C16-18-(even numbered)-alkyl fatty acid, compound with C16-18-(even numbered)-alkylamine

Endpoint summary

Description of key information

Results of reserve alkaline test and in vivo acute dermal irritation test in rabbits lead not to classify the registered substance. Non reversible effects within 21 days of application on the conjunctiva of one rabbit lead to consider the registered substance as it caused serious eye damage.  STOT-SE Cat.3 for respiratory irritation is not required. Exposure to via the inhalation route will be unlikely to occur.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 December 2012 - 21 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: CEGAV, Argenvilliers, France
- Age at study initiation: 2 to 4 months old on the day of treatment
- Mean body weight at study initiation: 2822 g (range: 2655 g to 3105 g)
- Fasting period before study: no
- Housing: individually housed in noryl cages
- Diet: 110 pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°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.

IN-LIFE DATES: 08 January 2013 to 21 January 2013.

Type of coverage:

semiocclusive

Preparation of test site:

other: clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.5 g/flank

Duration of treatment / exposure:

3 min, 1 h, 4 h.

Observation period:

1, 24, 48 and 72 h after removal of the dressing; if relevant, daily until reversibility of reactions

Number of animals:

Three males.

Details on study design:

TEST SITE
- Area of exposure: the test item was placed on a gauze pad, moistened with drinking water treated by reverse osmosis, which was then applied to a skin area of approximately 6 cm2.
During each exposure time, a dry gauze pad was applied to the opposite flank, in order to check that no alteration of the skin is induced by the semi-occlusive dressing and restraining bandage.
The untreated flank acted as control.
The gauze pad was held in place by a non-irritating semi-occlusive dressing and a restraining bandage. After required period of contact with the skin, the dressing was removed.
After removal of the dressing, any residual test item was wiped off by means of a moistened cotton pad with drinking water treated by reverse osmosis.
- Type of wrap if used: gauze pad held in place by a non-irritation semi-occlusive dressing and a restraining bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: using a dry cotton pad
- Time after start of exposure: at removal of each dressing (see Duration of exposure)

SCORING SYSTEM:

- Erythema and eschar formation:
0 no erythema
1 very slight erythema (barely perceptible)
2 well-defined erythema
3 moderate to severe erythema
4 severe erythema (beet redness) to slight eschar formation (injuries in depth)

- Edema formation:
0 no edema
1 very slight edema (barely perceptible)
2 slight edema (edges of area well-defined by definite raising)
3 moderate edema (raised approximately 1 millimeter)
4 severe edema (raised more than 1 millimeter and extending beyond area of exposure)

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

other: 24, 48, 72 h (mean)

Score:

1

Max. score:

1

Irritation parameter:

edema score

Basis:

animal #1

Time point:

other: 24, 48, 72 h (mean)

Score:

0

Max. score:

0

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

other: 24, 48, 72 h (mean)

Score:

0.3

Max. score:

1

Irritation parameter:

edema score

Basis:

animal #2

Time point:

other: 24, 48, 72 h (mean)

Score:

0

Max. score:

0

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

other: 24, 48, 72 h (mean)

Score:

1

Max. score:

2

Irritation parameter:

edema score

Basis:

animal #3

Time point:

other: 24, 48, 72 h (mean)

Score:

0

Max. score:

0

Other effects:

After a 3-minute exposure in 1/3 animals, dryness of the skin was observed on day 2.
After a 4-hour exposure, dryness of the skin was recorded from day 6 to day 8 in 1/3 animals and on day 4 in 1/3 animals.

Interpretation of results:

slightly irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: CLP Regulation

Conclusions:

The test item was slightly irritant when applied topically to rabbits.
However, the test item should not be classified as irritating to skin according to the criteria of CLP Regulation.

Executive summary:

The objective of this study was to evaluate the potential corrosive and irritant properties of the test item following dermal application on rabbits.

 

Methods

The test item was first applied for periods of 3 minutes, 1 hour and 4 hours to a single male New Zealand White rabbit.

After the 4-hour application, since the mean value from grading at 24, 48 and 72 hours after patch removal was < 2.3 for erythema and for edema, the test item was applied on the skin of two other animals for 4 hours.

 

A quantity of 0.5 g/flank was used.

The test item was placed on a gauze pad, moistened with drinking water treated by reverse osmosis, which was then applied to a skin area of approximately 6 cm². The gauze pad was held in place by a non-irritating semi-occlusive dressing and a restraining bandage. After the required period of contact with the skin, the dressing was removed.

Each animal was observed once a day for mortality and clinical signs. For each exposure period, cutaneous reactions were evaluated approximately 1 hour, 24, 48 and 72 hours after removal of the dressing and then daily until the reversibility of cutaneous reactions. The mean values of the scores for erythema and edema were calculated for each animal. Body weight was recorded on the day of treatment and at the end of the evaluation of cutaneous reactions.

On completion of the observation period, the animals were sacrificed then discarded without macroscopic post-mortem examination.

Results

No unscheduled deaths occurred during the study and no clinical signs were noted in any animals.

The body weight of the animals was unaffected by the test item treatment.

After a 4-hour exposure, a very slight or well-defined erythema was noted in all animals from day 1 until day 2, 4 or 5.

 

Dryness of the skin was recorded in one animal between day 6 and day 9 and in another animal on day 4.

 

Mean scores calculated for each animal over 24, 48 and 72 hours were as follows:

.          erythema: 1.0, 0.3, 1.0; showing no significant inflammation,

.          edema: 0.0, 0.0; 0.0; showing no significant inflammation.

 

Conclusion

The test item was slightly irritant when applied topically to rabbits.

However, the test item should not be classified as irritating to skin according to the criteria of CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 March 2013 -- 03 April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

Irritation parameters were not evaluated on day 22 for the first animal. As the more severe classification has been applied, this deviation was not considered to have compromised the validity of the study.

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

see above

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: breeder: CEGAV, Argenvilliers, France
- Age at study initiation: 2 to 4 months old on the day of treatment
- Mean body weight at study initiation: 2410 g and 2775 g
- Fasting period before study: no
- Housing: individually housed in noryl cages
- Diet: 110 pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: a period of 4 or 6 days before treatment .

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°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

IN-LIFE DATES: 18 March 2013 to 03 April 2013

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated right eye served as a control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.1 g/animal

Duration of treatment / exposure:

Not applicable: followed by rinsing.

Observation period (in vivo):

1, 24, 48 and 72 h; if relevant, daily until reversibility of reactions.

Number of animals or in vitro replicates:

Two males.

Details on study design:

REMOVAL OF TEST SUBSTANCE: No

SCORING SYSTEM: Draize scale.

- Conjunctival chemosis (lids and/or nictitating membranes):
0 no swelling
1 any swelling above normal (includes nictitating membranes)
2 obvious swelling with partial eversion of lids
3 swelling with lids about half-closed
4 swelling with lids more than half-closed

- Conjunctival redness (palpebral and bulbar conjunctivae, cornea and iris):
0 blood vessels normal
1 a number of blood vessels definitely hyperemic (injected)
2 diffuse, crimson colour, individual vessels not easily discernible
3 diffuse, beefy red

- Discharge:
0 absence of discharge
1 slight discharge (does not include small amounts normally found in inner canthus)
2 discharge with moistening of lids and hairs adjacent to lids
3 discharge with moistening of lids and hairs on wide area around the eye

- Iris lesions
0 normal
1 markedly deepened rugae, congestion, swelling, moderate circum-corneal hyperemia,or injection, any of these or combination of any thereof, iris still reacting to light (sluggish reaction is positive)
2 no reaction to light, haemorrhage, gross destruction (any or all of these)

- Cornea intensity of opacity (direct examination and, if necessary, with an UV lamp)
0 no ulceration or opacity
1 scattered or diffuse areas of opacity (other than slight dulling or normal lustre), details of iris clearly visible
2 easily discernible translucent area, details of iris slightly obscured
3 nacreous areas, no details of iris visible, size of pupil barely discernible
4 opaque cornea, iris not discernible through the opacity

- Cornea area of opacity (direct examination and, if necessary, with an UV lamp)
1 one quarter (or less) but not zero
2 greater than one quarter but less than a half
3 greater than one half but less than three quarters
4 greater than three quarters up to whole area

- Any other lesions observed were noted

TOOL USED TO ASSESS SCORE: UV lamp after instillation of 0.5% sodium fluorescein solution

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

other: 24, 48 and 72 h (mean)

Score:

3.3

Max. score:

4

Reversibility:

not fully reversible within: day 21

Remarks on result:

other: Day 22: irritation parameters not evaluated

Irritation parameter:

other: conjunctivae score (redness)

Basis:

animal #1

Time point:

other: 24, 48 and 72 h (mean)

Score:

3

Max. score:

3

Reversibility:

not fully reversible within: day 21

Remarks on result:

other: Day 22: irritation parameters not evaluated

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: 24, 48 and 72 h (mean)

Score:

1

Max. score:

1

Reversibility:

fully reversible within: day 20

Remarks on result:

other: Day 22: irritation parameters not evaluated

Irritation parameter:

cornea opacity score

Remarks:

(intensity)

Basis:

animal #1

Time point:

other: 24, 48 and 72 h (mean)

Score:

2

Max. score:

3

Reversibility:

fully reversible within: day 19

Remarks on result:

other: Day 22: irritation parameters not evaluated

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

other: 24, 48 and 72 h (mean)

Score:

1.7

Max. score:

2

Reversibility:

fully reversible within: day 10

Irritation parameter:

other: conjunctivae score (redness)

Basis:

animal #2

Time point:

other: 24, 48 and 72 h (mean)

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: day 10

Irritation parameter:

iris score

Basis:

animal #2

Time point:

other: 24, 48 and 72 h (mean)

Score:

0.7

Max. score:

1

Reversibility:

fully reversible within: day 4

Irritation parameter:

other: cornea score intensity

Basis:

animal #2

Time point:

other: 24, 48 and 72 h (mean)

Score:

2.7

Max. score:

3

Reversibility:

fully reversible within: day 8

Interpretation of results:

other: severely irritant

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item was severely irritant when administered by ocular route to rabbits.

In addition, the effects on the conjunctiva were not fully reversible within 21 days of application in one animal; it was considered that the test item caused serious eye damage.

According to the criteria of CLP Regulation, the test item should be classified category 1 and assigned the signal word "danger" and the hazard statement "H318: causes serious eye damage".

Executive summary:

The objective of this study was to evaluate the potential irritant properties of the test item for the eye following a single administration to rabbits.

This study was conducted in compliance with OECD Guideline No. 405 and the principles of Good Laboratory Practices.

Methods

The test item was first administered to a single male New Zealand White rabbit.

 

As mean value from grading at 24, 48 and 72 hours after instillation was = 2 for conjunctival edema (chemosis) and for conjunctival redness and = 1 for iris lesions and for corneal opacity, the test item was administered in the left eye of a second animal.

 

After administration to the second animal, as mean value from grading at 24, 48 and 72 hours after instillation was = 2 for conjunctival redness and = 1 for corneal opacity, no other animal was treated.

 

The test item was administered inthe conjunctival sac of the left eye. The right eye remained untreated and served as control.

A quantity of 0.1 g/animal was used.

For the first and second animals, a local anesthetic was used before the 24-hour reading and prior to treatment, respectively.

 

Just after the 1-hour scoring, both eyes were rinsed with a sterile isotonic saline solution (0.9% NaCl).

Due to important ocular reactions in both animals and in order to perform ocular reactions evaluation in good conditions, eyes were rinsed again before the 24-hour reading with sterile isotonic saline solution (0.9% NaCl).

 

Each animal was observed at least once a day for mortality and clinical signs. Ocular reactions were observed approximately 1 hour, 24, 48 and 72 hours after the administration and then daily until the reversibility of the ocular reactions or until day 21. The mean values of the scores for chemosis, redness of the conjunctiva, iris lesions and corneal opacity were calculated for each animal. Body weight was recorded on the day of treatment and at the end of the evaluation of ocular reactions or the day after the end of the evaluation of ocular reaction.

On completion of the observation period, the animals were sacrificed then discarded without macroscopic post-mortem examination.

Results

No unscheduled deaths occurred during the study.

One animal showed loud breathing on day 2. This could be considered as pain resulting from severity of ocular lesions. Thereafter chromorhynorrhea probably due to repeated administrations offluorescein solutionswas noted from day 15.

However no clinical signs indicative of systemic toxicity were observed in any animals. The body weight of the animals was unaffected by the test item treatment.

In the left treated eye of one animal, marked to severe chemosis and severe redness of the conjonctiva were observed from day 1 to day 4. Severe redness persisted on day 5. Slight to moderate conjunctiva reactions were noted between days 6 and 11. Thereafter, moderate to marked chemosis was recorded between days 12 and 19, whereas moderate to severe redness was observed from day 12 to day 16. Then, slight conjunctiva reactions persisted until the end of the observation period (day 21).

Iris lesions were noted from day 1 to day 19.

Moderate to marked corneal opacity was observed on days 2 and 3. Then, slight corneal opacity was recorded between days 4 and 14. Corneal opacity was graded marked again from day 15 to day 18.

In addition, marked blepharospasm was noted on day 2 and neovascularization was recorded from day 8 to day 18.

 

In the left treated eye of one animal, moderate chemosis and moderate to severe redness of the conjonctiva were noted from day 1 to day 3. Then, slight to moderate conjonctiva reactions persisted until day 9.

Iris lesions were noted from day 1 to day 3.

Moderate to marked corneal opacity was recorded from day 2 to day 4. Thereafter, slight corneal opacity was still present until day 7.

 

Lacrimation and whitish deposit were noted in the left eye of both animals on many occasions during the observation period.

 

Mean scores calculated for each animal over 24, 48 and 72 hours were as follows:

.       chemosis: 3.3 and 1.7 ; showing eye irritation in 1/2 animals,

.       redness of the conjunctiva: 3.0 and 2.7; showing eye irritation in 2/2 animals,

.       iris lesions: 1.0 and 0.7 ; showing eye irritation in 1/2 animals,

.       corneal opacity: 2.0 and 2.7; showing eye irritation in 2/2 animals.

 

However, as the effects on the conjunctiva were not fully reversible within 21 days of application in one animal; it was considered that the test item caused serious eye damage.

 

Conclusion

 

Under the experimental conditions of this study, the test item was severely irritant when administered by ocular route to rabbits.

In addition, the effects on the conjunctiva were not fully reversible within 21 days of application in one animal; it was considered that the test item caused serious eye damage.

According to the criteria of CLP Regulation,the test item should be classified category 1 and assigned the signal word "danger" and the hazard statement "H318: causes serious eye damage".

 

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Reserve alkaline test:

The dermal and ocular irritant potential of the registered substance was assessed using a non-animal testing based on alkaline reserve according to the method described in the two references : JF.Regnier and C.Imbert (1992), and V.Froment (1993).

This method can be used at first step of stepwise strategy for assessing cutaneous and ocular irritation.

An amount of the test substance is added to distilled water in a beaker. The solution or suspension is maintained for several hours under agitation and heating. The pH is directly measured and the sample is then titrated with diluted sodium hydroxyde or hydrochloric acid until neutrality is obtained. The acidic or alkaline reserve (R) is expressed as the number of grams of pure acid or base necessary to neutralise 100 g of product (%).

Under the test conditions, the aqueous test substance shows a slightly basic character demonstrated by a pH of 8.37 for a 100 g/L solution in water, an alkaline reserve R of 0.0081 (log R = -2.09) and a physicochemical index of 6.28.

As none of the criteria is fulfilled for both end-points, ACAA can be considered as neither potentially corrosive to the skin, nor severely irritant to the eye. Thus, an in vivo assessment could be performed.

Dermal irritation:

One acute dermal irritation study in rabbits was performed for the registered substance. The study was performed according to OECD 404 and under GLP.

 

The test item was first applied for periods of 3 minutes, 1 hour and 4 hours to a single male New Zealand White rabbit. After the 4-hour application, since the mean value from grading at 24, 48 and 72 hours after patch removal was < 2.3 for erythema and for edema, the test item was applied on the skin of two other animals for 4 hours.

The test item (0.5 g/flank) was placed on a gauze pad, moistened with drinking water and applied to a skin area of approximately 6 cm². After the required period of contact with the skin, the dressing was removed. Each animal was observed once a day for mortality and clinical signs. For each exposure period, cutaneous reactions were evaluated approximately 1 hour, 24, 48 and 72 hours after removal of the dressing and then daily until the reversibility of cutaneous reactions. The mean values of the scores for erythema and edema were calculated for each animal. Body weight was recorded on the day of treatment and at the end of the evaluation of cutaneous reactions.

No unscheduled deaths occurred during the study and no clinical signs were noted in any animals.

After a 4-hour exposure, a very slight or well-defined erythema was noted in all animals from day 1 until day 2, 4 or 5.

Dryness of the skin was recorded in one animal between day 6 and day 9 and in another animal on day 4.

  

In conclusion the test item was slightly irritant when applied topically to rabbits but should not be classified as irritating to skin according to the criteria of CLP Regulation. This difference of behaviour with the majority of the primary alkylamines that are classified corrosive or severely irritant is coherent with the salt form that could reduced the basicity of the compound. In addition, it is worthy to note that hydrogenated tallow alkylamines (i.e. the amine part of the salt) induced less severe effects on skin and is only classified as irritant. Eye irritation:

The objective of this study was to evaluate the potential irritant properties of the test item for the eye following a single administration to rabbits.This study was conducted in compliance with OECD Guideline No. 405 and the principles of Good Laboratory Practices.The test item was first administered to a single male New Zealand White rabbit.

 As mean value from grading at 24, 48 and 72 hours after instillation was = 2 for conjunctival edema (chemosis) and for conjunctival redness and = 1 for iris lesions and for corneal opacity, the test item was administered in the left eye of a second animal.

 Ocular reactions were observed approximately 1 hour, 24, 48 and 72 hours after the administration and then daily until the reversibility of the ocular reactions or until day 21. The mean values of the scores for chemosis, redness of the conjunctiva, iris lesions and corneal opacity were calculated for each animal.

No unscheduled deaths occurred during the study.One animal showed loud breathing on day 2. This could be considered as pain resulting from severity of ocular lesions. Thereafter chromorhynorrhea probably due to repeated administrations of fluorescein solutionswas noted from day 15.

In the left treated eye of one animal, marked to severe chemosis and severe redness of the conjonctiva were observed from day 1 to day 4. Severe redness persisted on day 5. Slight to moderate conjunctiva reactions were noted between days 6 and 11. Thereafter, moderate to marked chemosis was recorded between days 12 and 19, whereas moderate to severe redness was observed from day 12 to day 16. Then, slight conjunctiva reactions persisted until the end of the observation period (day 21).Iris lesions were noted from day 1 to day 19.

Moderate to marked corneal opacity was observed on days 2 and 3. Then, slight corneal opacity was recorded between days 4 and 14. Corneal opacity was graded marked again from day 15 to day 18.

In addition, marked blepharospasm was noted on day 2 and neovascularization was recorded from day 8 to day 18.

 In the left treated eye of one animal, moderate chemosis and moderate to severe redness of the conjonctiva were noted from day 1 to day 3. Then, slight to moderate conjonctiva reactions persisted until day 9.Iris lesions were noted from day 1 to day 3.Moderate to marked corneal opacity was recorded from day 2 to day 4. Thereafter, slight corneal opacity was still present until day 7.

Lacrimation and whitish deposit were noted in the left eye of both animals on many occasions during the observation period.

As the effects on the conjunctiva were not fully reversible within 21 days of application in one animal; it was considered that the test item caused serious eye damage.

Justification for classification or non-classification

 The available studies indicate that the registered substance should not be regarded as skin corrosive or irritant but should be classified irritant for serious eye damage Cat. 1, with hazard statement H318: Causes serious eye damage. STOT-SE Cat.3 for respiratory irritation is not required. Exposure to via the inhalation route will be unlikely to occur.