N,N'-bis{N-[N-(4-chlorophenyl)carbamimidoyl]carbamimidoyl}hexane-1,6-bis(aminium) diundec-10-enoate

Endpoint summary

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:

August 11-August 22, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

OTHER SPECIFICS:SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 001/CHUA/01 16
- Expiration date of the lot/batch: December, 2017
- Purity test date: January, 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature
- Stability under test conditions: Two years

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Mahaveera Enterprises,
AF. Plot No: 9 & 18, S. No: 127 & 150,
Peerzadiguda, Ghatkesar Mandal,
R.R. District, Hyderabad - 39, India
- Age at study initiation: 10 to 12 weeks
- Weight at study initiation: 2.2329 to 2.3519 Kg
- Housing: Housed individually in EN130S Noryl (Tecniplast) cages (approximately 653 mm x 653 mm).
- Diet (e.g. ad libitum): Teklad Certified Global High Fiber Rabbit Diet (Lot No. 2031C- 1 l 0415MA) from ENVIGO was provided ad libitum
- Water (e.g. ad libitum): Aquaguard filtered tap water was provided ad libitum.
- Acclimation period: Under laboratory conditions for a period of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 .9 to 21.4 °C
- Humidity (%): 57 to 67 %
- Air changes (per hr): > 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
- Concentration (if solution):

Duration of treatment / exposure:

4 h

Observation period:

72h

Number of animals:

3

Details on study design:

Test Item was used as delivered by the Sponsor. The test item, 0.5 g (per animal) was weighed on a Sartorius balance and moistened with distilled water and applied to the intact skin. The pH of the test item was measured before the study initiation date. A formulation of a 1 % (w/v) aqueous suspension was prepared for measuring the pH. The average pH was found to be 6.90 (from Solubility and pH Test).

Approximately 24 (± 2) hours before application of the test item, both the flanks were clipped with a clipper, exposing an area of approximately 100 cm" (10 cm x 10 cm).

Animals without any skin injury or overt signs of irritation were used in the test. A single male rabbit was treated first. As no severe skin reactions were observed until 48 hours (approximately) post exposure, the test was completed by exposing the remaining two male rabbits. The animals were exposed to the test item for a period of 4 hours (approximately). On the day of treatment, 0.5 g of test item was transferred to a surgical gauze patch, (approximately, 4 cm x 4 cm) and moistened with 0.5 mL of distilled water. The gauze patch was applied to the intact skin of the clipped area. The gauze loaded with test item was applied to the treatment site of the
clipped area (left flank), while the control site (right flank) was sham treated with distilled water. The patches were covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with non irritating adhesive tape.


The duration of exposure was 4 hours (approximately), after which the dressing was removed and the skin was gently wiped clean with cotton soaked in distilled water.

The skin reaction was assessed according to the numerical scoring system listed in OECD guidelines No. 404, "Grading of Skin Reactions" (28111 July 2015) (Refer Section 9), at approximately 1, 24, 48 and 72 hours post removal of the dressing, gauze patch and unabsorbed test item.

Irritant
· (Category 2) (applies to all authorities)

(1) Mean value of> 2.3 - < 4.0 for erythema/eschar or for oedema in at least 2 of 3 tested animals from grading at 24, 48 and 72 hours after patch removal or, if reactions are delayed, from grades on 3 consecutive days after the onset of dermal reactions, or
(2) Inflammation that persists to the end of the observation period normally 14 days in at least 2 animals, particularly taking into account alopecia (limited area), hyperkeratosis, hyperplasia, and scaling, or
(3) In some cases where there is pronounced variability of response among animals, with very definite positive effects related to chemical exposure in a single animal but less than the criteria above.

Mild irritant
(Category 3) (applies to only some authorities)

Mean value of> 1.5 - < 2.3 for erythema/eschar or for oedema from grading in at least 2 of 3 tested animals from grades at 24, 48 and 72 hours or, if reactions are
delayed, from grades on 3 consecutive days after the onset of dermal reactions (when not included in the irritant category above).

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

No corrosive effects were observed on the skin of treated animals.

Other effects:

The test item did not impart any coloration on the treated skin of any rabbit.

MORTALITY I CLINICAL SIGNS

Animal number	Sex	Test day
		0			1		2		3
		M	E		M	E	M	E	M	E
01	Male	1		1	1	1	1	1	1#	-
02		1		1	1	1	1	1	1#	-
03		1		1	1	1	1	1	1#	-

Key: M= Morning check, E= Evening check, 1 = Normal.

*Examinations were performed twice daily, # Animal sent for sacrifice after 72 hour.

No clinical signs were evident in any animal during the acclimatization.

SKIN IRRITATION SCORES - INDIVIDUAL MEAN VALUES AFTER 24, 48 AND 72 HOURS - LEFT FLANK( (TREATED -

SITE)

Animal number	01		02		03
Observation	Erythema	Oedema	Erythema	Oedema	Erythema	Oedema
Mean Score	0.00	0.00	0.00	0.00	0.00	0.00

Interpretation of results:

other: Non Irritant

Conclusions:

Since the mean score for erythema and oedema did not qualify for Category 2 or 3 of the classification criteria, CHUA is classified as per the Harmonized Integrated Classification System as "Non Irritant" to the rabbit skin.

Executive summary:

The acute dermal irritation/corrosion of CHUA in rabbits was investigated according to OECD test guideline Number 404. The test item was applied by topical (semi-occlusive) application, 0.5 g to the intact skin of left flank of a single male rabbit. As no severe skin reactions were observed upto 48 hours post test item application (approximately), the test was completed by exposing the remaining two male rabbits. The test item was left in contact with the skin for an exposure period of 4 hours. The scoring of skin reactions were done at approximately 1, 24, 48, and 72 hours post exposure for Animal No. 01, 02 and 03 (post removal of the dressing, gauze patch and unabsorbed test item).

The individual mean score for erythema/eschar formation for Animal No. 01., 02 and 03 at 24, 48 and 72 hours were 0.00, 0.00 and 0.00, respectively. The individual mean score of oedema for Animal No. 01, 02 and 03 at 24, 48 and 72 hours were 0.00, 0.00 and 0.00 respectively.

The test item did not induce any degree of erythema or oedema in Animal No. 01, 02, and 03 at approximately 1, 24, 48 and 72 hours observation post exposure (post removal of the dressing, gauze patch and unabsorbed test item). All the treated animals were sent for necropsy after 72 hour observation period. All animals survived till the end of the observation period. No clinical signs were observed in any of the animals throughout the acclimatization and experimental periods respectively. The body weights of all the animals were considered to be within the normal range of variability commonly observed for this species, strain and age. No abnormalities were detected in any of the treated animals during necropsy at terminal sacrifice.

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:

August 11- September 26, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

OTHER SPECIFICS:SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 001/CHUA/01 16
- Expiration date of the lot/batch: December, 2017
- Purity test date: January, 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature
- Stability under test conditions: Two years

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source:
- Age at study initiation:
- Weight at study initiation:
- Housing:
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: From: To:

Vehicle:

water

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

Duration of treatment / exposure:

21 days

Observation period (in vivo):

The eyes were examined at approximately 1, 24, 48, 72 hours, days 7, 14 and 21

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
- Time after start of exposure:

SCORING SYSTEM:

TOOL USED TO ASSESS SCORE: hand-slit lamp / biomicroscope / fluorescein

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.67

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

not fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritant / corrosive response data:

No corrosive effects were observed on the cornea of the treated rabbits.

Other effects:

The test item did not result in any staining of the treated eye of the rabbits. No abnormalities were detected internally in any of the treated animal during necropsy at terminal sacrifice.
Moderate corneal opacity and slightly conjunctivae edematous was present in left eye of Animal No. 01. Marked corneal opacity, conjunctiva edema slightly present in left eye of Animal No. 02 and 03 were observed externally during necropsy at terminal sacrifice.

EYE IRRITATION SCORES - MEAN VALVES AFTER 24, 48 AND 72 HOURS (TREATED - LEFT EYE) FOR INDIVIDUAL ANIMAL

Animal number	Sex	Eye reaction
		Corneal opacity	Iris	Conjunctivae	Chemosis
01	Male	1.00	1.00	1.67	3.00
02		1.67	1.33	1.33	3.00
03		1.67	1.33	1.33	3.00

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Since the treated animals exhibited redness of conjunctivae ≤ 1.67, corneal opacity ≤ 1.67, chemosis ≤ 3.00, lesions in iris ≤ 1.33 and the lesions in the eye were not reversed at day 21 observation in 3 out of 3 treated animals, hence the outcome of result qualify for category 1 of the classification criteria, hence CHUA is classified as per Harmonised Integrated Classification System as "irreversible effects" to rabbit eyes.

Executive summary:

The acute eye irritation/corrosion study of CHUBA in Rabbits was investigated according to OECD test guideline 405. The test item, 0.1g was placed in the conjunctiva! sac of the left eye of a single male rabbit. Eye reactions were observed up to 72 hours approximately post treatment, the test was completed using the remaining two male rabbits to confirm the findings of the initial test. The scoring of eye reactions was performed at 1, 24, 48, 72 hour, day 7, 14 and 21 for all the treated animals post test item instillation. The mean score was calculated across 3 scoring intervals (24, 48 and 72 hour post treatment) for each animal for corneal opacity, iris, conjunctival redness and chemosis. The individual mean score of opacity, iris, conjunctivae and chemosis for Animal No. 01 was 1.00, 1.00, 1.67 and 3.00, for Animal No. 02 and 03 were 1.67, 1.33, 1.33 and 3.00.

In Animal No. 01, instillation of test item in rabbit left eye caused redness of conjunctivae with some blood vessels definitely hyperaemic at 1 hour observation post test item instillation. Redness of conjunctivae with diffuse, crimson color; individual vessels not easily discernible was observed at 24 and 48 hour observation. Animal continued to exhibit conjunctival redness, the severity was slightly reduced to conjunctivae with some blood vessels definitely hyperaemic at 72 hour and the lesion fully reversed and appeared normal on day 7. Some swelling above normal (includes nictitating membranes) of chemosis was observed at 1 hour observation post test item instillation. Chemosis with obvious swelling with partial eversion of lids was observed at 24 hour observation. Swelling, with lids about half closed was observed at 48 hour and swelling, with lids about more than half closed was observed at 72 hour observation. The severity of Chemosis was slightly reduced to some swelling above normal (includes nictitating membranes) on day 7 and appeared normal on day 14 of observation. No opacity was observed at 1 hour and 24 hour observation whereas diffuse areas of opacity involving one quarter but not zero and details of iris clearly visible was observed at 48 hour and easily discernible translucent area of opacity with greater than one quarter, but less than half was observed at 72 hour observation. The severity increased to opaque cornea and iris not discernible through the opacity greater than three quarters, upto whole area on was observed on day 7, 14 and 21 observation. No lesion in iris was observed at 1, 24, hour and iris reactive to light (a sluggish reaction is considered to be an effect) was observed at 48 hour observation. At 72 hour the severity increased to no reaction to light and continued to exhibit on days 7, 14 and 21 observation.

In Animal No. 02 and 03, instillation of test item in rabbit left eye caused redness of conjunctivae with some blood vessels definitely hyperaemic at 1 hour observation post test item instillation. Redness of conjunctivae with diffuse, crimson color; individual vessels not easily discernible was observed at 24 and the severity was slightly reduced to conjunctivae with some blood vessels definitely hyperaemic at 48 and 72 hour observation. This lesion fully reversed and appeared normal on day 7. Some swelling above normal (includes nictitating membranes) of chemosis was observed at 1 hour observation post test item instillation. Chemosis with obvious swelling with partial eversion of lids was observed at 24 hour observation. Swelling, with lids about half closed was observed at 48 hour and swelling, with lids about more than half closed was observed at 72 hour observation. The severity of Chemosis was reduced to some swelling above normal (includes nictitating membranes) on day 7 and appeared normal on day 14 of observation. No opacity was observed at 1 hour observation whereas diffuse areas of opacity involving one quarter but not zero and details of iris clearly visible was observed at 24 hour and easily discernible translucent area of opacity with greater than one quarter, but less than half was observed at 48 and 72 hour observation. The severity of lesion increased to opaque cornea and iris not discernible through the opacity greater than three quarters, upto whole area was observed on day 7, 14 and 21 observation. No lesion in iris was observed at 1, 24, hour and no reaction to light was observed at 48 and 72 hour observation. This lesion was continued to exhibit on days 7, 14 and 21 observation. After 21 days of observation all the treated animals (Animal No. 01, 02 and 03) were sent for terminal sacrifice.

Based on the above findings, in 3 out of 3 animals, the eye lesions persisted till day 21 observation. The experiment was terminated on day 21 considering the test item had "irreversible effects" on the eye. No clinical signs were observed in any of the test animals throughout the acclimatization period and during experimental period. The body weights of all the animals were considered to be within the normal range of variability commonly observed for this species, strain and age. No abnormalities were detected internally in any of the treated animal during necropsy at terminal sacrifice. Moderate corneal opacity and slightly conjunctivae edematous was present in left eye of Animal No. 01. Marked corneal opacity, conjunctiva edema slightly present in left eye of Animal No. 02 and 03 were observed externally during necropsy at terminal sacrifice.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available