Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 September 1993 - 13 October 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

EC Number:

309-912-6

EC Name:

Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

Cas Number:

101357-15-7

Molecular formula:

This is a UVCB substance. See section 1.2 for individual components.

IUPAC Name:

Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (U.K.) Limited, Manston, Kent
- Age at study initiation: approximately five to six weeks old
- Weight at study initiation: males 136 to 162 g; females 121 - 150 g
- Housing: in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Diet (e.g. ad libitum): ad libitum. A pelleted diet (Rat and Mouse SQC Expanded Diet No. 1, Special Diets Services Limited, Witham, Essex, U.K.) was used.
- Water (e.g. ad libitum): ad libitum. Mains water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23 °C
- Humidity (%): 45 - 75 % relative
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From: To: 15 September 1993 - 13 October 1993

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

The test material was prepared at the appropriate concentrations as a suspension in Arachis oil B.P. The stability and homogeneity of the test material formulations were determined analytically and showed the formulations to be stable for at least ten days. Formulations were therefore prepared weekly and stored at 4 °C in the dark.
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.
The test material was administered at a treatment volume of 4 mL/kg in the vehicle.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The Nigrosine Base Ex concentration in the test samples was determined spectrophotometrically.

Samples
The test material formulations were dilute with acetone such that the final theoretical test material concentration was approximately 0.015 mg/mL.

Standards
Standard solutions were prepared in acetone at a nominal concentration of 0.015 mg/mL.

Procedure
The absorbance of the standard and sample solutions was measured at 554 nm in 10 mm cells using acetone as the reference medium.

Homogeneity Determinations
The test material formulations were mixed thoroughly and the samples were taken from the top, middle and bottom of the container, shaking between sampling. The sampling was performed in triplicate.

Stability Determinations
The test material formulations were sampled and analysed initially and then after storage at approximately 4 °C in the dark for 10 days.

Verification of Test Material Formulation Concentrations
The test material formulations were sampled and analysed within three days of preparation.


The results indicate that the prepared formulations were within ± 10 % of the nominal concentration.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The test material was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg/day of Arachis oil B.P.

The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

Examinations

Observations and examinations performed and frequency:

Clinical Signs
All animals were examined daily for overt signs of toxicity, ill-health or behavioural change.

Bodyweight
Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and on Days 7, 14, 21 and 28. Bodyweights were also recorded at necropsy.

Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study.

Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Laboratory Investigations
Haematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 29). Blood samples were obtained from the lateral tail vein. A few repeat samples were also obtained by cardiac puncture prior to necropsy. Animals were not fasted prior to sampling.

Haematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:

Haematocrit (Hct)
Haemoglobin (Hb)
Erythrocyte count (RBC)
Total leucocyte count (WBC)
Differential leucocyte count
Platelet count (PLT)
Erythrocyte indices: mean corpuscular haemoglobin (MCH)
mean corpuscular volume (MCV)
mean corpuscular haemoglobin concentration (MCHC)
Reticulocyte counts (Retic)
Methaemoglobin (Meth)

Clotting time (CT) was assessed by Hepato Quick time using samples collected into sodium citrate solution (0.11 mo1/L).


Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:

Blood urea Inorganic phosphorus
Total protein Creatinine
Albumin Alkaline phosphatase (AP)
Albumin/globulin ratio (by calculation) Alanine aminotransferase (ALAT)
Sodium Aspartate aminotransferase (ASAT)
Potassium Glucose
Chloride Total bilirubin
Calcium

Sacrifice and pathology:

Pathology
On completion of the dosing period all animals were killed by intra-venous administration of sodium pentobarbitone solution (Sagatal, 60 mg/mL) followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs from animals that were killed at the end of the study, dissected free from fat, were weighed before fixation:
Adrenals Brain
Gonads Heart
Kidneys Liver
Pituitary Spleen


Histopathology
Samples of the following tissues were removed from all animals and preserved in 10 % buffered formalin:


Adrenals Jejunum Salivary glands
Aorta (thoracic) Kidneys Sciatic nerve
Bone & Bone Marrow (femur) Liver Seminal vesicles
Bone & Bone Marrow (sternum) Lungs Skin (hind limb)
Brain Lymph nodes (cervical and mesenteric) Spleen
Caecum Muscle (skeletal) Stomach
Colon Oesophagus Testes
Duodenum Ovaries Thymus
Eyes Pancreas Thyroid/parathyroid
Gross lesions Pituitary Trachea
Heart Prostate Urinary bladder
Ileum Rectum Uterus

The following preserved tissues from all test and control group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 µm
and stained with haematoxylin and eosin.
Adrenals Testes
Spleen Mesentery
Heart Kidneys
Liver

Macroscopically observed lesions were also processed.
Initially microscopic examination was performed on control and high dose tissues only, but since there were indications of treatment-related hepatic and splenic changes, examination was subsequently extended to include sections of liver and spleen from all animals in the remaining dose groups.

Statistics:

Evaluation of Data
Data were processed to give group mean values and standard deviations where appropriate.
Absolute and relative organ weights, haematological and blood chemical data were analysed by one way analysis of variance incorporating ‘F-max' test for homogeneity of variance. Data showing heterogeneous variances were analysed using Kruskal Wallis non-parametric analysis of variance and Mann Whitney U-Test.
Probability values were calculated as:
p < 0.001 ***
p < 0.01 **
p < 0.05 *
p ≥ 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no deaths during the study. Animals showed no clinically observable signs of toxicity during the study.

Mortality:

no mortality observed

Description (incidence):

There were no deaths during the study. Animals showed no clinically observable signs of toxicity during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

All animals showed normally expected bodyweight development. Animals treated with the test material showed similar bodyweight gains to controls during the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no adverse effect on food consumption during the study.

Food efficiency:

no effects observed

Description (incidence and severity):

Animals treated with the test material showed similar weekly food efficiency to controls.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles revealed no intergroup differences in water consumption.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See below.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See below.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See below.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See below.

Histopathological findings: neoplastic:

not examined

Details on results:

Clinical signs:
Dark faeces were evident at all three treatment levels from Day 3 onwards; this is a common finding following oral administration of a dark-coloured test material to rats and is not indicative of toxicity.

Haematology:
Group mean values and standard deviations for test and control group animals are given in Tables 1 and 2 (statistically significant differences are indicated). High dose animals of both sexes showed a slight but statistically significant reduction in haemoglobin concentration and erythrocyte count compared with controls, and a concomitant increase in the mean corpuscular volume (MCV). Haematocrit was also reduced in these animals, although the intergroup difference failed to achieve statistical significance for the females.This was probably due to an increase in the relative number of reticulocytes in the blood, particularly in the females, although there was no clear evidence of reticulocytosis in the males at this dose level. In addition, high dose females showed a slight but statistically significant increase in mean corpuscular haemoglobin, together with a reduction in mean corpuscular haemoglobin concentration. These findings, taken with histopathological evidence of splenic haemosiderosis and splenic haematopoiesis, are consistent with haemolysis, although the animals could not be considered clinically anaemic. The changes in haematological parameters observed were largely within, or only marginally outside, the historical control ranges. A treatment-related methaemoglobinaemia was also identified at the high dose level with animals of both sexes showing a slight but statistically significant increase in the relative amount of methaemoglobin present in the blood compared with controls.No treatment-related haematological changes were detected at the remaining dose levels. High dose males showed a slight increase in neutrophils compared with controls but none of the individual values were abnormally high for rats of the strain and age used in this study and, in the absence of any convincing increase in the total leucocyte count, this finding was considered not to be toxicologically important. Intermediate and high dose males also showed a slight but statistically significant increase in clotting time compared with controls but again none of the individual values were abnormally high for rats of the strain and age used in this study and the intergroup differences were considered to be entirely due to lower than expected control values.

Blood Chemistry:
There were no treatment-related effects on the blood chemical parameters measured.Intermediate and high dose males showed a slight but statistically significant increase in plasma creatinine concentration compared with controls. None of the individual values were abnormally high for rats of the strain and age used in this study and, in the absence of any histopathological evidence of renal changes, these findings were considered not to be toxicologically important.The remaining statistically significant intergroup differences were confined to intermediate dose animals. These findings were not dose-related and, as such, were considered not to be treatment-related.

Necropsy:
All intermediate and high dose animals had abnormally pink adipose tissue at terminal kill.No macroscopic abnormalities were evident at the low dose level.Intermediate and high dose animals of either sex had black stomach contents at necropsy and several of these animals also had a stained non-glandular gastric region. Such findings are common when a dark-coloured test material is administered to rats, by gavage, and are not attributable to test material toxicity.

Organ Weights:
Group mean absolute and relative organ weights and standard deviations for test and control group animals are presented in Tables 3 to 6 (statistically significant differences are indicated). High dose animals of either sex showed a statistically significant increase in spleen weight, both absolute and relative to body-weight, compared with controls. High dose females also showed a slight but statistically significant increase in relative liver weight.No treatment-related organ weight changes were detected at the remaining dose levels.The remaining statistically significant intergroup differences were confined to low dose females. These findings were not dose related and, as such, were considered not to be treatment-related.

Histopathology:
A summary incidence table of histopathological findings is given In Table 7. Treatment-related hepatic and splenic changes were observed:
Liver: Periportal hepatocyte basophilla was observed in relation to treatment for rats of either sex dosed at 1000 mg/kg/day.
Spleen: Increased severities of extramedullary haemopoiesis and haemosiderin pigment deposition were reported in relation to treatment for rats of either sex receiving 1000 mg/kg/day.All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

Discussion:
The test material induced a methaemaglobinaemia in animals of bothr sexes dosed at 1000 mg/kg/day. In addition, haematological determinations at this dose level showed effects, when taken together with the splenic extramedullary haemopoiesis, and the increased splenic haemosiderosis (likely responsible for the increased spleen weights at this dose), suggest a chemically induced haemolysis. The nature of the haemolysis and methaemoglobinaemias is such that they are normally reversible following cessation of treatment with the causative agent. Furthermore, there was no evidence that these haematological changes indicative of an adverse process (haemolysis) were severe enough to cause hypoxic damage to tissues in the rat or any other outward in-life signs of anaemia, which were offset by increased haematopoiesis in these animals. Both haemolysis and methaemoglobinuria are both known to be caused by components used to manufacture the test article, through redox cycling (oxidative stress). The increased relative liver weight detected for females treated with 1000 mg/kg/day was probably associated with the periportal hepatocyte basophilia identified histopathologically, even though the microscopic changes were present in animals of either sex. Despite these liver changes there was, however, no convincing evidence of liver dysfunction at this dose level. The aetiology of the remaining treatment-related change at this dose level, the abnormally pink adipose tissue seen at terminal kill, is unclear at present. The abnormal discolouration was possibly due to accumulation of haemosiderin pigment or methaemoglobin in the adipose tissue but this is considered unlikely, especially as both substances typically discolour tissues brown and histopathological examination of adipose tissue failed to identify the presence of any pigment. It is more likely therefore that such discolouration results from bioaccumulation of test material metabolite(s) in the adipose tissue which, in the absence of any morphological changes, is of little toxicological concern.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

15 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1 Group Mean Haematological Values and Standard Deviations (SD) - Males

Dose Level (mg/kg/day)		Hb (g/dL)	RBC (10^12/L)	Hct (%)	MCH (pg)	MCV (fL)	MCHC (g/dL)	WBC (10^9/L)	Meth (%)	Differential (10^9/L)					CT (secs)	PLT (10^9/L)	Retic (%)
										Neut	Lymph	Mono	Eos	Bas
Control	Mean SD	15.3 0.5	7.52 0.33	43.6 1.4	20.4 0.4	58.0 1.9	35.2 0.6	15.2 1.8	1.51 0.87	1.82 0.89	13.29 1.51	0.03 0.06	0.06 0.09	0.00 0.00	25 1	978 252	4 2
15	Mean SD	15.1 0.4	7.30 0.36	42.7 1.4	20.7 0.5	58.6 1.2	35.4 0.4	12.7 0.5	0.59 0.61	2.01 0.28	10.63 0.69	0.00 0.00	0.10 0.11	0.00 0.00	26 1	1104 91	4 0
150	Mean SD	15.0 0.6	7.36 0.38	42.8 1.8	20.4 0.5	58.1 1.1	35.0 0.5	18.1 3.4	1.77 0.51	2.30 0.99	15.71 2.47	0.07 0.10	0.03 0.08	0.00 0.00	27** 1	1150 173	4 1
1000	Mean SD	13.8*** 0.2	6.60*** 0.10	39.7*** 0.8	20.9 0.4	60.2* 1.0	34.7 0.3	16.9 4..5	4.07*** 1.62	3.05* 1.15	13.70 3.46	0.08 0.12	0.05 0.10	0.00 0.00	28*** 1	1076 74	6 2

* = statistically different from control group value p<0.05

** = statistically different from control group value p<0.01

*** = statistically different from control group value p<0.001

Table 2 Group Mean Haematological Values and Standard Deviations (SD) - Females

Dose Level (mg/kg/day)		Hb (g/dL)	RBC (10^12/L)	Hct (%)	MCH (pg)	MCV (fL)	MCHC (g/dL)	WBC (10^9/L)	Meth (%)	Differential (10^9/L)					CT (secs)	PLT (10^9/L)	Retic (%)
										Neut	Lymph	Mono	Eos	Bas
Control	Mean SD	14.8 0.7	7.38 0.30	41.6 1.4	20.1 0.7	56.3 2.2	35.7 0.6	9.9 1.2	1.21 0.22	0.94 0.35	8.82 0.92	0.08 0.08	0.06 0.06	0.00 0.00	27 3	988 72	3 1
15	Mean SD	14.9 0.7	7.30 0.27	41.8 2.1	20.4 0.5	57.3 1.6	35.6 0.2	12.4 3.6	1.36 0.70	1.23 0.76	11.10 3.06	0.00 0.00	0.11 0.07	0.00 0.00	26 2	1020 46	3 2
150	Mean SD	14.7 0.3	7.32 0.34	41.2 0.9	20.1 0.8	56.3 1.7	35.7 0.4	8.4 1.5	1.60 0.71	1.07 0.69	7.26 4.54	0.03 0.05	0.06 0.13	0.00 0.00	27 1	1001 62	4 1
1000	Mean SD	13.8* 0.6	6.48*** 0.22	39.7 2.1	21.3** 0.3	61.2*** 1.2	34.7** 0.4	12.8 2.9	2.44** 0.44	1.03 0.65	11.57 2.33	0.10 0.13	0.06 0.14	0.00 0.00	26 1	1110 217	9*** 2

* = statistically different from control group value p<0.05

** = statistically different from control group value p<0.01

*** = statistically different from control group value p<0.001

Table 3 Group Mean Organ Weights and Standard Deviations (SD) - Males

Dose Level (mg/kg/day)		Organ weight (g)
		Adrenals	Brain	Gonads	Heart	Kidneys	Liver	Pituitary	Spleen
Control	Mean SD	0.0446 0.0082	1.9398 0.0772	4.0364 0.1427	1.6256 0.1915	2.4498 0.1393	14.7514 1.1256	0.0096 0.0011	0.8547 0.1127
15	Mean SD	0.0491 0.0075	1.9718 0.1064	4.2128 0.3621	1.4812 0.2590	2.4939 0.3821	14.2238 1.9802	0.0098 0.0018	0.8168 0.1077
150	Mean SD	0.0478 0.0096	1.9883 0.0765	3.9597 0.5100	1.3715 0.2161	2.2811 0.2595	13.2898 1.9012	0.0087 0.0008	0.7041 0.1406
1000	Mean SD	0.0462 0.0095	1.9837 0.0625	3.9796 0.2056	1.7059 0.2486	2.5502 0.1904	14.6910 0.5582	0.0101 0.0009	1.3585*** 0.1677

***significantly different from control group value p<0.001

 

Table 4 Group Mean Organ Weights and Standard Deviations (SD) - Females

Dose Level (mg/kg/day)		Organ weight (g)
		Adrenals	Brain	Gonads	Heart	Kidneys	Liver	Pituitary	Spleen
Control	Mean SD	0.0603 0.0165	1.8509 0.0554	0.1186 0.0149	0.9062 0.0790	1.6575 0.1615	7.8554 0.8467	0.0107 0.0010	0.5551 0.0652
15	Mean SD	0.0743* 0.0067	1.8609 0.0647	0.1258 0.0211	0.9640 0.0413	1.7904 0.1622	9.2305** 0.7362	0.0110 0.0014	0.6583* 0.0791
150	Mean SD	0.0623 0.0059	1.8310 0.0571	0.1133 0.0091	0.8809 0.0531	1.5607 0.1568	7.4615 0.6158	0.0103 0.0009	0.5012 0.0167
1000	Mean SD	0.0692 0.0047	1.8148 0.0986	0.1158 0.0176	0.8832 0.0981	1.7631 0.1217	8.5693 0.5878	0.0105 0.0021	0.7275*** 0.0623

**significantly different from control group value p<0.01

***significantly different from control group value p<0.001

 

Table 5 Group Mean Relative Organ Weights (% of Bodyweight) and Standard Deviations (SD) - Males

Dose Level (mg/kg/day)		Relative Organ weight (%)
		Adrenals	Brain	Gonads	Heart	Kidneys	Liver	Pituitary	Spleen
Control	Mean SD	0.0119 0.0022	0.5154 0.0223	1.0727 0.0510	0.4329 0.0611	0.6504 0.0286	3.9162 0.2526	0.0025 0.0002	0.2279 0.0368
15	Mean SD	0.0127 0.0013	0.5148 0.0355	1.0992 0.0751	0.3881 0.0793	0.6472 0.0602	3.6916 0.2531	0.0025 0.0003	0.2123 0.0194
150	Mean SD	0.0133 0.0020	0.5589 0.0467	1.1077 0.1101	0.3836 0.0507	0.6369 0.0294	3.7037 0.2738	0.0025 0.0002	0.1957 0.0287
1000	Mean SD	0.0122 0.0027	0.5214 0.0205	1.0476 0.0889	0.4474 0.0588	0.6692 0.0316	3.8606 0.1433	0.0026 0.0003	0.3577*** 0.0499

***significantly different from control group value p<0.001

 

Table 6 Group Mean Relative Organ Weights (% of Bodyweight) and Standard Deviations (SD) - Females

Dose Level (mg/kg/day)		Relative Organ weight (%)
		Adrenals	Brain	Gonads	Heart	Kidneys	Liver	Pituitary	Spleen
Control	Mean SD	0.0266 0.0071	0.8182 0.0510	0.0522 0.0052	0.3995 0.0236	0.7301 0.0445	3.4604 0.2536	0.0047 0.0004	0.2447 0.0249
15	Mean SD	0.0300 0.0028	0.7505 0.0308	0.0506 0.0072	0.3890 0.0241	0.7199 0.0311	3.7151 0.1579	0.0045 0.0007	0.2648 0.0254
150	Mean SD	0.0286 0.0025	0.8415 0.0533	0.0522 0.0061	0.4043 0.0235	0.7148 0.0484	3.4202 0.2023	0.0047 0.0005	0.2303 0.0132
1000	Mean SD	0.0306 0.0024	0.7999 0.0247	0.0512 0.0086	0.3891 0.0375	0.7773 0.0435	3.7788* 0.2309	0.0046 0.0010	0.3210*** 0.0277

*significantly different from control group value p<0.05

***significantly different from control group value p<0.001

 

Table 7 Histopathological Findings - Summary Incidence

	Males				Females
	Dose Level (mg/kg/day)				Dose Level (mg/kg/day)
	Control	15	150	1000	Control	15	150	1000
No. of Animals	5	5	5	5	5	5	5	5
Heart
Focal myocarditis No data Absent Minimal Slight	0 2 2 1	5 0 0 0	5 0 0 0	0 2 3 0	0 2 3 -	5 0 0 -	5 0 0 -	0 4 1 -
Kidneys
Groups of basophilic/dilated tubules No data Absent Minimal	0 1 4	5 0 0	5 0 0	0 4 1	0 1 4	5 0 0	5 0 0	0 3 2
Liver
Mononuclear cell foci Minimal Slight Moderate   Basophilic periportal hepatocytes Absent Slight Moderate	3 2 0         5 0 0	5 0 0         5 0 0	5 0 0         5 0 0	1 3 1         2 2 1	4 1 -         5 0 -	5 0 -         5 0 -	5 0 -         5 0 -	5 0 -         4 1 -
Spleen
Extramedullary haemopoiesis Minimal Slight Moderate Marked   Pigment deposition Minimal Slight Moderate	3 2 0 0     5 0 -	4 1 0 0     5 0 -	4 1 0 0     5 0 -	0 0 3 2     0 5 -	5 0 0 -     2 3 0	5 0 0 -     1 4 0	5 0 0 -     1 4 0	2 2 1 -     0 0 5
Statistical Information
Mode of death Terminal kill	5	5	5	5	5	5	5	5

Applicant's summary and conclusion

Conclusions:

Repeat-dose toxicity: a fully compliant 28-d toxicity study is available (Wragg MS and Brooks PN 1994), performed according to OECD guideline 407 (1995).

The study established a NOAEL at 150 mg/kg/day, because treatment at the limit dose of 1000 mg/kg/day caused haematological effects, changes in spleen weight (and relative liver weight in females only) and histopathological changes in the spleen and liver consistent with methaemoglobin formation and haemolysis, although the animals did not become anaemic.

Executive summary:

Repeat-dose toxicity: a fully compliant 28-d toxicity study is available (Wragg MS and Brooks PN 1994), performed according to OECD guideline 407 (1995).

The study established a NOAEL at 150 mg/kg/day, because treatment at the limit dose of 1000 mg/kg/day caused haematological effects, changes in spleen weight (and relative liver weight in females only) and histopathological changes in the spleen and liver consistent with methaemoglobin formation and haemolysis, although the animals did not become anaemic.

Methaemoglobinaemia and haemolysis are known to be caused by aniline and nitrobenzene which are used to make nigrosine. Aniline is a documented component of Nigrosine (Section 1). Aniline and nitrobenzene redox cycle and their presence in erythrocytes results in the reduction haemoglobin to methemoglobin, and also oxidative stress resulting damage to, and ultimately, lysis of the erythrocyte (hemolysis). Once exposure ceases, and the responsible chemical species are cleared from the body, full recovery is made from the methemoglobinaemia and hemolysis. It is important to note that exacerbation of methaemoglobin formation, and sequalae thereof, does not occur with increased duration of dosing. For further information see Kiese, 1996 .Muller et al 2006, Stolk and Smith 1966, ECB 2004.

The criteria for labelling for specific organ toxicity (STOT) under the CLP Regulation are not met. However humans are known to be more sensitive than rats to these mechanisms resulting in methemglobinaemia and hemolysis, and specific consideration was given to the classification and labelling for hemolysis by the EU Working Group on Haemolytic Anaemia (Muller et al, 2006). The findings of the working group were accepted by the European Commission in 2004. The haemolysis observed in Wragg and Brooks does not trigger the criteria set out in Muller et al (2006) where classification for specific organ toxicity would be reguired. In addition interspecies assessment factors would also accommodate the differences in sensitivity when deriving DNELs, and there were no effects in the endpoints measured in the developmental toxicity study up to the limit dose of 1000 mg/kg. The only change noted at 150 mg/kg/day was abnormal discolouration of adipose tissue, considered likely to represent presence of test substance metabolite(s).