Registration Dossier

Administrative data

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 February 2018 to 08 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Version / remarks:
OECD GUIDELINES FOR TESTING OF CHEMICALS (No.: 402, 09th Oct. 2017)
Deviations:
yes
Remarks:
Due to technical reason, relative humidity values (minimum of 28%) out of the target range 30-70% were observed, however these minor differences in the environmental parameter were considered not to adversely affect the study.
Qualifier:
according to guideline
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Version / remarks:
Commission Regulation (EC) No 440/2008, B.3 (L 142, 30 May 2008)
Deviations:
yes
Remarks:
Due to technical reason, relative humidity values (minimum of 28%) out of the target range 30-70% were observed, however these minor differences in the environmental parameter were considered not to adversely affect the study.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1200 (Acute Dermal Toxicity)
Version / remarks:
OPPTS 870.1200 (EPA 712-C-98-192, August 1998)
Deviations:
yes
Remarks:
Due to technical reason, relative humidity values (minimum of 28%) out of the target range 30-70% were observed, however these minor differences in the environmental parameter were considered not to adversely affect the study.
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Nitrosodiphenylamine
EC Number:
201-663-0
EC Name:
Nitrosodiphenylamine
Cas Number:
86-30-6
Molecular formula:
C12H10N2O
IUPAC Name:
N-nitroso-N-phenylaniline
Test material form:
solid: pellets

Test animals

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, 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 239 g and 260 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: Individual and group caging
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: 19.0–24.2°C
Relative humidity: 28–45%
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 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).

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:
water
Remarks:
to moisten area only.
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 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:
24-hour exposure period
Doses:
A single dermal application of 2000 mg/kg bw
No. of animals per sex per dose:
One female rat was dosed initially (sentinel animal) than 2 female animals were dosed 48 hours later.
Control animals:
no
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 pentobarbital sodium (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.
Statistics:
The method used was not intended to allow the calculation of a precise LD50 value.
Body weight and body weight gain are summarised in tabular form. Clinical signs and necropsy findings are described and summarised in tabular form.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD0
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: no mortality occurred
Mortality:
The test item did not cause mortality at the dose level of 2000 mg/kg bw.
Clinical signs:
There were no systemic clinical signs noted in any animal throughout the study.
Body weight:
There was no treatment 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.
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 days observation period.
The test item coloured the treatment area to yellowish on Day 1 (3/3 animals) and Day 2 (2/3) animals.

Any other information on results incl. tables

Clinical Observations

DOSE LEVEL: 2000 mg/kg bw                                                                                                                                      SEX: FEMALE

Cage No.

Animal No.

Observations

Observation days

Frequency

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

30’

1h

2h

5h

1

3126

Symptom Free

+

+

+

+

-

-

+

+

+

+

+

+

+

+

+

+

+

+

16/18

Skin colour (yellowish-treated area)

-

-

-

-

+

+

-

-

-

-

-

-

-

-

-

-

-

-

2/18

2

3127

Symptom Free

+

+

+

+

-

+

+

+

+

+

+

+

+

+

+

+

+

+

17/18

Skin colour (yellowish-treated area)

-

-

-

-

+

-

-

-

-

-

-

-

-

-

-

-

-

-

1/18

3

3128

Symptom Free

+

+

+

+

-

-

+

+

+

+

+

+

+

+

+

+

+

+

16/18

Skin colour (yellowish-treated area)

-

-

-

-

+

+

-

-

-

-

-

-

-

-

-

-

-

-

2/18

Remarks:            + = present                           - = absent

                          h = hour                Treatment day = Day 0

                          ‘ = minute

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

 

Body Weight Data

DOSE LEVEL: 2000 mg/kg bw                                                                                               SEX: FEMALE

Cage No.

Animal No.

Body weight (g)

Days

Body Weight Gain (g)

0

7

14

0-7

7-14

0-14

1

3126

260

271

282

11

11

22

2

3127

247

279

294

32

15

47

3

3128

239

250

257

11

7

18

Mean:

248.7

266.7

277.7

18.0

11.0

29.0

Standard deviation:

10.6

15.0

18.9

1.1

4.0

15.7

 

Macroscopic Findings

DOSE LEVEL: 2000 mg/kg bw                                                                                                                                  SEX: FEMALE

Cage No.

Animal No.

Necropsy Date /

Necropsy Day

External Observations

Internal Observations

Organ/Tissue

1

3126

06 March 2018

Day 14

No external observations

No internal observations

Not applicable

2

3127

08 March 2018

Day 14

No external observations

No internal observations

Not applicable

3

3128

08 March 2018

Day 14

No external observations

No internal observations

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 N-NITROSODIPHENYLAMINE was found to be greater than 2000 mg/kg body weight in female Crl:WI rats.
According to the GHS criteria, N-NITROSODIPHENYLAMINE can be ranked as "Unclassified" for acute dermal exposure.
Executive summary:

An acute dermal toxicity study was performed with the test item N-NITROSODIPHENYLAMINE in female Crl:WI Wistar rats, in compliance with OECD Guideline No. 402 (2017), Commission Regulation (EC) No 440/2008, B.3 and OPPTS 870.1200.

 

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 has been designed such that the minimum number of animals were used. Single animal at dose level of 2000 mg/kg body weight (bw) was used in a range-finding phase in female rats, followed by two animals to confirm the expected 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 2-week observation period (Day 14).

 

The results of the study were summarised as follows:

 

Mortality

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 days observation period.

The test item coloured the treatment area to yellowish on Day 1 (3/3 animals) and Day 2 (2/3) animals.

 

Body weight and body weight gain

There was no treatment 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 N-NITROSODIPHENYLAMINE was found to be greater than 2000 mg/kg body weight in female Crl:WI rats.

 

According to the GHS criteria, N-NITROSODIPHENYLAMINE can be ranked as "Unclassified" for acute dermal exposure.