Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998-Mar-05 to 1998-Apr-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Reaktiv Marineblau FC 63805

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, Gartenstr. 27, D-33178 Borchen
- Age at study initiation: approximately 6 weeks
- Housing: fully air-conditioned rooms in macrolon cages (type IV) on soft wood granulate in groups of 5 animals
- Diet (e.g. ad libitum): ssniff R(M-H (V15354), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: ad least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

In-life phase:
Start of the study: March 05, 1998
Necropsy (final value): April 02, 1998
Necropsy (recovery value): April 16, 1998

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was dissolved in the stated concentrations in deionized water at the following dates
1. 1998-Mar-05
2. 1998-Mar-19

VEHICLE
- Concentration (in % (w/v)) in vehicle:0, 1.25, 5, 20 (according to doses of 0, 62.5, 250, 1000 mg/kg bodyweight)
- Amount of vehicle (if gavage): 5 ml/ kg bodyweight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
stability is guaranteed for 15 days according to report of Analytical Toxicology dated Feb. 17, 1998
dose concentratons are verified in the first and last week of treatment by HPLC
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5/sex for 62.5 and 250 mg/kg bw
10/sex for 0 and 1000 mg/kg bw
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: acute oral toxicity testing of Reactive Navy FC 63805 in rat yielded a LD50 above 2000 mg/kg bw in male and female animales
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 1/day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before first treatment, 1/week during exposure, at study termination

BODY WEIGHT: Yes
- Time schedule for examinations: before start of exposure, 2/weeks during the 28 d of exposure

FOOD CONSUMPTION:
- Food consumption was determined 2/week and food consumption per 100 g body weight/ d was calculated

WATER CONSUMPTION: Yes
- Time schedule for examinations: water consumption was determined 1/week over a period of 16 h and is given as water consumption/animal/16 h

HAEMATOLOGY: Yes
- Time schedule for collection of blood: termination of the study and for satellite groups after the additional 14 days follow up period
- Anaesthetic used for blood collection: Yes (narcosis with intraperitoneal injection of 67 + 6.7 mg/kg bw ketamine-hydrochloride + xylazine)
- Animals fasted: No
- How many animals: all animals
- Parameters checked: erythrocytes count, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHM), leucocyte count, thrombocyte count, differential leucocyte count and red cell morphology, coagulation time
additional in control and 100 mg/kg bw/day groups only: reticulocyte count, Heinz bodies

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: termination of the study and for satellite groups after the additional 14 days follow up period
- How many animals: all animals
- Parameters checked: sodium, potassium, inorganic phosphorus, uric acid, bilirubin total, bilirubin direct, creatinine, glucose, urea, calcium, chloride, aspartate aminotransferase (ASAT/GOT), alanine aminotransferase (ALAT/GPT), alkaline phosphatase (AP), gamma-glutamyltranspeptidase (GGT), cholesterol, triglycerides, total protein, albumin

URINALYSIS: Yes
- Time schedule for collection of urine: overnight from day 26 to day 27 as well as from day 35 to day 36
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: appearance, color, volume, specific weight, pH-Value, hemoglobin, protein, glucose, bilirubin, urobilinogen, ketone bodies
additionally in control and 1000 mg/kg bw/day groups only: sediment

-OTHER: neurotoxicological examination, including "open field" observation, assessment of sensory function, motor activity, forelimb and hindlimb strength
at study termination additionally: sensory stimuli (auditory, visual and proprioceptive) including startle reflex (click response), response to approach with the finger to the nose of the animal and righting reflex; pupillary constriction
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
After exsanguination, all animals were necropsied. Skin, orificed, eyes, teeth, oral mucosa and internal organs were macroscopically examined.
The lungs, including part of the trachea were removed and then fixed endotracheally with formalin solution.
Organ weights and organ to body weight ratios were measured/calculated for: heart, liver, kidneys, adrenal glands, spleen, testes, epididymides, thymus and brain

HISTOPATHOLOGY: Yes
following tissues or organs (or pieces of them) were preserved in a suitable fixative: heart, lungs, liver, spleen, kidneys, stomach, jejunum, colon, brain, thymus, trachea, thyroid glands with parathyroid glands, testes, epididymides, ovaries, uterus, urinary bladder, lymph nodes iliac, lymph nodes mandibular, adrenal glands, prostate gland, bone marrow (sternum), N. ischiadicus, spinal cord (cervical)
Statistics:
The following parameters were compared statistically with the control group values at the level of significance p=0.05:
body weight at the designated measurement times, hematological data, clinical chemistry parameters, urine analysis (volume, pH-value and specific weight), absolute organ weights and organ to body weight ratios, neurotoxicological measurements (motor actitvity, forelimb and hindlimb grip strength)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological examinations at the end of dosing revealed a slight increases in thrombocyte counts of the high dose group males and females. However, as this result was statistically significant only in males, was no longer present in the recovery animals and no other, correlating, effects were seen, it was not considered to be related to treament with the test substance.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Reversible discoloration of the urine was considered to be treatment-related but without any toxicological relevance.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Discoloration of the kidneys.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathologically, acute inflammatory cell infiltration with slight secretory disorder of the stomach mucous membrane (fundus) was found in high dose group males and females. This change was not accompanied by destructive processes and, as the recovery test shows, disappeared within an appropriate time after discontinuation of treatment. This effects is most likely related to the high salt-concentration entered directly to the stomach mucosa by giving repeatedly a high concentration of the dissolved test material via a gastric tube. It is hence not relevant for human hazard assessment.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No deaths were encountered. Behavior and state of health remained unaffected by the administration of the test compound.

NEUROTOXICOLOGICAL EXAMINATIONS
Neurotoxicological measurements including ,open field" observations, assessment of sensory function, motor activity, forelimb and hindlimb grip strength were not influenced by the administration of the test compound in all groups.


BODY WEIGHT AND WEIGHT GAIN
Coincidental intergroup variation in the males resulted in significantly increased body weights, but were of no toxicological relevance. Females of the recovery group had a slightly higher body weight than control animals, which was considered coincidental.

HAEMATOLOGY
There were no toxicologically relevant compound related hematological findings. At the end of dosing an increase in thrombocyte count was statistically significant in males, but not in females and returned to control level during the 14d recovery period,.

CLINICAL CHEMISTRY
Observed changes were considered to be of no toxicological relevance, since:
- significantly increased total bilirubin and bilirubin direct levels, only observed in the male higher dosage group missed hematological or histological correlate findings.
- other changes in blood parameters (urea, cholesterol, ALAT, alkaline phosphatase) were decreases, while only increases point to organ damages.
- changes were only marginal.

URINALYSIS
Urine from animals of the high dose was yellow-brown (3 males, 1 female) or blue-violet (2 males). Reversible discoloration of the urine was considered to be caused by the staining properties of the dye but without any toxicological relevance.

ORGAN WEIGHTS
Increased absolute weight of liver, kidney and brain in the males of the high dosage group was in line with the increased body weight of this group. Relative organ weight was not affected.

GROSS PATHOLOGY
Brown or olive discoloration of the kidneys was found in 2 females of the middle dose and 4 females and 5 males of the high dosage group. Discolouration remained in 4 females and 1 male in the high dosage satellite group at the end of the 14 d recovery period. There was no histopathological finding which could be related to the discoloration of the kidneys.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological, acute inflammatory cell infiltration with slight secretory disorder of the stomach mucous membrane (fundus) was found in high dose group males and females. This change was not accompanied by destructive processes and, as the recovery test shows , disappeared within an appropriate time after discontinuation of treatment.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no toxicologically relevant treatment-related effects were seen during the entire study period.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Repeated doses of Reactive Navy FC 63805 did not cause any toxicologically relevant adverse effects for human hazard assessment. The inflammatory reaction of the stomach mucous membrane (fundus) in male and female rate at the dose level of 1000 mg/kg bw/d was considered to be due to the high salt load by administering repeatedly a high concentration of the dissolved test material via a stomach tube directly onto the gastric mucosa. As this will not be a way of exposure for humans, this effect is irrelevant for hazard assessment. Consequently, 1000 mg/kg bw/d is considered to be the NOAEL.
Executive summary:

Groups of male and female rats received Reactive Navy FC 63805 by oral gavage at dose levels of 0, 62.5, 250 and 1000 mg/kg body weight per day for 28 days. On day 29 five males and five females from each group were killed and necropsied. The remaining animals of the control and high dose group were killed and necropsied after a recovery period of 14 days.

Behaviour and state of health were observed daily in all groups. Body weights and food consumption were recorded twice weekly, and water consumption once weekly. Once before the first treatment and thereafter once a week detailed clinical observations were performed in all animals outside the home cage in a standard arena („open field"). Additionally, the animals were examined for opacity of the refracting media of the eyes, damage to the oral mucosa, and impairment of dental growth. Neurotoxicological measurements including assessment of sensory function, motor activity, forelimb and hindlimb grip strength were conducted at the end of the treatment period. Haematological examinations and clinical chemistry were carried at the end of the treatment period and after the recovery period. Urine analysis was carried out at the end of the treatment period and after the recovery period. During necropsy the animals were examined for macroscopically visible abnormalities, the main organs weighed and the organ to body weight ratios calculated. Many organs and tissues were processed for histopathological examination and checked for microscopically visible changes.

Body weights, haematological and clinical chemistry data, urine data (pH-value, volume, specific weight), absolute and relative organ weights, neurotoxicological measurements (motor activity, forelimb and hindlimb grip strength) were analysed with the aid of a statistical program to show differences compared to the controls.

No deaths occurred throughout the study. Behaviour and state of health remained unaffected by the administration of the test compound in all dose groups. Neurotoxicological parameters remained unaffected by the administration of the test compound in all groups. Body weight development, food and water consumption remained unaffected by the administration of the test compound in all groups. Haematological examination showed a slight increase in thrombocyte counts which was considered to be toxicologically irrelevant. Clinical chemistry examinations revealed no compound-related changes in any dose group. The urine was discoloured brownish yellow (3 males, 1 female) or blue violet (2 males) in the high dose group at the end of the treatment period. This effect which proved to be reversible within the recovery period was considered compound-related but without any toxicological relevance. Examination of urine sediments revealed no compound-related changes in any dose group. Examination of the organ weights revealed no compound-related changes in any dose groups. At necropsy, compound-related brown or olive discoloration of the kidneys was seen in two intermediate dose females and in four high dose males and five high dose females at the end of the treatment period and in one male and four females of the highest dose at the end of the recovery period. Histopathologically, the test compound caused an acute inflammatory cell infiltration with slight secretory disorder of the stomach mucous membrane (fundus) in high dose group males and females. This change was not associated with destructive processes and reversible. It was considered to be due to the high salt load by administering repeatedly a high concentration of the dissolved test material via a stomach tube directly onto the gastric mucosa. As this will not be a way of exposure for humans, this effect is irrelevant for hazard assessment. There were no other histological organ changes which could be related to the administration of the test compound.

Consequently, 1000 mg/kg bw/d is considered to be the NOAEL.