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Description of key information

In a reliable study performed with a surrogate for the submission substance no evidence for sensitising properties were observed.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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:
2016-02-02 to 2016-03-04
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]

It is hypothesized that the target chemical and the following chemicals as source chemicals should exhibit comparable toxicity profiles:
-6-(isononanoylamino)hexanoic acid, compound with 2,2`,2``-nitrilotriethanol
-3,5,5-trimethylhexanoic acid
It is proposed to use the toxicity data of the mentioned source chemicals to fulfill the data requirement for the target chemical. The first chemical is a salt of the target chemical. 3,5,5-trimethylhexanoic acid is a presumed metabolite.

The underlying scientific rationale for the use of corresponding salt as source chemical is apparent. The target chemical is a weak acid due to the terminal carboxylic acid moiety and can be neutralized/dissolved in aqueous system by reaction with base such as 2,2`,2``-nitrilotriethanol. The proposed source chemical can be formally described as carboxylate of the target chemical. As the carboxylate and carboxylic acid are inter-convertible, it is apparent that source and target chemicals are inter-convertible and should exhibit comparable toxicity profile. The base 2,2`,2``-nitrilotriethanol is a well-investigated substance and is considered to be less relevant for the proposed read-across consideration.

The underlying scientific rationale for the use of 3,5,5-trimethylhexanoic acid as source chemical is based on the metabolism consideration. Upon resorption, the target chemical is expected to undergo a degradation process, resulting in the systemic release of 3,5,5-trimethylhexanoic acid, thereby providing the justification for the read-across especially for the mid- and long term toxicities such as repeated dose toxicity and reproduction toxicity.

The proposed approach applies for all exposure routes (oral/dermal/inhalation), because both the target chemical and source chemicals are expected to be bioavailable by all exposure routes: the inter-conversion between carboxylic acid and carboxylate is likely to occur prior to resorption; and the systemic release of the presumed metabolite is less dependent on exposure route.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]

Target chemical:
6-(Isononanoylamino)hexanoic acid; CAS: 71902-23-3
Source chemicals:
6-(isononanoylamino)hexanoic acid, compound with 2,2`,2``-nitrilotriethanol; CAS: 85702-79-0
3,5,5´-trimethylhexanoic acid; CAS: 3302-10-1

The target chemical is a mono-substituent substance, the analytical purity being >99%. The source chemicals are either the raw material (3,5,5´-trimethylhexanoic acid) or further chemically processed products (dissolved in water with 2,2`,2``-nitrilotriethanol) of the target chemical. A toxicity difference due to different impurity profiles is not likely to occur.

3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]

Justification for the use of 6-(isononanoylamino)hexanoic acid, compound with 2,2`,2``-nitrilotriethanol as source chemical:

- The given source chemical is an ionic compound that results from the neutralization reaction of the target chemical and 2,2`,2``-nitrilotriethanol. When it is dissolved in an aqueous system or in a biological fluid, an immediate dissociation occurs to give the target chemical and the base, thereby explaining the expected comparable toxicity profile to that of target chemical.
A significant toxicity contribution of 2,2`,2``-nitrilotriethanol is not expected. 2,2`,2``-nitrilotriethanol is a well investigated substance. It is of low toxicity and the available kinetic data are demonstrative of efficient elimination mechanisms in animal models.
- In order to verify the expected toxicity comparability, the given source and the target chemicals were investigated under identical testing conditions. Both substances exhibited comparable findings after 7-day oral application to rat:
-liver and kidney enlargement
-decrease of eosinophil counts
-peroxisome proliferation in the liver
The decrease of eosinophil counts is possibly a transient effect, associated with the peroxisome proliferation stimulating effect of test compounds. No such findings were present after 28-day treatment of the source chemical.
- Comparable findings were obtained in the 28-day oral toxicity study for the given source chemical and in the above mentioned two 7-day repeated oral toxicity studies. Further special histopathological investigation revealed the alpha-2µ-globulin accumulation in male kidneys and the peroxisome proliferation in liver.
- In the available skin sensitization data the given source chemical was applied using water as vehicle. The dissociation into the target chemical and 2,2`,2``-nitrilotriethanol is expected to have occurred prior to resorption, so that the animals must have been exposed to the target chemical.

Justification for the use of 3,5,5-trimethylhexanoic acid as source chemical:

- 3,5,5-trimethylhexanoic acid is the presumed metabolite. It is also the presumed metabolite of 6-(isononanoylamino)hexanoic acid, compound with 2,2`,2``-nitrilotriethanol.
This view is based on the results of the metabolites investigation in degradation samples obtained in a Zahn-Wellens test. The ß-oxidation at the terminal carboxylic acid moiety in combination with hydrolysis at amide bond could be assumed as the degradation pathway leading to 3,5,5-trimethylhexanoic acid as a stable metabolite.
Also the hydrolysis at the amide moiety is thinkable. The hydrolysis products would be then 3,5,5-trimethylhexanoic acid and 6-aminocaproic acid. The latter compound is a drug known as amicar and is expected to be far more rapidly eliminated than 3,5,5-trimethylhexanoic acid. A significant toxicity attribution of 6-aminocaproic acid cannot be derived.
One literature article was found, describing further biotransformation of 3,5,5-trimethylhexanoic acid in rat: gamma-lactone of 3,5,5-trimethylhexanoic acid is formed, which may be the ultimate toxicant for the alpha-2µ-globulin accumulation in kidneys
- The findings in the 28-day oral toxicity study on the 3,5,5-trimethylhexanoic acid are comparable to those found in the repeated dose toxicity studies of the target chemical and 6-(isononanoylamino)hexanoic acid, compound with 2,2`,2``-nitrilotriethanol:
- liver and kidney enlargement
- peroxisome proliferation in the liver
- The alpha-2µ-globulin accumulation in kidneys.


4. DATA MATRIX

Data matrix and other information see the attached read-across justification in chapter 13
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was performed for regulatory purpose outside EU
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Procured from approved breeder, National Institute of Nutrition
- Age at study initiation: 8 to 9 weeks
- Weight at study initiation: 311.53 g to 321.24 g
- Housing: individually in a standard polypropylene cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:
Healthy young adult animals for pre-study and main study were acclimatized for a period of five and twelve days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1°C to 22.9°C
- Humidity (%): 46% to 63%
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12/12
Route:
epicutaneous, semiocclusive
Vehicle:
water
Concentration / amount:
5.0% v/v, 100% for intradermal and topical induction, respectively.
Day(s)/duration:
7
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%
Day(s)/duration:
on day 22
Adequacy of challenge:
other: 100%
No. of animals per dose:
10 Males - Vehicle Control
20 Males - Test Item
Details on study design:
RANGE FINDING TESTS:

MAIN STUDY
A. INDUCTION EXPOSURE
day1: Intrademal injection:
- Site: 3 pair
- Concentrations:
50:50 volume ratio of FCA in distilled water
5% (v/v) Test item in distilled water
5% (v/v) Test item , emulsified in a 50:50 Volume of FCA and distilled water

day 8: Topical application:
100 % (as such) Test item

B. CHALLENGE EXPOSURE
- Day(s) of challenge: day 22
- Concentrations: 100 % (as such) Test item
- Evaluation (hr after challenge): 24±2 and 48±2 hours
Challenge controls:
Distilled water
Positive control substance(s):
yes
Remarks:
2-Mercaptobenzothiazole in study No.: BIO-TX 1685
Positive control results:
The positive control 2-Mercaptobenzothiazole showed a mean skin sensitisation rate of 60% at approximately 24 and 48 hours post patch removal in challenge phase.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no skin reaction was observed
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no skin reaction was observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no skin reaction was observed
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no skin reaction was observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Remarks on result:
not measured/tested
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Remarks on result:
not measured/tested

No treatment related change in body weight and percent change in body weight with respect to Day 1 was noted in all the groups. All animals showed physiologically normal increase in body weights.

No gross pathological changes were noted in any of the treated animals in all the groups.

Interpretation of results:
GHS criteria not met
Conclusions:
The skin sensitisation of the submission substance was assessed based on the analogue approach using 6-(Isononanoylamino)hexanoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) as read-across supporting substance. Based on the above results of the experiment and under experimental conditions employed, it can be concluded that the test item 6-(Isononanoylamino)hexanoic acid, compound with 2,2′,2″-nitrilotriethanol (1:1) did not show any skin sensitisation or allergic potential in Guinea pigs. Hence, the submission substance is not classified as a sensitizer according to the Globally Harmonized System (GHS) of Classification and Labelling of Chemicals.
Executive summary:

The skin sensitisation of the submission substance was assessed based on the analogue approach using 6-(Isononanoylamino)hexanoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) as read-across supporting substance.

The test item,6-(isononanoylamino)hexanoic acid, compound with 2,2′,2″-nitrilotriethanol (1:1) obtained from Clariant India Private Limited was evaluated for the skin sensitization potential in Guinea pigs as per theOECD Guideline for Testing of Chemicals, Section 4, Number 406, “Skin Sensitization”.

The study was conducted in two phase viz., pre-study and main study.

Pre-study: For pre-study, a total of 4 Guinea pigs were used, 2 Guinea pigs for intradermal injection and 2 Guinea pigs for topical application. Approximately 24 hours before treatment, the fur from the shoulder region and both flank region was clipped for intradermal injection and topical application, respectively. For intradermal injection, 2.5% and 5% (v/v) in distilled water was administered to one animal, and 7.5% and 10% in distilled water was administered to another animal. Pairs of injection, i.e. 0.1 mL/injection were administered at the shoulder region during intradermal injection. For topical application, test concentrations of 25% and 50% v/v in distilled water was applied topically at left and right flank of one animal and 75 % v/v in distilled water and undiluted test item was applied to the left and right flank of another animal.The occlusive patches were removed after 24 hours of contact period and washed with normal saline swabs and dried with absorbent cotton.

After intradermal injection, thetest itemrevealed no erythema at 24 hours and very slight erythema (barely perceptible) at 48 hours in 5.0% (v/v) test item in distilled water. After removal of patch in topical application, thetest itemdid not reveal erythema or oedema with 100% (as such) test item at 24 and 48 hours observation period. Hence, 5.0% v/v of test item in distilled water and 100% (as such) test item was selected for intradermal and topical induction, respectively for the main study, and 100% (as such) test item was selected for challenge phase for main study.

Main study: The main study comprised of two groups, group G2 (10 males) designated as vehicle control (distilled water for intradermal injection, topical application, and for challenge application distilled water and undiluted test item) and G3 (20 males) designated as treatment group 5.0% v/v test item for intradermal induction, 100% as such (undiluted) test item for topical application, and distilled water and undiluted test item for challenge application. Main study included an intradermal induction on Day 1, topical induction on Day 8 and challenge on Day 22. The designated sites for respective phases of the experiment were clipped closely using an electric hair clipper approximately 24 hours prior to initiation of the treatment.

On Day 1 (induction phase), the animals were administered intradermally with 3 pairs of injection at the shoulder region with 0.1 mL/injection. Post intradermal induction, all animals in groups G2 and G3 were scored for skin reactions approximately at 24 and 48 hours as per the Draize (1959) scoring system. Skin reaction like very slight erythema (barely perceptible) was observed at site 2 of intradermal injection in treated group (G3) approximately at 48 hour observation. However, no skin reaction observed at site 2 of vehicle control group (G2) approximately at 24 and 48 hours observation period.

On Day 7, fur from the area of the intra-dermal injection sites on the shoulder region was clipped in both vehicle control and treatment group animals. On Day 8 (induction phase), the filter paper (2 cm x 4 cm) was saturated with distilled water and 100 % as such (undiluted) test item. The filter paper saturated with distilled water was applied to G2 groups and filter paper saturated with 100% as such test item was applied to G3 groups. The soaked filter paper was topically applied to the previously injected sites to the respective groups. The patch was held in place with non-irritating adhesive tape and further wrapped with crepe bandage by an occlusive dressing. The test patch was held in their position for 48±2 hours. After 48±2 hours of contact period the test patch was removed and the area was cleaned with normal saline swab and dried with absorbent cotton. The skin reactions were observed at 1 and 24 hours of post removal of the test patch. The animals did not reveal any skin reactions in G2 and G3 groups.

On Day 22 (challenge phase), the filter paper (2 cm x 4 cm) was saturated with vehicle control and 100% as such (undiluted) test item. The soaked filter paper was applied to the G2 and G3 groups over the pre-clipped area. The vehicle control and test item was applied on the anterior left and posterior left flank region respectively in G2 and G3 groups respectively. After 24±2 hours of contact period, the test patches were removed and the areas was cleaned with normal saline swabs and dried with absorbent cotton. The skin reactions were observed at 24±2 and 48±2 hours after removal of the test patch and the skin reactions were observed and recorded according to Magnusson and Kligman grading scale. There was no skin reactions observed.

No treatment related changes were noted in body weight and percent change in body weight with respect to Day 1 in all the groups. All animals showed physiologically normal increase in body weights.

All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality during observation period. No clinical signs of toxicity and mortality were noted in any of the animals in all the groups.

Body weights were recorded on Day 1 and at termination. At the end of the observation period all the animals were sacrificed under carbon dioxide anaesthesia

No gross pathological changes were noted in any of the animals in all the groups.

Conclusion

Based on the observed results of the experiment and under experimental conditions employed, it can be concluded that the test item 6-(isononanoylamino)hexanoic acid, compound with 2,2′,2″-nitrilotriethanol (1:1) did not show any skin sensitisation or allergic potential in Guinea pigs. Hence, the test item is not classified as a sensitizer according to the Globally Harmonized System (GHS) of Classification and Labelling of Chemicals.

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

The study was performed using 6-(Isononanoylamino)hexanoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1)-a surrogate for the submission substance- and was rated as reliable without restriction (reliability category 1).

The test item,6-(isononanoylamino)hexanoic acid, compound with 2,2′,2″-nitrilotriethanol (1:1) obtained from was evaluated for the skin sensitization potential in Guinea pigs as per theOECD Guideline for Testing of Chemicals, Section 4, Number 406, “Skin Sensitization”. On Day 22 (challenge phase), the filter paper was saturated with vehicle control and 100% as such (undiluted) test item. After 24±2 hours of contact period, the test patches were removed and the areas was cleaned with normal saline swabs and dried with absorbent cotton. The skin reactions were observed at 24±2 and 48±2 hours after removal of the test patch and the skin reactions were observed and recorded according to Magnusson and Kligman grading scale. There was no skin reactions observed.


Migrated from Short description of key information:
In a reliable study performed with a surrogate for the submission substance no evidence for sensitising properties were observed.

Justification for selection of skin sensitisation endpoint:
Only reliable study performed with surrogate for submission substance

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the results of available data the submission substance does not have to be classified for sensitising effects on skin according to the criteria of Regulation (EC) No. 1272/2008 (CLP) as well as Council Directive 67/548/EEC.