concentration / amount:

10%/0.4 ml

Day(s)/duration:

Day 14/6 h

Adequacy of challenge:

not specified

No. of animals per dose:

Test article-20 animals
Positive control-6 animals
Negative control-10 animals
Dose range-4 animals
Naive challenge - 4 animals

Details on study design:

RANGE FINDING TESTS: Prior to intiation of the study, the irritation potential was determined. Four unexposed animals (2 males and 2 females) were exposed to four concentrations of the test material by the technique described in site preparation treatment. In this test, both sides of the animal were shaved and exposed to four concentrations of the material. The procedure described in the table I for primary challenge was used fro grading of the response, except that only 24 hour grades were obtained. The highest non-irritating concentration was that concentration in vehicle that induced responses not exceeding two + and two 0 grades in the group of four animals.
Before beginning the study, it was decided in accordance with the sponsor that the dose chosen for induction and challenge was 5%.


SITE PREPARATION AND TREATMENT:
The left shoulder of each animal was clipped free of hair with an Oster Model A2 clipper twenty-four hours prior to the 1st, 2nd and 3rd application of the test material. The shaved area was approximately 5x10 cm, i.e. 10% of the body surface.
The test material was applied to 20 guinea pigs (10 males and 10 femelas). The material was applied beneath a 37X40 mm Parke-Davis Readi-Bandage, (Park-Davis and Company, Greenwood, South Carolina) and covered with dental dam. The patch and dam were held in place with clips attached to the sides of the guinea pig restrainer.
The patches were allowed to remain in place for six hours, after which the rubber dams and patches were removed. The treated sites were examined after each dosing day and scored at 24 and 48 hours according to table I. This procedure was performed once a week for three weeks, a total of three six-hour inductions. A concurrent positive control group consisting of 6 guinea pigs(3 males and 3 females) was treated with (0.3%) 1-chloro-2,4-dinitrobenzene. An additional group of 10 guinea pigs (5 males and 5 females) was treated with vehicle (80% ethanol).
The intial body weight of all animals was recorded.


MAIN STUDY
A. INDUCTION EXPOSURE
Frequency and duration of administration:
- three (3) inductions: test item 25% (experimental group)
- three (3) inductions: DNCB 0.3% (positive control animals)
- three (3) inductions: Ethanol 80% (vehicle control animals)


Fourteen days after the last induction sensitizing exposure, the animal were challenged im the same manner on a naive site on the left side. Four naive pigs (2 males and 2 females) were induced with the test material at a 10 % concentration, on challenge day in the same manner. Local and systemic effects were observed. Twenty four hours after the challenge, all animals were depilated with Neet Cream Hair Remover (Whitehall Laboratories, Inc., New-York, 10017). The depilatory was placed on the test sites and surrounding areas and left for no more than 30 minutes. A minimun of two hours afetr depilation test sites were graded. the grading was repeated 24 hours later (48 hours)
B. CHALLENGE EXPOSURE
- One (1) challenge: Test item 10% (naive animals)
- One (1) challenge: Test item 10% (experimental animals)
- One (1) challenge: DNCB 0.3% (positive control animals)
- One (1) challenge: Ethanol 80% (vehicle control animals)

Challenge controls:

- One (1) challenge: DNCB 0.3% (positive control animals)
- One (1) challenge: Ethanol 80% (vehicle control animals)

Positive control substance(s):

yes

Remarks:

0.3% 1-chloro-2,4-dinitrobenzene (DNCB)

Positive control results:

A positive response was elicited in the animals receiveing the positive control article, 1-Chloro-2,4 dinitrobenzene (DNCB)

Group:

positive control

Remarks on result:

positive indication of skin sensitisation

Group:

negative control

Remarks on result:

other: Slight patchy erythema was observed at 24 and 48 hours after challenge in one animal in the negative control group.

Hours after challenge:

24

Group:

test chemical

Remarks on result:

positive indication of skin sensitisation

Remarks:

Positive responses, greater than those responses observed in the naive animals were observed in the experimental group at 24 and 48 hours after challenge.

Hours after challenge:

48

Group:

test chemical

Remarks on result:

positive indication of skin sensitisation

Remarks:

Positive responses, greater than those responses observed in the naive animals were observed in the experimental group at 24 and 48 hours after challenge.

Results:

A positive response was elicited in the animals receiving the positive control article, 1 -chloro-2,4 dinitrobenzene (DNCB).

Slight patchy erythema was observed at 24 and 48 hours after challenge in one animal in the negative control group.

Responses were observed at 24 and 48 hours in the naive group at 24 and 48 hours after challenge.

Positive responses, greater than those responses observed in the naive animals were observed in the experimental group at 24 and 48 hours after challenge.

Delayed contact hypersensitivity in Guinea Pigs Incidence and severity of responses at challenge.

	Challenge
	Naive site
	24 hours		48 hours
Test Article	Incidence	Severity	Incidence	Severity
Test item (5%)	9/20	0.8	10/20	0.9
Naive animals ( test item (5%)	0/4	0.4	0/4	0.1
DNCB (0.3%)	6/6	2.8	6/6	2.7
ethanol (80%)	0/10	0.1	0/10	0.1

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test article was found to cause delayed hypersensitivity in guinea pigs under the tested conditions. Therefore the substance is classified as cateogry 1B according to CLP Regulation

Executive summary:

In a preliminary dose-range-finding study to detrmine the highest non-irritating dose, four animals (2males and 2 females) were exposed to four different concentrations of the test item.

Based upon the results of the dose-range-finding study and in discussion with the sponsor, the dose chosen for induction was 25% and the dose chosen for challenge was 10%.

In order to determine if the test article is capable of causing delayed contact hypersensitivity, the test item was dermally applied to twenty guinea pigs (ten males and ten females) for a total of three six hour insult periods.

Another group of six guinea pigs (three males and three females) was treated with 1 -chloro-2,4, dinitrobenzene at a 0.3% concentration for a total of three six-hour insult periods. An additional group of ten guinea pigs (five males and five females) was treated with vehicle for a total of three six-hour insult periods.

Fourteen days after the last induction period, all animals were challenged at a naive site. A positive response was elicited in the animals receieving the positive control article, 1 -chloro-2,4 dinitrobenzene (DNCB). Slight patchy erythema was observed at 24 and 48 hours after challenge in one animal in the negative control group. responses were observed in the naive group at 24 and 48 hours after challenge. Positive reponses, greater than those responses observed in the naive animals, were observed in the experimental group at 24 and 48 hours after challenge.

Based upon the observations made in the delayed contact hxpersensitivity study in guinea pigs, the test item, induced and challenged at 5% concentration, caused delayed contact hypersensitivity in guinea pigs.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Justification for classification or non-classification

The test article was found to cause delayed hypersensitivity in guinea pigs under the tested conditions. Therefore the substance is classified as cateogry 1B ( H317: May cause an allergic skin reaction.) according to CLP Regulation.