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Acute Toxicity: dermal

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Administrative data

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 - 29 November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
yes
Remarks:
Due to technical oversight the environmental parameters were recorded only once on 10 November 2018.
Qualifier:
according to guideline
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Deviations:
yes
Remarks:
Due to technical oversight the environmental parameters were recorded only once on 10 November 2018.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1200 (Acute Dermal Toxicity)
Deviations:
yes
Remarks:
Due to technical oversight the environmental parameters were recorded only once on 10 November 2018.
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-Propanone, reaction products with diphenylamine
EC Number:
270-192-0
EC Name:
2-Propanone, reaction products with diphenylamine
Cas Number:
68412-48-6
Molecular formula:
Variable.
IUPAC Name:
N-cyclohexylcyclohexanamine; propan-2-one
Test material form:
solid: particulate/powder
Details on test material:
Name: AMINOX®
Chemical name: 2-Propanone, reaction products with diphenylamine
Batch/Lot number: EL5D06K347
Appearance: Green to brown powder
CAS number: 68412-48-6
Purity: 100% (as a UVCB)
Retest date: 05 April 2018
Storage conditions: Room temperature
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
Specific details on test material used for the study:
Name: AMINOX®
Chemical name: 2-Propanone, reaction products with diphenylamine
CAS number: 68412-48-6
Batch/Lot Number: T7C16001
Description: Green to brown flakes
Purity: 100% (as a UVCB)
Expiry date: 14 March 2019
Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH%)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WIWistar rats
Sex:
female
Details on test animals or test system and environmental conditions:
Species and strain: Crl: WIWistar rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld, Germany
Hygienic level at arrival: SPF
Hygienic level during
the study: Standard housing conditions
Justification of strain: The Wistar rat is one of the standard rodent species used in acute toxicity studies.
Number of animals: 3 animals
Sex: Female, nulliparous and non-pregnant.
Age of animals at study start: ~9 weeks old
Body weight range at dosing: Between 238 g and 246 g
Acclimation time: 5 or 7 days

Husbandry
Animal health: Only healthy animals were used for the study. The staff Veterinarian certified the health status.
Room-Box: 242/3
Housing / Enrichment: Individual and group caging. Rodents were housed with deep wood sawdust bedding to allow digging and other normal rodent activities.
Cage type: Type II. polypropylene/polycarbonate

Bedding: Lignocel 3/4-S Hygienic Animal Bedding (produced by J. Rettenmaier & Söhne GmbH+Co.KG, Germany) was available to animals during the study. The quality of the bedding was guaranteed by the supplier. Details of bedding quality are archived with the raw data.
Nesting: Nest building material Arbocel Crinklets natural (produced by J. Rettenmaier & Söhne GmbH+Co.KG, Germany) was available to animals during the study. The quality of the nest building material was guaranteed by the supplier. Details of nest building material quality are archived with the raw data.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.2–24.2°C
Relative humidity: 33–62%
Ventilation: 15-20 air exchanges/hour
Enrichment: Rodents were housed with deep wood sawdust bedding to allow digging and other normal rodent activities.
Temperature and relative humidity were recorded twice daily during the study.

Food and Water Supply
Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany, ad libitum, and tap water from the municipal supply, as for human consumption from a 500 mL bottle, ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study and the water was considered fit for human consumption.
The batch of feed employed in the study was as follows:
• 639 38520, expiry date: 30 April 2019.
The supplier provided an analytical certificate for the batch used. A copy of the certificate will be archived with the raw data.

Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). The quality control results are retained in the archives at Citoxlab Hungary Ltd.

Animal Identification
Animals were individually identified using numbers written on the tail with an indelible pen. The numbers were given on the basis of Citoxlab Hungary Ltd.' s Master File for each animal allocated to the treatment groups. The cages were identified by cards containing information about study code, sex, dose group, cage number and individual animal number.

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
The back of each animal was shaved (approximately 10% area of the total body surface) approximately 24 hours prior to treatment. The test item was applied to the shaved skin as a single dose and remained in contact with the skin for the 24-hour exposure period. Sufficient amount of water was used to moisten the test item to ensure good contact with the skin. Sterile gauze pads were placed on the skin of rats to cover the test item. These gauze pads were kept in contact with the skin using a patch with adhesive hypoallergenic plaster. The entire trunk of the animal was then wrapped with semi occlusive plastic wrap for 24 hours.
At the end of the exposure period, the treated area of skin with the test item was washed with water at body temperature.
Duration of exposure:
14 days
Doses:
Dose range-finding test
Initially one animal was dosed at the selected limit dose (2000 mg/kg bw). As the animal survived, the second and third animals received the same dose.

Main test
A single dermal application of 2000 mg/kg bw was made and was followed by a 14-day observation period.
No. of animals per sex per dose:
3 animals were dosed with 2000 mg/kg bw
Control animals:
yes, concurrent no treatment
Details on study design:
Observations
Clinical Observations
Clinical observations were performed on the day of treatment at 30 minutes, 1, 2 and 5 hours after application of the test item and once each day for 14 days thereafter. Observations included the skin and fur, eyes and mucous membranes, the respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Skin Irritation
Adverse skin reactions at the site of application were recorded daily following the removal of the dressing.

Measurement of Body Weight
The body weights were recorded on Day 0 (before the test item administration) and on Days 7 and 14 (before necropsy).

NECROPSY
Macroscopic examination was performed on all animals. All animals were anaesthetised with sodium pentobarbital (details in 3.3) and exsanguinated. Following confirmation of death, after examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed. All macroscopic changes were recorded.


Formulation
The powdered test item was administered as a single dose without dilution, as supplied by the Sponsor. Sufficient water was used to moisten the test material to ensure good contact with the skin.

Justification of the dose:
In an acute oral toxicity study (study code: 17/174-001P), adverse effects were noted up to 2000 mg/kg bw (hunched back in 3 animals out of 6). Thus in this dermal acute study, one female animal was treated initially at 2000 mg/kg bw in the range finding test followed by 2 more animals treated in the main study.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
>= 2 000 mg/kg bw
Based on:
test mat.
Mortality:
The test item did not cause mortality at a dose level of 2000 mg/kg bw.
Clinical signs:
other: There were no systemic clinical signs noted in any animal throughout the study.
Gross pathology:
There was no evidence of any gross macroscopic changes at a dose level of 2000 mg/kg bw.
Other findings:
Local Dermal Signs
No adverse local dermal signs were observed after treatment with the test item or during the 14-day observation period.

Any other information on results incl. tables

DOSE LEVEL: 2000 mg/kg bw, Treatment on Day 0

Cage No.

 

Animal Number

 

Observations

 

Observation days

 

Frequency

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

30’

1h

2h

5h

1

 

5890

 

Symptom Free

 

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

18/18

 

2 / 1

 

5891

 

Symptom Free

 

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

18/18

 

3 / 1

 

5892

 

Symptom Free

 

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

18/18

 

 

Remarks:

+ = present

h = hour (s)

' = minute

Frequency of observation = number of occurrence of observation / total number of observations

 

Body Weight DataDOSE LEVEL: 2000 mg/kg bw, Treatment on Day 0

SEX: FEMALE

 

 

Cage No.

Animal Number

Body weight (g)

Body Weight Gain (g)

0

7

14

0-7

7-14

0-14

1

5890

244

245

253

1

8

9

2 / 1

5891

246

248

256

2

8

10

3 / 1

5892

238

246

261

8

15

23

Mean:

242.7

246.3

256.7

3.7

10.3

14.0

Standard deviation:

4.2

1.5

4.0

3.8

4.0

7.8

 

Macroscopic FindingsDOSE LEVEL: 2000 mg/kg bw, Treatment on Day 0

SEX: FEMALE

 

Cage No.

Animal Number

Necropsy Date/ Necropsy Day

External Observations

Internal Observations

Organ/Tissue

1

5890

27 November 2018 Day 14

No external observations recorded

No internal observations recorded

Not applicable

2 / 1

5891

29 November 2018 Day 14

No external observations recorded

No internal observations recorded

Not applicable

3 / 1

5892

29 November 2018 Day 14

No external observations recorded

No internal observations recorded

Not applicable

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The acute dermal median lethal dose (LD50) of the test item AMINOX® was found to be greater than 2000 mg/kg body weight in female Crl: WI rats.
According to the GHS criteria, AMINOX® can be ranked as "Unclassified" for acute dermal exposure.
Executive summary:

An acute dermal toxicity study was performed with the test item AMINOX® in female Crl: WIWistar rats, in compliance with OECD Guideline No.: 402 (2017), Commission Regulation (EC) No 440/2008, B.3 and OPPTS 870.1200 [1-3].

This study was being performed with vertebrate animals as no in vitro alternative is available. The Sponsor confirmed previously that the specific regulatory purpose of this study did not allow a waiving of this dermal acute study, taking account of the OECD guidance document 237.

The study was designed such that the minimum number of animals were used. One female animal was initially treated at a dose level of 2000 mg/kg body weight (bw) in a range-finding phase. Due to the absence of clinical signs, a further 2 females were then treated to confirm the non-lethal dose level. The test item was applied as a single dermal 24-hour exposure followed by a 14-day observation period.

Clinical observations were performed on all animals at 30 minutes, 1, 2, 5 hours after dosing and daily for 14 days thereafter. Body weight was measured on Day 0 (prior to dosing) and on Days 7 and 14 (before necropsy). Gross macroscopic examination was performed on all animals at necropsy at the end of the 14-day observation period (Day 14).

The results of the study were summarised as follows:

Mortality

The test item did not cause mortality at the dose level of 2000 mg/kg bw.

Systemic clinical signs

There were no systemic clinical signs noted in any animal throughout the study.

Local dermal signs

No adverse local dermal signs were observed after treatment with the test item or during the 14-day observation period.

Body weight and body weight gain

There were no test item related effects on body weight or body weight gain during the observation period. Body weights were within the range commonly recorded for this strain and age.

Necropsy

There was no evidence of any macroscopic changes at a dose level of 2000 mg/kg bw.

 

Conclusions

The acute dermal median lethal dose (LD50) of the test item AMINOX® was found to be greater than 2000 mg/kg body weight in female Crl: WI rats.

According to the GHS criteria, AMINOX® can be ranked as "Unclassified" for acute dermal exposure.