Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 August 2017 to 25 August 2017
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 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
OECD Guidelines for Testing of Chemicals No. 423. Acute Oral Toxicity – Acute Toxic Class Method. Adopted: 17 December 2001
Deviations:
yes
Remarks:
Due to technical reason temperature values (maximum of 25.6°C) out of the target range 19-25°C 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.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:
yes
Remarks:
Due to technical reason temperature values (maximum of 25.6°C) out of the target range 19-25°C 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 870.1100 (Acute Oral Toxicity)
Version / remarks:
EPA Health Effects Test Guidelines (OPPTS 870.1100), United States, EPA 712-C-02-190 (2002)
Deviations:
yes
Remarks:
Due to technical reason temperature values (maximum of 25.6°C) out of the target range 19-25°C 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:
acute toxic class 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
Hygienic level at arrival: SPF
Hygienic level during the study: Standard housing conditions
Number of animals: 6 animals, 3 animals / group
Sex: Female, nulliparous and non-pregnant.
Age of animals at dosing: Young healthy adult rats, 8 weeks old
Body weight at treatment: 184 – 198 g
Acclimation period: at least 5 days

Husbandry
Animal health: Only healthy animals were used for the test. The staff Veterinarian certified their health status.
Number of animal room: 522/2
Housing: 3 animals / cage
Cage type: Type II polypropylene/polycarbonate
Bedding: LIGNOCEL ¾ S certified wooden chips (J. Rettenmaier & Söhne GmbH + Co. KG 73494, Holzmühle, Rosenberg, Germany) was available to animals during the study.
Nesting: ARBOCEL crinklets natural nest building material (J. Rettenmaier & Söhne GmbH + Co. KG, 73494, Holzmühle, Rosenberg, Germany) was available to animals during the study.
Lighting period: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 21.6 – 25.6°C
Relative humidity: 35 – 68%
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.

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.: 262 21592, expiry date: 31 January 2018), ad libitum, except for the night before treatment. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Animals received tap water from the municipal supply from 500 mL bottles, ad libitum. The water was fit for human consumption and was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
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 test item was dusted in order to enhance the formulation. Formulation container was magnetic stirred continuously up to the end of dose administration procedures.

Vehicle: Corn oil
Lot number: A0384372
Expiry date: 31 May 2019
Dose volume: 10 mL/kg bw
Produced by: Acros Organics, Belgium

The initial dose level was selected by the Study Director to be that which is most likely to produce mortality in some of the dosed animals. Based on the preliminary toxicological information in the SDS, limit dose of 2000 mg/kg bw was selected as a starting dose.
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
6 animals (3 per group)
Control animals:
no
Details on study design:
ADMINISTRATION OF THE TEST ITEM
Justification of the dose:
The initial dose level was selected by the Study Director to be that which is most likely to produce mortality in some of the dosed animals. Based on the preliminary toxicological information in the SDS, limit dose of 2000 mg/kg bw was selected as a starting dose.
Initially, 3 female animals were treated with N-NITROSODIPHENYLAMINE at dose level of 2000 mg/kg bw. No mortality was observed, therefore further 3 animals were treated at the dose level of 2000 mg/kg bw. As only 1 animal died in this second dose group, further testing was not required according to the test guidelines (OECD 423, Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1.Tris).

Procedure
A single oral gavage administration was followed by a fourteen-day observation period in the surviving animals or until the day of death as applicable. 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 returned 3 hours after the treatment.

OBSERVATIONS
Clinical Observations
Clinical observations were performed on all animals at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter in all surviving animals or until the day of death 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) and weekly thereafter or on the day of death.

NECROPSY
Macroscopic examination was performed on all animals. The animals were sacrificed by exsanguination under pentobarbital anaesthesia (Release 30% inj.; Lot No.: 106075, Expiry Date: 31 July 2018, Produced by: Wirtschaftsgenossenschaft deutscher Tierärzte eG; Siemensstr. 14, 30827 Garbsen, Germany). After examination of the external appearance, the cranial, thoracic and the abdominal cavities were opened and the organs and the tissues were observed. Macroscopic abnormalities were recorded.
Statistics:
The method used was not intended to allow the calculation of a precise LD50 value.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: 1 animal died
Mortality:
N-NITROSODIPHENYLAMINE caused the death of 1/6 animals (Day 1, ID: 377) at a dose level of 2000 mg/kg bw.
Clinical signs:
At dose level of 2000 mg/kg bw, decreased activity (score 1 or 2, up to Day 2), hunched back (up to Day 2), incoordination (score 1 or 2, up to Day 1), piloerection (up to Day 2) in all animals and diuresis (between Day 2 and Day 4) in all surviving animals were observed.
From Day 5 all surviving animals were symptom-free.
Body weight:
There were no effects on body weights or body weight gains that could be attributed to treatment with of N-NITROSODIPHENYLAMINE.
Gross pathology:
In the rat, dosed at 2000 mg/kg bw, that was found dead on Day 1, the lungs (all lobes) were collapsed and dark red (diffuse).
There was no evidence of the macroscopic observations in the surviving animals dosed at 2000 mg/kg bw and terminated on Day 14.
Other findings:
No further findings reference in the study report.

Any other information on results incl. tables

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

374

Symptom Free

-

-

-

-

-

-

-

-

-

-

+

+

+

10/20

Activity decreased

1

1

1

1

1

1

1

1

-

-

-

-

-

8/20

Hunched back

+

+

+

+

+

+

+

-

-

-

-

-

-

7/20

Incoordination

-

-

-

1

1

2

1

-

-

-

-

-

-

4/20

Piloerection

-

-

+

+

+

+

+

-

-

-

-

-

-

5/20

Diuresis

-

-

-

-

-

-

-

+

+

+

-

-

-

3/20

375

Symptom free

-

-

-

-

-

-

-

-

-

-

+

+

+

10/20

Activity decreased

1

1

1

1

1

2

1

-

-

-

-

-

-

7/20

Hunched back

+

+

+

+

+

+

+

+

-

-

-

-

-

8/20

Incoordination

-

-

-

1

2

2

1

-

-

-

-

-

-

4/20

Piloerection

-

-

+

+

+

+

+

-

-

-

-

-

-

5/20

Diuresis

-

-

-

-

-

-

-

+

+

+

-

-

-

3/20

376

Symptom free

-

-

-

-

-

-

-

-

-

-

+

+

+

10/20

Activity decreased

1

1

1

1

1

2

1

-

-

-

-

-

-

7/20

Hunched back

+

+

+

+

+

+

+

+

-

-

-

-

-

8/20

Incoordination

-

-

-

1

1

2

1

-

-

-

-

-

-

4/20

Piloerection

-

-

+

+

+

+

+

-

-

-

-

-

-

5/20

Diuresis

-

-

-

-

-

-

-

+

+

+

-

-

-

3/20

2

377#

Activity decreased

-

1

1

1

1

2

 

5/6

Hunched back

+

+

+

+

+

+

6/6

Incoordination

-

-

1

1

1

2

4/6

Piloerection

-

-

+

+

+

+

4/6

Found dead

-

-

-

-

-

-

+

 

-

378

Symptom free

-

-

-

-

-

-

-

-

-

-

+

+

+

10/20

Activity decreased

-

1

1

1

1

2

2

1

-

-

-

-

-

7/20

Hunched back

+

+

+

+

+

+

+

+

-

-

-

-

-

8/20

Incoordination

-

-

1

1

1

2

1

-

-

-

-

-

-

5/20

Piloerection

-

-

-

+

+

+

+

+

-

-

-

-

-

5/20

Diuresis

-

-

-

-

-

-

-

+

+

+

-

-

-

3/20

379

Symptom free

-

-

-

-

-

-

-

-

-

-

+

+

+

10/20

Activity decreased

-

1

1

1

1

1

1

1

-

-

-

-

-

7/20

Hunched back

+

+

+

+

+

+

+

+

-

-

-

-

-

8/20

Incoordination

-

-

1

1

1

1

1

-

-

-

-

-

-

5/20

Piloerection

-

-

-

+

+

+

+

+

-

-

-

-

-

5/20

Diuresis

-

-

-

-

-

-

-

+

+

+

-

-

-

3/20

Remarks:              + = present           - = absent

                               h = hour                ‘ = minute

                               # = Found dead

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

                               Severities: 1 = Slight/Small/Few; 2 = Moderate/medium; 3 = Marked/Large/Many

 

BODY WEIGHT DATA

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

Cage No.

Animal Number

Body weight (g)

Days

Day/Body Weight (g)

Death

Body Weight Gain (g)

-1

0

7

14

-1-0

0-7

7-14

-1-14

1

374

200

186

220

247

-

-14

34

27

47

375

214

194

227

248

-

-20

33

21

34

376

201

184

216

243

-

-17

32

27

42

2

377#

212

198

-

-

1/187

-14

-

-

-

378

211

198

229

240

-

-13

31

11

29

379

202

192

223

235

-

-10

31

12

33

Mean:

206.7

192.0

223.0

242.6

-

-14.7

32.2

19.6

37.0

Standard deviation:

6.3

5.9

5.2

5.3

-

3.4

1.3

7.8

7.3

- = No data

# = Found dead

 

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

374

22 August 2017

Day 14

No external observations recorded

No internal observations recorded

Not applicable

375

22 August 2017

Day 14

No external observations recorded

No internal observations recorded

Not applicable

376

22 August 2017

Day 14

No external observations recorded

No internal observations recorded

Not applicable

2

377#

12 August 2017

Day 1

No external observations recorded

Collapsed

Lungs

Discoloration, red, diffuse, all lobes

378

25 August 2017

Day 14

No external observations recorded

No internal observations recorded

Not applicable

379

25 August 2017

Day 14

No external observations recorded

No internal observations recorded

Not applicable

# = Found dead

Applicant's summary and conclusion

Interpretation of results:
Category 5 based on GHS criteria
Conclusions:
Under the conditions of the study, the acute oral LD50 value of the test item N-NITROSODIPHENYLAMINE was found to be above 2000 mg/kg bw in female Crl:WI rats.
According to the GHS criteria, N-NITROSODIPHENYLAMINE can be ranked as "Category 5" for acute oral exposure.
Executive summary:

The single-dose oral toxicity of N-NITROSODIPHENYLAMINE 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 Wistar rats.

 

Two groups of 3 female Crl:WI rats were treated with the test item at a dose level of 2000 mg/kg bw (Group 1 and Group 2).

 

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 formulated in corn oil at a concentration of 200 mg/mL at a dose volume of 10 mL/kg bw.

 

Initially, three females (Group 1) were treated at a dose level of 2000 mg/kg bw. As no mortality was observed, a confirmatory group (Group 2) was treated at the same dose level and 1 animal died on Day 1 from this group. As no other 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.

 

Clinical observations were performed at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter or until the day of death. Body weight was measured on Days -1, 0, 7 and 14 (before necropsy), or on the day of death. All animals were subjected to a necropsy and a macroscopic examination.

 

The results of the study were summarized as follows:

Mortality

N-NITROSODIPHENYLAMINE caused the death of 1/6 animals (Day 1, ID: 377) at a dose level of 2000 mg/kg bw.

 

Clinical Observations

At dose level of 2000 mg/kg bw, decreased activity (score 1 or 2, up to Day 2), hunched back (up to Day 2), incoordination (score 1 or 2, up to Day 1), piloerection (up to Day 2) in all animals and diuresis (between Day 2 and Day 4) in all surviving animals were observed.

From Day 5 all surviving animals were symptom-free.

 

Body Weight and Body Weight Gain

There were no effects on body weights or body weight gains that could be attributed to treatment with N-NITROSODIPHENYLAMINE.

 

Necropsy

In the rat, dosed at 2000 mg/kg bw, that was found dead on Day 1, the lungs (all lobes) were collapsed and dark red (diffuse).

There was no evidence of the macroscopic observations in the surviving animals dosed at 2000 mg/kg bw and terminated on Day 14.

 

Conclusion:

Under the conditions of the study, the acute oral LD50 value of the test item N-NITROSODIPHENYLAMINE was found to be above 2000 mg/kg bw in female Crl:WI rats.