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

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

813-944-0

Cas Number:

1884575-91-0

Molecular formula:

C44H70Cl2N10O4

IUPAC Name:

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

Test material form:

solid: particulate/powder

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

Test animals

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

Test system

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).

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 2

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 3

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 4

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 5

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Results 6

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.