2-Propanone, reaction products with diphenylamine

Endpoint summary

Description of key information

Acute Toxicity: Oral

LD50 > 2000 mg/kg bw in female CRL:(WI) rats.

Acute Toxicity: Dermal

LD50 > 2000 mg/kg bw in female CRL: (WI) rats.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 June 2017 to 05 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

OECD GUIDELINES FOR TESTING OF CHEMICALS (423, adopted at 17th Dec. 2001)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1.tris

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

EPA Health Effects Test Guidelines (OPPTS 870.1100), United States, EPA 712-C-98-190 (1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

No further details specified in the study report.

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Species and strain: CRL:(WI) Wistar rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
Hygienic level at arrival: SPF
Hygienic level during the study: Standard housing conditions
Number of animals: 6 animals
Sex: Female, nulliparous and non-pregnant
Age of animals at dosing: Young healthy adult rats, 9 weeks old
Body weight at treatment: 203 – 232 g
Acclimatization period: at least 12 days

Husbandry
Animal health: Only healthy animals were used for the test. The Veterinarian certified health status.
Number of animal room: 522/9
Housing: 3 animals / cage
Cage type: Type II polypropylene/polycarbonate
Bedding/Nesting: “Lignocel 3/4-S Hygienic Animal Bedding” and “Arbocel crinklets natural” nest building material produced by J. Rettenmaier & Söhne GmbH & Co.KG (D-73494 Rosenberg, Germany) were available to animals during the study.
Lighting period: 12 hours daily, from 6.00 am to 6.00 pm
Temperature: 22 ± 3 °C
Relative humidity: 30-70%
Ventilation: 15 – 20 air exchanges/hour
Enrichment: Animals were housed by group to allow social interaction and with deep wood sawdust bedding to allow digging and other normal rodent activities.
The 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 (Batch no.: 285 17890, expiry date: 30 August 2017), ad libitum, and tap water from the municipal supply, as for human consumption from 500 ml bottles, 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.
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).

Animal Identification
Animals were individually identified using numbers written on the tail with an indelible marker 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, with information about study code, sex, dose group, cage number and individual animal numbers.

Route of administration:

oral: gavage

Vehicle:

DMSO

Details on oral exposure:

Formulation
The test item was freshly formulated at a concentration of 200 mg/mL in the vehicle, in the Pharmacy of CiToxLAB Hungary Ltd. on the day of administration. The formulation container was stirred continuously up to finishing the treatment.

Vehicle Selection
The selection of the vehicle was made during trial formulations with the test item. The final choice of vehicle was approved by the Sponsor.
On the basis of the trial formulations with the test item, the vehicle used was DMSO.
Vehicle: Dimethyl sulfoxide (DMSO)
Batch number: STBG8411
Expiry date: 29 February 2020
Dose volume: 10 mL/kg bw

Justification of the dose:
A limit of 2000 mg/kg bw dose was selected by the Sponsor.
Initially, three females (assigned to Group 1) were treated at a dose level of 2000 mg/kg bw. The test item did not cause mortality in this group and a second group (Group 2) was treated at the same dose level. No mortality was observed in the confirmatory group, therefore no further testing was required according to OECD 423 and Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1.tris.

Doses:

A limit of 2000 mg/kg bw dose was selected by the Sponsor.

No. of animals per sex per dose:

Initially, three females (assigned to Group 1) were treated at a dose level of 2000 mg/kg bw. The test item did not cause mortality in this group and a second group (Group 2) was treated at the same dose level.

Control animals:

no

Details on study design:

Procedure
A single oral gavage administration was followed by a 14-day observation period. On the night before treatment, the animals were fasted. The food but not water was withheld during an overnight period. Animals were weighed just before treatment. The test item was administered by oral gavage in the morning. The food was made available again at about 3 hours after the treatment.

OBSERVATIONS
Clinical Observations
Clinical observations were performed at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter, as applicable. Individual observations were performed on the skin, fur, eyes, mucous membranes, 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.

Body Weight Measurement
The body weight was recorded on the day before treatment (Day -1), on the day of the treatment (Day 0), Day 7 and Day 14.

NECROPSY
All animals were subjected to a necropsy and a macroscopic examination. The animals were exsanguinated after verification of narcosis following an injection of pentobarbital sodium (Release; Lot No.: 106075, Expiry Date: July 2018, Produced by: Wirtschaftsgenossenschaft deutscher Tierärzte eG, Siemensstr. 14, 30827 Garbsen, Germany). After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed. All gross macroscopic changes were recorded for each animal.

Statistics:

Not specified.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

AMINOX® did not cause mortality at a dose level of 2000 mg/kg bw.

Clinical signs:

other: Clinical signs were observed in 3/6 animals treated at the dose level of 2000 mg/kg bw with AMINOX® which included hunched back. There was no other test item related effects on the animals.

Gross pathology:

There was no evidence of the macroscopic observations at necropsy at a dose level of 2000 mg/kg bw.

Other findings:

No further findings detailed in the study report.

CLINICAL OBSERVATIONS

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

Cage No.

Animal Number

Observations

Observation days

Frequency

0

1

2

3

4

5

6

7-14

30’

1h

2h

3h

4h

6h

1

9631

Symptom free

+

+

-

-

-

+

+

+

+

+

+

+

+

17/20

Hunched back

-

-

+

+

+

-

-

-

-

-

-

-

-

3/20

9632

Symptom free

+

+

-

-

-

+

+

+

+

+

+

+

+

17/20

Hunched back

-

-

+

+

+

-

-

-

-

-

-

-

-

3/20

9633

Symptom free

+

+

-

-

-

+

+

+

+

+

+

+

+

17/20

Hunched back

-

-

+

+

+

-

-

-

-

-

-

-

-

3/20

2

9634

Symptom free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

9635

Symptom free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

9636

Symptom free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

Remarks:              + = present           - = absent

                          h = hour              ‘ = minute

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

 

BODY WEIGHT DATA

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

Cage No.	Animal Number	Body weight (g) Days				Body Weight Gain (g)
		-1	0	7	14	-1-0	0-7	7-14	-1-14
1	9631	243	227	235	250	-16	8	15	7
	9632	233	217	233	231	-16	16	-2	-2
	2633	239	227	237	243	-12	10	6	4
2	9634	241	232	240	261	-9	8	21	20
	9635	212	203	227	239	-9	24	12	27
	9636	220	207	230	247	-13	23	17	27
Mean:		231.3	218.8	233.7	245.2	-12.5	14.8	11.5	13.8
Standard deviation:		12.6	11.8	4.7	10.2	3.1	7.3	8.3	12.5

 

NECROPSY FINDINGS

DOSE 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	9631	04 July 2017 Day 14	No external observations recorded	No internal observations recorded	Not applicable
	9632	04 July 2017 Day 14	No external observations recorded	No internal observations recorded	Not applicable
	9633	04 July 2017 Day 14	No external observations recorded	No internal observations recorded	Not applicable
2	9634	05 July 2017 Day 14	No external observations recorded	No internal observations recorded	Not applicable
	9635	05 July 2017 Day 14	No external observations recorded	No internal observations recorded	Not applicable
	9636	05 July 2017 Day 14	No external observations recorded	No internal observations recorded	Not applicable

 

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the acute oral LD50 value of the test item AMINOX® was found to be above 2000 mg/kg bw in female CRL:(WI) rats.

Executive summary:

The single-dose oral toxicity of AMINOX® was performed according to the acute toxic class method (OECD 423 and Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1.Tris) in CRL:(WI) rats.

 

Initially, three females (assigned to Group 1) were treated at a dose level of 2000 mg/kg bw. The test item did not cause mortality in this group and a second group (Group 2) was treated at the same dose level. No mortality was observed in the confirmatory group, therefore no further testing was required according to OECD 423 and Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1.tris.

 

A single oral treatment was carried out by gavage for each animal after an overnight food withdrawal. Food was made available again 3 hours after the treatment. The test item was administered formulated in Dimethyl sulfoxide (DMSO) at a concentration of 200 mg/mL at a dosing volume of 10 mL/kg bw.

 

Clinical observations were performed at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Body weight was measured on Days -1, 0 and 7 and Day 14.

All animals were subjected to a necropsy and a macroscopic examination.

 

Results

Mortality

AMINOX® did not cause mortality at a dose level of 2000 mg/kg bw.

 

Clinical Observations

Clinical signs were observed in 3/6 animals treated at the dose level of 2000 mg/kg bw with AMINOX® which included hunched back. There was no other test item related effects on the animals.

 

Body Weight and Body Weight Gain

One animal at dose level 2000 mg/ kg bw (No: 9632) showed a reduced body weight gain in the second week of the observation period. This change was considered incidental and not ascribed to treatment. There was no other treatment related effects on body weight or body weight gain during the observation period.

 

Macroscopic Findings

There was no evidence of the macroscopic observations at necropsy at a dose level of 2000 mg/kg bw.

 

Conclusion:

Under the conditions of this study, the acute oral LD50 value of the test item AMINOX® was found to be above 2000 mg/kg bw in female CRL:(WI) rats.

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Study was not conducted in accordance to GLP or recognised guidelines

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

GLP compliance:

no

Test type:

other:

Limit test:

yes

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

Not specified

Route of administration:

oral: unspecified

Vehicle:

other: methylcellulose

Details on oral exposure:

Prior to oral exposure rats were fasted for 24 hours

Doses:

Single oral dose of 5 g/kg on day 1

No. of animals per sex per dose:

5 male and 5 female rats were each dosed with 5 g/kg of the test substance

Control animals:

not specified

Details on study design:

A group of ten Long Evans rats (5 male and 5 female) weighing 200 – 250 g were fasted for 24 hours and given AMINOX C#4711 at 5 g/kg orally. The test material was administered as a suspension in 0.25% methylcellulose at a volume of 10 ml/kg. The rats were observed for mortality for 14 days

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Mortality:

None

Clinical signs:

other: None clinical signs specified

Gross pathology:

Not specified

Other findings:

No other findings

None of the treated rats died during the fourteen day period of observation. Necropsy upon termination of the study revealed no visible drug related lesions.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: other: Section 191.1(b) (1) of the Federal Hazardous Substances Act.

Conclusions:

AMINOX C#4711 would not be a toxic substance according to section 191.1(b) (1) of the Federal Haze- Substances Act (LD50 > 5 g/kg)

Executive summary:

The aim of the study was to assess the toxicity potential of Aminox C#4711 via the oral route. 10 rats (5 male and 5 female) were administered Aminox C#4711 5 g/kg orally on day 1 and observed for 14 days. None of the rats died during the 14 day period and necropsy did not reveal any visable drug related lesions. Therefore it can be concluded that the acute oral toxicity of Aminox C#4711 is LD50 > 5g/kg and is not considered toxic according to section 191.1(b) (1) of the Federal Haze-Susbtances Act.

Results from the study concluded that Aminox C#4711 is not considered a toxic substance as none of the treated rats died during the 14 day perood of observation. Necropsy also revealed that there was no visible test substance related lesion. Therefore the acute oral LD50 > 5g/kg

Reference 3

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Study was not conducted in accordance with GLP or a recognised test guideline

Qualifier:

no guideline available

GLP compliance:

no

Remarks:

Predates GLP

Test type:

other:

Limit test:

yes

Species:

rat

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

All animals were housed in metal cages with raised wire mesh floors, food and water were available at all times.

Route of administration:

oral: unspecified

Vehicle:

other: Distilled water

Details on oral exposure:

Animals were force fed test substance which was suspended in distilled water.

Doses:

Single dose of 400mg (equivalent of 1.69 g/kg/bw) on day 1

No. of animals per sex per dose:

Not specified

Control animals:

not specified

Details on study design:

Distilled water suspension of the samples in various amounts were made and force fed to adult albino rats, in an attempt to determine the minimum amount of the sample which could cause death within 3 days.

Statistics:

None

Sex:

not specified

Dose descriptor:

LD50

Effect level:

> 1 690 mg/kg bw

Based on:

test mat.

Mortality:

None

Clinical signs:

other: Not specified

Gross pathology:

Not specified

Other findings:

Not specified

400mg (equivalent to 1.69 g/kg/bw) was force fed to several rats but surivived the 3 day study period.

Interpretation of results:

other: Inconclusive

Remarks:

Criteria used for interpretation of results: not specified

Conclusions:

Minimum leath dose was not obtained. 400mg (equivalent to 1.69 g/kg/bw) was force fed to several rats but surivived the 3 day study period.

Executive summary:

Study was conducted on albino rats to determine the minimum lethal dose of Aminox A-3250. The maximum amount of test substance that was administered to several rats was 400 mg (equivalent to 1.69 g/kg/bw). All rats survived the 3 day study period, LD50 > 1.69 g/kg/bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

K1

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Study not conducted in accordance with GLP or a recognised test guideline

Qualifier:

according to guideline

Guideline:

other: Section 162.8 (c) of Regulation for the Enforcement of Federal Insecticide, Fungicide and Rodenticide Acte, United States Dept. of Agriculture. March 1948

GLP compliance:

no

Remarks:

Predates GLP

Test type:

other:

Limit test:

no

Species:

rabbit

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

Not specified in report

Type of coverage:

occlusive

Vehicle:

other: Distilled water

Duration of exposure:

Exposure of 24 hours.

Doses:

200mg/kg/bw

No. of animals per sex per dose:

10

Control animals:

not specified

Details on study design:

The hair from one side of the rabbit was removed using OIster small animal clipper model #2. The submitted samples were tested at a dosage of 200 mg/kg/bw.
The calculated dosage was wetted with distilled water and then placed onto gauze squares measuring 2 x 4 inches and two layers in thickness. The squares were immediately placed on the bare skin of each rabbit and securely help in place with waterproof adhesive tape. Care was taken to completely cover each patch securely so as to minimize evaporation and to ensure continuous contact with the base skin for 24 hours. Additional observations were made after 72 hours.

Sex:

not specified

Dose descriptor:

LD50

Effect level:

> 200 mg/kg bw

Based on:

test mat.

Mortality:

None

Clinical signs:

other: None

Gross pathology:

No data

Other findings:

None

All animals survived the 24 hours test period and remained alive and well. There were no toxic manifestations exhibited by any animal under the conditions of the study.

Interpretation of results:

practically nontoxic

Remarks:

Migrated information Criteria used for interpretation of results: not specified

Conclusions:

All animals survived the 24 hours test period and remained alive and well. There were no toxic manifestations exhibited by any animal under the conditions of the study.

Executive summary:

The aim of the study was to determine whether Aminox A-3520 is toxic via the dermal absorption. The test was conducted in accordance to Section 162.8 (c) of Regulation for the Enforcement of Federal Insecticide, Fungicide and Rodenticide Acte, United States Dept. of Agriculture. March 1948. All animals survived the 24 hours test period and remained alive and well, not showing any signs of toxicity. To conclude Aminox A-3520 was not deemed to be toxic via the dermal route and the LD50 > 200 mg/kg/bw

Reference 2

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

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

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.

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.

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.

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.

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

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

K1

Additional information

Acute Toxicity: Oral

The single-dose oral toxicity of AMINOX® was performed according to the acute toxic class method (OECD 423 and Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1.Tris) in CRL:(WI) rats.

Initially, three females (assigned to Group 1) were treated at a dose level of 2000 mg/kg bw. The test item did not cause mortality in this group and a second group (Group 2) was treated at the same dose level. No mortality was observed in the confirmatory group, therefore no further testing was required according to OECD 423 and Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1.tris.

A single oral treatment was carried out by gavage for each animal after an overnight food withdrawal. Food was made available again 3 hours after the treatment. The test item was administered formulated in Dimethyl sulfoxide (DMSO) at a concentration of 200 mg/mL at a dosing volume of 10 mL/kg bw.

Clinical observations were performed at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Body weight was measured on Days -1, 0 and 7 and Day 14.

All animals were subjected to a necropsy and a macroscopic examination.

 

Results

Mortality: AMINOX® did not cause mortality at a dose level of 2000 mg/kg bw.

Clinical Observations: Clinical signs were observed in 3/6 animals treated at the dose level of 2000 mg/kg bw with AMINOX® which included hunched back. There was no other test item related effects on the animals.

Body Weight and Body Weight Gain: One animal at dose level 2000 mg/ kg bw (No: 9632) showed a reduced body weight gain in the second week of the observation period. This change was considered incidental and not ascribed to treatment. There was no other treatment related effects on body weight or body weight gain during the observation period.

Macroscopic Findings: There was no evidence of the macroscopic observations at necropsy at a dose level of 2000 mg/kg bw.

 

Conclusion: Under the conditions of this study, the acute oral LD50 value of the test item AMINOX® was found to be above 2000 mg/kg bw in female CRL:(WI) rats.

Acute Toxicity: Dermal

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 noin vitroalternative 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.

Justification for classification or non-classification

Acute Toxicity: Oral

The test substance is not classified under the CLP criteria.

Acute Toxicity: Dermal

The test substance is not classified under the CLP criteria.