Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Acute toxicity studies were performed on n-nitrosodiphenylamine by oral and dermal route.

Based on the results, the LD50 for both routes are higher than 2000 mg/kg/day.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
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
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
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.
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.
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.

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

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
The study is considered as reliable (klimish score of 1).

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Link to relevant study records
Reference
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
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
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.
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.
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.

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

 

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
discriminating dose
Value:
2 000 mg/kg bw
Quality of whole database:
The study is considered as reliable (klimish score of 1).

Additional information

Acute toxicity study by oral route (Tarcai 2018) :

The single-dose oral toxicity of n-nitrosodihenylamine 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 substance caused the death of 1/6 animals at a dose level of 2000 mg/kg bw. 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.

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. 

There were no effects on body weights or body weight gains that could be attributed to treatment with the test substance.

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

Acute toxicity study by dermal route (Tarcai 2018) :

An acute dermal toxicity study was performed with the test item n-nitrosodihenylamine in female Crl:WI Wistar rats, in compliance with OECD Guideline No. 402 (2017). 

Test item did not cause mortality at the dose level of 2000 mg/kg bw. There were no systemic clinical signs noted in any animal throughout the study. 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. 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. There was no evidence of any macroscopic changes at a dose level of 2000 mg/kg bw. 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.

Justification for classification or non-classification

Based on the available data, no classification for the acute toxicity is required for Nitrosodiphenylamine according to the Regulation EC n°1272/2008.