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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-04-11 to 1994-05-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
equivalent or similar 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
Details on test material:
Hüls AG, produced 02 February 1993
Purity 98.5 %
Sample No. 0099 (internal)
Sample ID 3633/81495

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ORGANISMS
- Source: Harlan Winkelmann, Borchen (Germany)
- Age: 6 - 8 weeks
- Weight at study initiation:    range of group mean weights, males: 190-200 g   range of group mean weights, females: 146-155 g
- Number of animals: total 30 males, 30 females

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
ADMINISTRATION / EXPOSURE 
- Vehicle: corn oil
- Total volume applied: 2 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration (weight-%) in corn oil was determined by high-temperature GC
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
15; 50; 150 mg/kg bw day
Basis:
other: actual dose by gavage
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: 14 days recovery (satellite groups only)
SATELLITE GROUPS AND REASONS THEY WERE ADDED: 
additional 150 mg/kg bw d  and control group for recovery study
DEVIATIONS FROM PROTOCOL: 
No fixation of tibia during necropsy
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS AND FREQUENCY: 
- Clinical signs: twice daily (weekends: once daily); detailed once a week
- Mortality: twice daily (weekends: once daily)
- Body weight: before first treatment, weekly thereafter until day of  necropsy
- Food consumption: weekly for each cage (5 rats/cage)
- Water consumption: daily for each cage
- Ophthalmoscopic examination: control and high dose groups during  acclimatization and prior to terminal bleeding
- Haematology: all animals twice for toxicokinetics during study plus  once (terminal) for serum chemical and haematological investigations:    
sodium, potassium, calcium, aspartate aminotransferase, alanine  aminotransferase, glucose, triglycerides, cholesterol, total bilirubin,  blood 
urea nitrogen, creatinine, total protein, albumin    red blood cell count, total white blood cell count, platelet count,  haemoglobin, haematocrit, 
erythrocyte indices (mean corpuscular volume,  mean corpuscular haemoglobin concentration), differential white blood  cell count, reticulocyte 
count
- Urinalysis: end of study; non-satellite groups additionally on days 3  (males) and 4 (females)   volume, specific gravity, pH, colour,    
semiquantitative: protein, glucose, ketone, urobilinogen, blood  ingredients   urine sediment analysis: leucocytes, erythrocytes, bacteria, epithelial  
cells (squamous), oxalate crystals, triple phosphate crystals, urate  crystals
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
- Macroscopic:    weights of adrenals, kidneys, liver, spleen, testes   adrenals, aorta (thoracic), anus, brain, caecum, coagulation gland,  colon, 
concha (tattooed), duodenum, epididymides, eyes, exorbital  lacrimal glands, gross lesions, heart, ileum, jaw (upper), jejunum,  kidneys, larynx, 
liver, lungs, lymph nodes (skin, cervical & mesenteric), mammary  gland, muscle (skeletal), ovaries, oesophagus, pancreas, pituitary,  prostate, 
rectum, salivary glands, sciatic nerve, seminal vesicles, skin,  spinal cord (cervical), spleen, stomach, testes, thymus, thyroid /  parathyroid, tongue, 
trachea, urinary bladder, uterus, vagina   bone marrow smears
- Microscopic: eyes, kidney, liver, lung, lymph nodes, oesophagus,  Peyer's patches, spleen, uterus
OTHER EXAMINATIONS: toxicokinetics: see separate report and entry
Sacrifice and pathology:
- Gross pathology: 
No macroscopic lesions considered to be related to  treatment were observed. There were rare cases of ophthalmia / ulceration  of the cornea due
 to blood sampling and one subcutaneous purulent  alteration due to application failure. 
- Histopathology: Findings consisted of spontaneous lesions in males and  females of all groups such as hydrometriosis of the uterus, calcification  
of Peyer's patches, hyaline casts in the kidney and multifocal  lymphocytes in the lung. Acute and chronic lesions of the eyes due to  bloodletting 
were observed occasionally. In the oesophagus subacute or  chronic traumatization due to application failure was observed in some  animals. There  were also pigmentation in the lymph nodes cervicales  caused by tattooing ears.   
Kidneys: No lesions except hyaline casts, also in controls; no lesions  in recovery group   
Liver: Extramedullary haematopoiesis in animals of all groups,  considered normal; no lesions in recovery group   
Spleen: Treatment related slight increase of haematopoiesis in 4/5 high  dose males and 2/5 high dose females
- Other: Several clinical signs in one intermediate dose female could be  attributed at necropsy to an application failure.
Statistics:
STATISTICAL METHODS: 
- Kruskal Wallis non parametric analysis of variance, in case of  significance followed by Wilcoxon, Mann, and Whitney U tests: body  weights, body 
weight changes, organ weights, differential blood count,  urine analysis data 
- one way analysis of variance (ANOVA) incorporating Bartlett's test for homogeneity of variance and if indicated followed by  Kruskal Wallis or 
Scheffe Test: haematological data (except differential  blood count) and serum clinical chemistry data
- Median (geometric mean), minimum, maximum: differential blood count

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
no mortalities; Piloerection, alopecia, and transient squatting position were observed in control and dose groups. High dose animals additionally revealed reduced activity at the start of the study
Mortality:
mortality observed, treatment-related
Description (incidence):
no mortalities; Piloerection, alopecia, and transient squatting position were observed in control and dose groups. High dose animals additionally revealed reduced activity at the start of the study
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significantly decreased absolute bodyweight was observed in high dose males throughout the study. No statistically significant differentes coneerning weight and weight gain were observed during recovery. However, high dose males showed a clear weight gain
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Food conversion rate was clearly increased in male high dose group
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
High dose males revealed a statistically significant decrease in red blond cell count (RBC). RBC in high dose females was equally reduced, though not significant. After recovery, RBC was still decreased in high dose males.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
significant increase was observed in serum sodium concentration in high dose animals of either sex.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urine of all dose groups revealed a change in colour to yellow-brown, darker than urine colour of controls. A statistically significant increase in oxalates in high dose males wasobserved.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
High dose males revealed a statistically significant increase in relative weight of spieen. After the two weck recovery period, a significant increase of absolute spieen weight was still observed in high dose males
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Substance-related slight increase of haematopoiesis in the spieen was observed in 4 out of 5 male animals of the high dose group
Details on results:
OXIC RESPONSE/EFFECTS BY DOSE LEVEL: 
- Mortality and time to death: No mortalities in either dose group
- Clinical signs:    
Control group: slight alopecia for a maximum of 4 (males) and 19  (females) consecutive days; piloerection slight in females and 
moderate  in males   
Low dose: Alopecia in one female; piloerection slight to moderate in  females (maximum 10 consecutive days) and throughout the  study in males;  
squatting position and closed eyes in either sex (maximum 5 consecutive  days)   
Intermediate dose: Piloerection slight to mode rate in all animals  throughout the study; alopecia in one female; squatting position for several 
times  n all animals; closed eyes in 7 animals (maximum 3  consecutive days); slightly abnormal gait and lop-sided head in  one male   
High dose: Slight to moderate piloerection (maximum 14 consecutive days  in females / throughout the study in males); Squatting  position in all 
females (maximum 5 consecutive days) at begin of study,  repeatedly and  transient in all males throughout the study; closed eyes for 1 (first)  day 
in females and for up to 5 days in all males; reduced activity in all  males on days 1 and 2; lethargy in  two animals (1 day); tonic convulsions  on day  2 and absence of auditory startle reflex in one male   
Recovery groups: Complete absence of piloerection in all animals within  several days after end of treatment
- Body weight gain: No statistically significant differences in either  dose group of either sex.   Absolute body weights only affected significantly in 
all treated males  (decrease) on days 7 and 21 and in high dose males additionally on day 28  (11.3 % below control). A gain in this latter group was
the only  significant observation in body weights  during recovery.
- Food/water consumption: Food conversion rate was clearly increased in  high dose males. Low and high dose females showed a  less pronounced  
increase. Food conversion of high dose males decreased during recovery.  No overt intergroup differences in water  consumption were observed. 
- Ophthalmoscopic examination: Cornea damages in several animals due to  repeated blood sampling; no signs of test substance 
related effects in  either dose group
- Clinical chemistry:    
Sodium: statistically significant increases in all high dose animals  and in low dose males, close to historical control data 
maximum in control males, still statistically increased after recovery in high dose  males   
Total bilirubin: Experimental difficulties (lipaemia, values close to  detection limit) were met. No clear differences could be observed  in  treated 
versus control groups.   
Calcium and creatinine: Increased only in females of intermediate dose  group   
Glucose: The range of historical control data was slightly exceeded in  seven dosed animals (one low, two intermediate, four high  dose animals  
including two recovery)    
Other parameters: No clear pattern of change   
Recovery group: For cholesterol, total bilirubin, and alkaline  phosphatase small, yet significant differences were observed for both   sexes
- Haematology:    
Red blood cell count: decreased significantly in males and  insignificantly in females of high dose group, improvement with males during recovery 
still left a significant decrease    
Reticulocytes: significantly increased in high dose females   
Eosinophiles: significant increase in high dose females   
Recovery group: significant increase in haemoglobin and consequently in  MCV and MCH of dosed females   
Other: Deviations in two individual intermediate dose animals could in  one case be explained by application failure
- Urinalysis:    
Colour: change to yellow-brown, darker colour in treated animals, not  dose dependent   
Urine sediment analysis: dose-dependent increase in oxalates,  statistically significant in high dose males with individual values beyond   the range  of the historical control data also for three  intermediate dose males. Recovery left oxalates of two individuals beyond  the range of the historical 
control data. High presence of oxalates in  urine was also observed in one or two individuals each of all female control and high dose groups 
including both recovery groups.  
Triplephosphates were significantly increased and erythrocytes  significantly decreased in high dose males.   
Urine volume: significantly increased in high dose females   
Urine pH: significantly decreased in high dose females (6.80, control  8.20) and one intermediate dose female. Unusual presence of glucose in  
urine and high presence of ketone was also observed in the high dose  female with the lowest pH of urine.
- Organ weights:    Relative kidney weights were insignificantly increased in high dose  animals.   Relative spleen weights were increased statistically 
significantly in  high dose males and insignificantly, not dose related in intermediate and  high dose females. Absolute spleen weights were 
decreased in low dose  females.  In the high dose male recovery group, absolute weight of spleen and  relative weight of adrenals were increased.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
clinical signs
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
clinical signs
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Upon 28-day oral gavage administration a NOAEL of 50 mg/kg bw/d was observed in Wistar rats.
Executive summary:

Tetrahydronaphthalene (THN) was administered by gavage to three groups of five male and five female Wistar rats, HsdfWin:WU strain. Administration was performed for 4 weeks, at dose levels of 15, 50 and 150 mg/kg/day. A control group of five males and five females was dosed with the vehicle (corn oll) alone. A control and a high dose recovery group each consisting of five males and five females, respectively, were observed for a period of 14 days after the end of the dosing period. Clinical signs, bodyweight, food and water consumption were monitored during the study. Blood chemistry and haematology as well as a urine analysis were evaluated for all animals at the end of the study. Ophthalmological examinations were performed on animals of the control and high dose groups prior to the start and at the end of the study.

Oral administration of the test material tetrahydronaphthalene to rats for a period of 28 days at a maximum dose leveI of 150 mg/kg/day resulted in treatment-related effects such as reduced bodyweight, increased relative spleen weight, increased haematopoiesis in spleen, reduced red blood cell count and an increase of reticulocytes suggesting an adverse effect an circulating erythrocytes. These adverse effects are regarded as transient and were reversible when the administration of the test substance was stopped. Minor and equivocal changes were observed in some clinical chemistry and urine analysis parameters. No such effects except for the equivocal changes in clinical chemistry and urine analysis were demonstrated in animals treated with 50 mg/kg/day, and no changes at all were observed at a dose level of 15 mg/kg/day. Therefore, the "No Observed Adverse Effect Level" (NOAEL) was considered to be 50 mg/kg/day for both sexes.