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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 April 1995 - 17 August 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to GLP requirements and OECD method. Very extensive and well documented report
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
other: The study has been conducted and the final report is formatted in accordance with US EPA (TSCA), Federal Register, Vol. 50, No. 188, 27th September 1985
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): AHTN
- Physical state: pure active substance

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Eighty nine male and ninety one female rats of the Crl:CD(SD)BR strain (VAF plus), supplied by Charles River (UK) Limited, Margate, England, were approximately 28 days old on arrival. They were acclimatised for 19 days, towards the end of which period they were re-examined and confirmed to be suitable for experimental use.
At the start of treatment the males weighed 224 - 301g and the females weighed 157 - 214g, and were approximately 46-48 days old.
The animals were housed singly, in grid-bottomed cages, suspended over cardboard-lined excreta trays.
The experimental room (F6) was air conditioned and recorded temperature was generally within the target range of 19 - 25°C. The temperature dropped below the target range on a few occasions (16- 18°C) and exceeded It once (26°C). These deviations were minor and were considered not to have affected the validity or integrity of the study. Relative humidity was within the range 30 - 70%. Fluorescent lighting was controlled automatically to give a cycle of 12 hours light (0600 to 1800 hours) and 12 hours dark.
Tap water and SQC Rat and Mouse Maintenance Diet No. 1 were available ad libitum.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: dietary admixture
Details on oral exposure:
The test article was administered orally, by dietary admixture, for 13 weeks. As the necropsy procedures at the end of the treatment period took three days to complete, those animals in the treated groups intended for termination after 13 weeks continued to receive the test article in the diet until the day before necropsy.
On completion of the treatment period (from the first day of week 14), the last 3 surviving males and last 3 surviving females in the control and high dose groups were maintained for a 4 week treatment-free period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of diet indicated that desired homogeneity was reached. A gas chromatographic method of analysis with flame ionization was developed and used to analyze samples taken from diets prepared for weeks 1, 7, and 13 of the study. Diets were generally within +/-15% of nominal, stable for at least 8 days, and no AHTN was detected in control samples. The concentrations of AHTN in the test diets were adjusted weekly. The mean achieved daily intakes of AHTN were 1.6, 5.0, 15.2 and 50.9 mg/kg bw for males and 1.5, 5.1, 15.1 and 50.8 mg/kg bw for females
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Unrestricted access to the diet
Doses / concentrations
Remarks:
Doses / Concentrations:
1.5, 5, 15 and 50 mg/kg/day
Basis:
nominal in diet
No. of animals per sex per dose:
15 males and 15 females per dose
Control animals:
yes
Details on study design:
The study was designed with 5 groups, with 15 animals per group and sex.
The observations analysed were: bodyweights at the start of the study; bodyweight gains over the intervals weeks -1 to 4, week 4 to 13, weeks -1 to 13 and weeks 13 to 17;
haematology, blood chemistry and urinalysis (volume and specific gravity only) determined in weeks 7 and 13; and absolute and relative bodyweight/brain weight related organ weights (percent).
Average food consumption over the intervals weeks 1 to 4, 5 to 13 and 14 to 17 were derived and analysed.

Examinations

Observations and examinations performed and frequency:
Mortality: The animals were checked twice daily for mortality or signs of morbidity.
Clinical signs: All animals were checked daily for clinical signs of toxicity or changes in condition or behaviour.
Bodyweights: Bach animal was weighed on one occasion during the pretreatment period on the day before the start of treatment, and then weekly thereafter throughout the treatment and treatment-free periods, up to and including the day before the start of the necropsy phase.
Food consumption: The food consumed by each animal was recorded for 6 days in each week throughout the treatment and treatment-free periods and group mean weekly intakes were calculated. This was performed to the same schedule as determination of bodyweight.
Ophthalmological: All animals were examined on one occasion before the start of treatment. Subsequently, all animals from each of the control and high dose groups were examined on one occasion during week 13, and at the end of the treatment-free period.
Urinalysis: On one occasion during weeks 6 and 12 of treatment, and at the end of the treatment-free period, overnight urine samples were collected from all animals, under food and water deprivation.
Blood: Samples for haematology and blood chemistry investigations were obtained from all animals on one occasion during weeks 7 and 13 of treatment, and at the end of the treatment-free period, by tail venepuncture.
Coagulation: tests were performed in week 13, and at the end of the treatment-free period, by tail venepuncture.
Sacrifice and pathology:
On completion of the treatment and treatment-free periods, the appropriate animals were killed and a macroscopic examination was performed. The weights of designated organs were recorded and a range of tissues was preserved for histopathological examination. In view of the macroscopic findings noted at necropsy, frozen liver sections from all animals in the control and high dose groups were cryostat sectioned and examined for red fluorescence consistent with porphyrin deposits under ultraviolet light of wavelength 300-405nm.
Statistics:
The data were subjected to an analysis of variance (ANOVA). The absolute residuals from this analysis were further subjected to an ANOVA to assess whether there were any between group differences in variances (Levene’s test).
If the Levene’s test indicated that there was no evidence of inequality of variance (ie p>0.01) then pair wise tests of all treated groups versus control were performed using Williams’ test. For the comparison of the high dose against the control, a two-sided test was performed. If the comparison of the high dose with the control was not significant, then testing was stopped, otherwise the process continued (now using one sided tests) with the lower doses until a non-significant comparison was encountered. At this point the testing ceased and the dose with which the non-significant comparison was associated was declared to be the ‘no effect’ level for the variable concerned.
If the Levene’s test indicated that there was evidence of differences between group variances or if the data was judged to be unsuitable for a parametric analysis, then a non-parametric approach to the data analysis was used. This involved the Kruskal-Wallis non-parametric ANOVA and Shirleys’ non-parametric equivalent of Williams’ test. The version of Shirley’s test used incorporated the modification to the ranking procedure suggested by Williams (1986). The testing scheme with Shirley’s test was the same as that described for Williams’ test above, two sided tests for the high dose, one sided tests otherwise and terminating the testing process when a non-significant result is obtained,
For variables measured pre-dose, the analysis was performed using ANOVA to assess any between group differences. 1f this test was statistically significant, then it was followed by all pair wise comparisons using Students t-test. If indicated by Levene’s test, an equivalent non-parametric method was used.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
decreased
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
brown discolouration
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see any other inform
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
green discoloration of liver, mesenteric lymph nodes & lachyrmal glands
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
Intake: Close approximation to the nominal doses. Mean values over weeks 1 - 13 of treatment for groups 2, 3, 4 and 5, respectively, were 1.6, 5.0, 15.2 and 50.9 mg/kg/day for males, and 1.5, 5.1, 15.1 and 50.8 mg/kg/day for females.
 
Food consumption: The consumption of males and females in all groups given AHTN was similar to that of respective controls throughout the whole study.
 
Mortality: There were no mortalities during the study.
 
Clinical signs:No clinical signs attributable to the administration of AHTN were noted during the study.
 
Mean bodyweight gain: The gain of males and females given 50 mg/kg/day was statistically significantly lower than that of respective controls throughout the treatment period. During the treatment-free period, the mean bodyweight gain of females in the high dose group was similar to that of controls, while males in the same group showed a higher bodyweight gain compared to controls.
 
Ophthalmoscopy: No ophthalmologic abnormalities attributable to administration of AHTN were noted for the high dose animals examined during week 13, or for those examined at the end of the treatment-free period.
 
Heamatology: The principal haematological findings included a reduction in red cell count, haemoglobin concentration and packed cell volume, which were observed at week 7 and 13 for males and females given 50 mg/kg/day. However, the differences from controls were only slight and were considered to be of no toxicological importance.
The slightly prolonged prothrombin time noted at week 13 for males and females given 5, 15 and 50 mg/kg/day was also slight and was considered to be of no toxicological importance.
These findings were absent following cessation of treatment.
 
Blood Chemistry: Among the blood chemical parameters showing an effect of treatment with AHTN, the most noticeable were cholesterol and triglyceride concentration. A reduction in plasma cholesterol was observed at weeks 7 and 13 for males and females given 15 and 50 mg/kg/day. Plasma triglyceride concentration was also reduced in all dose groups in week 7 and at 5, 15 and 50 mg/kg/day in week 13.
Other treatment-related findings were; a higher A/G ratio noted at week 13 for males and females given 15 mg/kg/day, and at weeks 7 and 13 for females given 50 mg/kg/day, related to a slight reduction in total protein; a lower plasma glucose concentration at week 7 in males given 15 and 50mg/kg/day, and males in all dose groups at week 13. All of these findings were slight and were considered to be of no toxicological importance. Moreover, these findings were absent following cessation of treatment.
A brown colouration of the urine was observed at weeks 6 and 12 for a small proportion of males given 50mg/kg/day. This was not evident at the end of the treatment-free period. The significance of this finding is unclear. There were no effects on urine composition or cellularity.
 
Sacrifice: For those animals killed on completion of the 13 week treatment period:

The mean absolute and bodyweight related liver weights of females given 15 mg/kg/day, and males and females given 50 mg/kg/day were higher than those of respective controls. Statistical significance was attained for the bodyweight-related liver weight for males and females given 50 mg/kg/day. There were no differences in liver weight at the end of the treatment-free period.
Although attributed to administration of AHTN, in the absence of any histopathological changes in the liver, the above-mentioned differences in liver weight were considered to be of no toxicological importance and may represent an increased demand for liver function
 
Abnormal green coloured livers were observed in eleven males and four females given 50 mg/kg/day. Similar findings were noted in the mesenteric lymph nodes of ten males and three females in the same group, and the lachrymal glands of one female given 5 mg/kg/day, four females given 15 mg/kg/day and eight females given 50 mg/kg/day. In addition, an abnormally shaped liver was noted for two males given 50 mg/kg/day.
At the end of the treatment-free period, only one male in the high dose group showed green coloured mesenteric lymph nodes, with two females in the same group showing green coloured lachrymal glands.
These findings were clearly related to administration of AHTN, although there were no associated histopathological findings.
 
No abnormalities attributable to administration of AHTN were noted during histopathological examination.
There were no corresponding microscopic findings in those animals which showed macroscopic abnormal colouration of the liver, mesenteric lymph nodes or lachrymal glands.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: decreased body weight gain
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: haematological parameters

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The daily administration of AHTN to rats by dietary admixture at doses of 1.5, 5, 15 or 50mg/kg/day was generally well tolerated. There were no mortalities or adverse clinical signs. Bodyweight gain was reduced in males and females given 50mg/kg/day, but there was evidence of an improvement on cessation of treatment. Food consumption was unaffected in all dose groups. There were no ophthalmological abnormalities.
Haematological findings included a slight reduction in red cell count, haemoglobin concentration and packed cell volume, almost exclusively for males and females given 50mg/kg/day, and a slightly prolonged prothrombin time for males and females given 5, 15 and 50mg/kg/day. Changes in blood chemical parameters included a reduction in plasma cholesterol for males and females given 15 and 50mg/kg/day, and triglyceride concentration in all dose groups at week 7 and at 5, 15 and 50mg/kg/day at week 13. In addition, a higher A/G ratio was noted at week 13 for males and females given 15mg/kglday, and at weeks 7 and 13 for females given 50mg/kg/day, and a lower glucose concentration was noted at week 7 in males given 15 and 50mg/kg/day, and males in all dose groups at week 13. These findings in haematology and blood chemistry, which were absent following cessation of treatment, were considered to be insufficient to have compromised the animals and to be of no toxicological importance.
A brown colouration of the urine was observed for a small proportion of males given 50mg/kg/day, which was not evident at the end of the treatment-free period. The significance of this finding is unclear.
The liver weights of females given 15mg/kg/day, and males and females given 50mg/kg/day were higher than those of respective controls. In the absence of any histopathological changes in the liver, this may represent an increased demand for liver function. There were no differences in liver weight at the end of the treatment free period. Abnormal green coloured livers, mesenteric lymph nodes and lachrymal glands were observed among males and females given 50mg/kg/day, and in the lachrymal glands of a small number of females given 5 and 15mg/kg/day. These findings were also observed at the end of the treatment-free period. Microscopic examination of unstained sections of frozen liver under ultra-violet illumination did not reveal any fluorescence that might have been consistent with porphyrin accumulation. These findings were clearly related to administration of AHTN, although there were no associated histopathological findings and they were considered to be of no toxicological importance.
No abnormalities attributable to administration of AHTN were noted in any tissue during histopathological examination.
Executive summary:

Groups of rats received by dietary admixture nominal doses of 0 (control), 1.5, 5, 15 and 50 mg AHTN/kg bw/day for 13 weeks.

After the treatment period, 3 females and 3 males from the control and the high dose groups were maintained for a treatment-free period of 4 weeks.

 

Observations included mortality and clinical signs (daily), body weight and food consumption (weekly), ophthalmoscopy (at week 13 and at the end of the treatment-free period, only controls and high dose animals), urinalysis (at weeks 6 and 12 of treatment and at the end of the treatment-free period), haematology and clinical chemistry (at weeks 7 and 13 of treatment and at the end of the recovery period), macroscopy, organ weights and histopathology (on all tissues from controls and high dose animals, on all gross lesions, and on lungs, liver, kidneys and male and female reproductive and accessory organs from all animals).

There were no mortalities during the study and no clinical signs or ophthalmological.

 

During treatment, the mean body weight gain of males and females given 50 mg/kg bw/day was statistically significantly lower than that of controls (78 and 88% of controls, respectively).

Body weight at week 13 was only statistically significant decreased with 15% in the male high dose group. This improved upon cessation of treatment. Treatment with AHTN did not affect food consumption.

 

A reduction in red cell count, haemoglobin concentration and packed cell volume were observed at weeks 7 and 13 for males and females at 50 mg/kg bw/day. At 7 weeks but not at 13 weeks the animals at that dose showed polychromasia and anisocytosis. These findings were also observed in some animals of the two mid dose groups. A small but statistically significant prolonged prothrombin time was noted at week 13 for males and females given 5, 15 and 50 mg/kg bw/day but the values were all within the standard deviation (except for the high dose males) of controls and the range of historical controls, and not clearly related to dose. Males at 50 mg/kg bw/day had statistically significantly higher white blood cell counts (WBC) at weeks 7 and 13. After the treatment free period, no statistically significant differences were observed, but females and males at the high dose group still had slightly prolonged prothrombin time and higher WBC, respectively.

 

At weeks 7 and 13, in the male high dose group, higher alkaline phosphatase (19% (week 7) and 38% (week 13) increase) and alanine aminotransferase (17% (week 7) and 36% (week 13) increase) activities were found.

A higher A/G ratio was noted at week 13 for males and females given 15 mg/kg bw/day and at weeks 7 and 13 for males and females given 50 mg/kg bw/day, in males related to a small but statistically significant reduction in total protein at the two highest doses after 7 weeks and at all doses after 13 weeks. No significant differences were seen for these parameters after the treatment-free period.

A lower plasma glucose concentration was seen at week 7 in males given 15 and 50 mg/kg bw/day, and in males of all dose groups at week 13.

A reduction in plasma cholesterol was observed at weeks 7 and 13 for males and females given 15 and 50 mg/kg bw/day.

Plasma triglyceride concentration was dose-relatedly reduced in all dose groups in week 7 and at 5, 15 and 50 mg/kg bw/day in week 13, at least in the two highest dose groups, a clear dose response was seen. No values were significantly different from controls after the treatment-free period.

A brown colouration of the urine was observed at weeks 6 and 12 for 5/15 males given 50 mg/kg bw/day (not seen after the treatment-free period). There were no effects on urine composition or cellularity.

The only organ weight changes attributable to AHTN were the increased absolute and relative liver weights in males and females. The absolute liver weight was at the highest dose increased with 14 and 7%, respectively in males and females (not statistically significant).

The relative liver weight was significantly increased with 32% (males) and 13% (females) after exposure to 50 mg/kg bw/day. The liver changes were not seen after the treatment-free period and there was no increase in liver weight relative to brain weight. There were no histopathological changes in the liver. This indicates that the difference in liver/body weight could be due to the decreased body weight gains rather than an effect on the liver.

Upon macroscopy, abnormal green to dark brown coloured livers were observed in 11/12 males and 4/12 females given 50 mg/kg bw/day. Similar findings were noted in the mesenteric lymph nodes of 10/12 males and 3/12 females in the same group. No such discolouration was seen in the lower dose groups.

A green colouration was seen in the lachrymal glands of females only at 50 mg/kg bw/day (8/12), 15 mg/kg bw/day (4/12), and 5 mg/kg bw/day (1/12). There were no associated histopathological findings in these organs. In addition, in liver there was no porphyrin accumulation. At the end of the treatment-free period, only 1/3 males in the high dose group showed green coloured mesenteric lymph nodes, and 2/3 females in the same dose group showed green coloured lachrymal glands.

Uterine distension reflects normal cyclic change and should not be considered as an adverse effect.

Source: EU Risk Assessment Report AHTN, ECB (May 2008).