reaction product of: C.I. Leuco Sulfur Black 1 with (3-chloro-2-hydroxypropyl)trimethylammonium chloride

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:

From 25. Sep. 1995 to 13. Nov. 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1996

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)

Version / remarks:

1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

424-510-1

EC Name:

-

Cas Number:

220150-59-4

Molecular formula:

not applicable for UVCB substance

IUPAC Name:

Reaction products of Phenol, 2,4-dinitro-, sulfurized, leuco derivatives and (3-chloro-2-hydroxypropyl)trimethylammonium chloride

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

HanIbm: WIST (SPF) (recognised by the international guidelines as a recommended test system)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Biological Research Laboratories Ltd., Wölferstrasse 4, 4414 Füllinsdorf, CH
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 135 to 154 g; females: 110 to 128 g
*- Fasting period before study:
- Housing: individually in Makrolon type-3 cages with wire mesh tops and granualted softwood bedding (Lignocel, Schill AG, 4132 Muttenz, CH). Before use, the bedding was autoclaved at 120 °C for 50 minutes
- Diet: ad libitum; pelleted standard Kliba no. 343, batch no. 90/95 Rat Maintenance Diet ('Kliba', Klingentalmühle AG, 4303 Kaiseraugst, CH)
- Water: ad libitum; community tap water from Itingen, CH
- Acclimation period: one week under Optimal Hygiene Conditions, behind a barrier system, after health examination
- Randomisation: computer-generated random algorithm
- Identification: individual cage number and corresponding ear tattoo

DETAILS OF FOOD AND WATER QUALITY:
- Food: contaminant analyses reported suitability
- Water: representative bacteriological, chemical and contaminant analyses reported suitability

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40 to 70 %
- Air changes: 10 to 15 per hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

bi-distilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test article was weighed into a glass beaker on a tared Mettler PK 300 balance and the vehicle was added. The mixture (w/v) was prepared using a homogeniser. Homogeneity of the test article in the vehicle was maintained during treatment using a magnetic stirrer.
- Each test animal received 10 mL/kg bw treatment containing their respective concentrations of test item

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability of the test item / vehicle mixtures were determined in samples taken during acclimatisation (25. Sep. 1995) and during week 3 of the treatment (16. Oct. 1995). The analyses were performed in the Analytical Laboratories of RCC Unweltchemie AG, CH, according to a method which was supplied by the sponsor.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

0 mg/kg bw/day: 10 males, 10 females
50 mg/kg bw/day: 5 males, 5 females
200 mg/kg bw/day: 5 males, 5 females
1000 mg/kg bw/day: 10 males, 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
- In an acute oral toxicity study and a 5-day dose-range finding study in which the test item was administered orally by gavage at 0, 200 and 1000 mg/kg bw to rats, there were no deaths during the 5-day treatment period, and no clinical signs or effects of treatment on body weight or food consumption were observed. Adrenal weights were statistically significantly lower in the mid-dose group, but macroscopic examination revealed changes only within the range of spontaneous alterations which may be seen in rats of this age and strain.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: yes
- Time schedule: twice daily
- Parameters observed: mortality, viability, cageside observations

BODY WEIGHT: yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: yes. The food consumption was recorded once during the acclimatisation period and weekly thereafter using an online electronic recording system consisting of a Mettler PM 4600 or PK 4800 balance connected to the RCC computer.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: yes
- The food consumption was calculated per rat and per food consumption interval. It expresses the average food consumed per animal and per day over the food consumption interval. Formulae: FC = C / AD where FC = food consumption(g / animal / day), C = measured food consumption per animal over the consumption interval in g food) and AD = total consumption days during the consumption interval, considering date of death if applicable. RFC = (FC / BW) x 100 where RFC = relative food consumption (g / day / kg bw), FC = food consumption (g / animal / day) and BW = body weight of animal on closest date to food weighing.

FOOD EFFICIENCY: no

WATER CONSUMPTION AND COMPOUND INTAKE: no

OPHTHALMOSCOPIC EXAMINATION: yes
- Time schedule for examinations: at 4 weeks (25. Oct. 1995) and at 6 weeks (08. Nov. 1995)
- Dose groups that were examined: at 4 weeks: all animals; at 6 weeks: all surviving recovery animals of groups 1 and 4
- A description of any abnormality was recorded 10 to 90 minutes after the application of a mydriatic solution (Ciba Vision AG, Niederwangen). The cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Miroflex 2 Ophthalmoscope (Eisenhut Vet. AG, 4123 Allschwil, CH)

HAEMATOLOGY: yes
- Time schedule for collection of blood: after 4 weeks (30. Oct. 1995) and after 6 weeks (13. Nov. 1995); between 06.30 and 08.35 am to reduce biological variation caused by circadian rhythms.
- Anaesthetic used for blood collection: yes, light ether anaesthesia
- Animals fasted: yes, in metabolic cages for approximately 18 hours with access to water ad libitum
- How many animals: at 4 weeks: all animals; at 6 weeks: all surviving recovery animals of groups 1 and 4
- Parameters in Table 1 of "Any other information on materials and methods" section were examined
- The following anticoagulants were used during blood collection: EDTA-K2 (haematology); Lithium heparin 30 I.U./mL (methaemoglobin); sodium citrate, 3.8 % (coagulation; 1 part anticoagulant to 9 parts blood)
- The following commercial reference controls were used to monitor the performance of the method: Haematology: Eightcheck-3WP (normal range), Eightcheck-L-3WP (low abnormal range), and Ret-check (reticulocyte control) (TOA Medical Electronics Co., Ltd. Kobe, Japan); Methaemoglobin: CalDye Reference Standard, and IL Multi-4 CO-Oximeter Control (Instrumentation Laboratory, Lexington, MA, USA); Coagulation: IL Control Plasma (normal range) and IL Control Plasma (abnormal range) (Instrumentation Laboratory, SPA, Milano, Italy)

CLINICAL BIOCHEMISTRY: yes
- Time schedule for collection of blood: after 4 weeks (30. Oct. 1995) and after 6 weeks (13. Nov. 1995); between 06.30 and 08.35 am to reduce biological variation caused by circadian rhythms.
- Anaesthetic used for blood collection: yes, light ether anaesthesia
- Animals fasted: yes, in metabolic cages for approximately 18 hours with access to water ad libitum
- How many animals: at 4 weeks: all animals; at 6 weeks: all surviving recovery animals of groups 1 and 4
- Parameters in Table 2 of "Any other information on materials and methods" section were examined
- The following anticoagulant was used during blood collection: Lithium heparin 15 I.U./mL
- The following commercial reference controls were used to monitor the performance of the method: Clinical biochemistry: Qualitrol HS-N (normal range), Qualitrol HS-P (high range) (E. Merck, Darmstadt, Germany), and Lyotrol N (normal range) and Lyotrol P (abnormal range) for the assay control of phospholipids.

URINALYSIS: yes
- Time schedule for collection of urine: after 4 weeks (30. Oct. 1995) and after 6 weeks (13. Nov. 1995); during the 18-hour fasting period for blood sampling; into a specimen vial
- Metabolism cages used for collection of urine: yes
- Animals fasted: yes
- Parameters in Table 3 of "Any other information on materials and methods" section were examined
- The following commercial reference controls were used to monitor the performance of the method: Urinalysis: Lyphochek Quantitative Urine Control - Normal I and Abbnormal II - (for the assay control of Osmolarity, pH, Protein, Glucose, Ketone, Bilirubin, Blood and Urobilinogen) (Bio-Rad, ECS Division, Anaheim, California, USA).

NEUROBEHAVIOURAL EXAMINATION: no

IMMUNOLOGY: no

Sacrifice and pathology:

NECROPSY
- After 4 weeks (30. Oct. 1995): all surviving animals of treatment groups not designated for recovery testing
- After 6 weeks (13. Nov. 1995): animals designated for recovery testing (5 males and 5 females from groups 1 and 4)
All animals were weighed and necropsied and descriptions of all macroscoplc abnormalities were recorded. Prior to necropsy, the animals were fasted for approxintately 18 hours, but free access to water was provided. Necropsies were performed by experienced prosectors supervised by an experienced veterinary pathologist. At the end of the treatment or recovery period the animals designated according to the necropsy schedule were anesthetized by intraperitoneal injection of NARCOREN (Rhone Merieux GmbH, 88471 Laupheim, Germany) at a dose of 2.0 ml/kg bw (equivalent to 320 mg sodium pentobarbitone/kg bw), weighed and sacrificed by exsanguination.

ABSOLUTE AND RELATIVE ORGAN WEIGHTS: yes
- The following organ weights were recorded on the scheduled dates of necropsy: adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thyroid including parathyroid gland.
- The organ to terminal body weight ratios as well as the organ to brain weight ratios were determined.
- The determination of the terminal body weight was performed immediately prior to necropsy using an online electronic recording system consisting of a Mettler PM 4000 balance connected to a computer system.

GROSS PATHOLOGY: yes
- Histological examination was performed on: adrenal glands, heart, kidneys, liver, lungs, spleen, stomach and testes from all rats of groups 1 and 4, as well as all gross lesions from all rats. Organ samples were processed, embedded, cut at an approximate thickness of 4 micrometers, and stained with hematoxylin and eosin.

HISTOPATHOLOGY: yes
- Representative tissue specimens were taken from the following organs and tissues and fixed in phosphate-buffered neutral 4% formaldehyde solution (10% formalin), then were processed, embedded, cut at an approximate thickness of 4 micrometers, and stained with hematoxylin and eosin.
Adrenal glands, aorta, bone - sternum and femur; bone marrow - femoral and sternal; brain, epididymides, esophagus, eyes with optic nerve and Harderian gland, heart, joint - femorotibial, kidneys, large intestine - caecum, colon and rectum; lacrimal glands - exorbital, larynx, liver, lungs, lymph nodes - mesenteric and mandibular; mammary gland area, nasal cavity, ovarles, pancreas, pituitary gland, prostate gland, salivary glands - mandibular and sublingual; sciatic nerve, seminal vesicles, skeletal muscle, skin, small intestine - duodenum. jejunum and ileum; spinal cord - cervical, midthoracic and lumbar segments; spleen, stomach, testes, thymus, thyroid glands with parathyroid glands, tongue, trachea, urinary bladder, uterus, vagina and all gross lesions.

Statistics:

The following statistical methods were used to analyse the body weights, food consumption, organ weights and all ratios and clinical laboratory data:
- When the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The Fisher's exact test was applied to the ophthalmoscopy data.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- In groups 3 and 4 (200 and 1000 mg/kg bw/day, respectively) hunched posture was observed, which in rats is commonly associated with pain or discomfort. Hunched posture was first noted on day 2 and was last seen on day 10 in group 3, on day 12 in group 4 males and on day 11 in group 4 females. All rats in these groups showed this sign on 3 or more days, with a mean incidence of 5 days in group 3 (200 mg/kg bw/day) and 6 days in group 4 (1000 mg/kg bw/day).
- There were no treatment-related clinical signs in group 2 (50 mg/kg bw/day).
- No clinical signs were noted during the recovery period for group 4 (1000 mg/kg bw/day).
- One female from group 2 (50 mg/kg bw/day), which died spontaneously, displayed poor condition, hunched posture, ruffled fur, emaciation, labored respiration and lateral recumbency prior to death.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- One female in group 2 (50 mg/kg bw/day) died spontaneously due to urinary tract infection and calculus.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- Although group mean body weight in all male treated groups was approximately 5 % less than control at the end of the treatment period, there was only one statistically significant difference (p < 0.05) during week 3 noted at 1000 mg/kg bw/day.
- The rate of weight gain in group 4 males (1000 mg/kg bw/day) did not increase when treatment was withdrawn after 28 days, confirming that treatment did not affect body weight gain.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- Occasional statistically significant differences between group means showed no treatment-related trends and are not toxicologically significant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Incidental findings, commonly detected in untreated rats of this strain and present in similar frequency in treated and untreated rats in this study, included persistent pupillary membranes, anterior synechia, posterior lens opacity, persistent hyaloid vessels and corneal opacity.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- All statistically significant differences in haematology parameters were considered to be incidental or unrelated to the treatment and well within limits of the historical control data for rats of this strain and age.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- All statistically significant differences in clinical biochemistry parameters were considered to be incidental or unrelated to the treatment and well within limits of the historical control data for rats of this strain and age.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

- Higher incidence of urine colouration in males of group 3 (200 mg/kg bw/day) and in both sexes of group 4 (1000 mg/kg bw/day), ranging from a deep yellow (four males) in group 3 to yellow green (two males, two females) to light green (three males) to green (two males) to deep green (one male, two females) in group 4. It is possible that the test article is absorbed and renally excreted as an unchanged or metabolised compound.
- At termination of the treatment-free recovery period, no abnormal colouration of the urine sample was observed, except deep yellow urine colouration in animals 8, 27 and 28.
- All other statistically significant differences in urinalysis parameters were considered to be incidental or unrelated to the treatment and well within limits of the historical control data for rats of this strain and age.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant differences in organ weights or organ weight to body weight ratios was observed.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Bluish discolouration of various segments of the gastrointestinal tract in several rats of group 4 (1000 mg/kg bw/day) at the end of the treatment period, which was attributed to colouration by the test item.
- The macroscopic findings recorded were mostly unremarkable and within the range of spontaneous alterations which may be seen in rats of this age and strain. They included dilated renal pelves, reddish discoloration in various organs and dilated uterine horns.
- Isolated findings of note were: an epididymal nodule (#2, control), bluish foci in the lungs (#19, male group 3), liver nodule (#28, male recovery group 4), urinary bladder stone with discolored foci in the kidneys (#45, female group 2) and discolored liver lobe (#49, female group 3).
- In female no. 45 (group 2, 50 mg/kg bw/day), which died spontaneously, a urinary bladder calculus and discoloured foci in the kidneys were seen.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- A moderate degree of alveolar inflammation associated with black pigment deposition was noted the lungs of animal #19 and was due to accidental instillation of the test article into the lungs. No effect on the microscopic appearance of any organ associated with test item treatment.
- Histological correlates to the gross pathological findings were: sperm granuloma (#2), post-necrotic focal fibrosis in the liver (#28) and ectopic hepatic tissue (#49). These findings were considered spontaneous in nature.
- Animal #45 had a severe degree of cystitis, purulent inflammation of the ureters and purulent pyelonephritis which were considered the cause of death. These findings were not considered to be related to treatment with the test article.
- In female rat no. 45 (group 2, 50 mg/kg bw/day), which died spontaneously, severe cystitis, purulent inflammation of the ureters and purulent pyelonephritis were considered to be the cause of death.
- The remainder of the recorded microscopic findings did not vary significantly in incidence or severity between control and treated groups, nor between rats sacrificed at the end of the treatment period or following the recovery phase. They were all of a spontaneous nature and within the normal range of background morphologic alterations which may be recorded in Wistar rats of this strain at these ages.

Other effects:

not specified

Effect levels

Dose descriptor:

NOEL

Remarks:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

NOEL = NOAEL (oral, rat, 28 days) = 50 mg/kg bw/day

Executive summary:

The repeated dose toxicity of the substance was evaluated in a subacute 28-day oral toxicity study according to the OECD Guideline 407 (1981) and method B.7 of the Directive 92/69 EEC. The substance was administered daily by oral gavage to SPF-bred Wistar rats of both sexes for a period of 28 days, followed by a 14-day recovery period. Test animals were randomly assigned into four groups at dose levels of 0, 50, 200 and 1000 mg/kg bw/day (5 males and 5 females per dose level) whereby the control group received only the vehicle (bi-distilled water) and test animals were sacrificed after a 28-day treatment period. Two additional, parallel groups received either 0 or 1000 mg/kg bw/day (5 males and 5 females per dose level) and were monitored for a 14-day recovery period following the 28-day treatment period. These animals were sacrificed after the recovery period.

Observations for clinical signs and mortality were recorded daily; body weight and food consumption measurements were taken weekly. Ophthalmoscopic examinations, blood samples and urine samples were taken at 4 weeks (both treatment and recovery groups) and 6 weeks (recovery groups only). Necropsies were performed on all test animals. Post-mortem organ weights and macroscopic and microscopic histological parameters were recorded.
All test animals administered 200 and 1000 mg/kg bw/day demonstrated hunched postur, which in rats commonly is associated with pain or discomfort, was seen intermittently. This was first noted 24 h after the first dose and was last seen on day 10 in group admistered at 200 mg/kg and on day 12 in group administered at 1000 mg/kg.

No other clinical signs, mortality, body weight changes and food consumption abnormalities of toxicological significance were recorded.

There were no treatment-related effects of toxicological significance recorded in haematology, clinical biochemistry, urinalysis or ophthalmic parameters. Reversible urine discolouration was observed in 12 out of 20 test animals administered the highest dose.

No histological effects or organ weight differences of toxicological significance were observed. Bluish dicolouration was observed in various sections of the gastrointestinal tract in several test animals of the treatment-only group administered 1000 mg/kg bw/day, and only one test animal of the treatment plus recovery group (1000 mg/kg bw/day). No test animals of 0, 50 or 200 mg/kg bw/day demonstrated discolouration of the gastrointestinal tract.
Based on the hunched posture observed at 200 and 1000 mg/kg bw/day, the NOEL and NOEAL for the substance in male and female rats was determined to be 50 mg/kg bw/day.