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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1996

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Source: Ajinomoto Co., Inc (Tokyo, Japan)
- Physical state: odourless white crystalline powder
- Analytical purity: 99.8%
- Storage: in the diet at 4°C until use

Test animals

Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories International, Inc.
- Age at study initiation: 5 weeks
- Other: Specific pathogen free
- Housing: polycarbonate cages with wood-chip bedding (5rats/cage)
- Diet : basal diet: CRF-1 ad libitum (Oriental Yeast Inc., Tokyo)
- Water : tap water ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24+/-1°C
- Humidity (%): 55+/-5%
- Air changes (per hr): air-conditioned room
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
HMHC was added to CRF-J powdered basal diet to give final concentrations of 0 (control), 1.25 and 2.5%.
RATIONALE:
These dose levels were selected on the basis of the results of a 13 weeks subchronic toxicity study at doses of 0.31, 0.62. 1.25, 2.5 and 5% in the diet which was performed prior to the present study (Ikezaki et al, 1994). Because the dietary dose level of 5% proved to exert significant toxicity, it was concluded that the maximum tolerable dose is 2.5% in the diet.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The amounts of food consumed were measured in order to calculate the actual intakes of HMHC.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2.5%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1.25%
Basis:
nominal in diet
No. of animals per sex per dose:
50 males and 50 females; 3 groups
Control animals:
yes, plain diet
Details on study design:
- Rats were randomly allocated to three groups (0 (control), 1.25 and 2.5%.), each consisting of 50 males and 50 females.
- Post-exposure recovery period: 3 weeks
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes, daily

BODY WEIGHT: Yes, once a week for the first 13 wk of the study and then once every 4 wk.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes, daily
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY: Yes, blood taken from abdominal aorta
Sacrifice and pathology:
SACRIFICE
- After treatment, all surviving animals were given basal diet for a further 3 wk and then killed under ether annaesthesia after a 24-hr fast.

GROSS PATHOLOGY: Yes
- After careful gross examination, a complete autopsy was performed.
- Brain, lungs, heart. liver, spleen, adrenal glands. kidneys and testes of each animal were weighed.
- Moribund or dead animals were also autopsied completely to determine the development of tumours.

HISTOPATHOLOGY: Yes
- Tumour masses, as well as all organs and/or tissues were fixed in 10% buffered formalin and paraffin-embedded sections were routinely prepared and stained with haematoxylin and eosin.
Statistics:
Differences between mean values were analysed statistically using analysis of variance followed by Dunnett's test or Scheffe's test.
The incidences of tumours were compared with the Fisher's exact probability test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group a significant depression of body weight gain occured in male (weeks 76-104) and female rats (weeks 84-100).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No statistical significant differences between control and tested animals.
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The total intake of HMHC was clearly dose related. Final survival rate of female rats in the 2.5% group was 74% and slightly lower than those in the two other groups (Table 1).
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Increased parameters in 2.5% HMHC treated male rats. As the haemoglobin molecule contains 8% histidine, these effects might be expected. Indeed, an histidine-rich diet can induce excessive erythropoiesis in rats.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Description (incidence and severity):
No toxicological changes were histologically evident in the tested animals.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Non-neoplastic changes were observed frequently in all groups including the controls, without any significant differences between the groups.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Lack of any increase in neoplastic lesions.
Details on results:
GROWTH OF ANIMALS/BODY WEIGHT
The growth curves indicate that in the 2.5% group a significant depression of body weight gain occured for the 76-104-week period in male rats and for week 84-100 in females. Compared to the control group, this corresponds with 4.9% and 6.3% decrease respectively. The mean body weights of both sexes of the 1.25% group were essentially comparable with the controls, although significant differences (P < 0.05) were sporadically noted in male rats at a few time points.

CLINICAL SIGNS AND MORTALITY
Data for final survival rate and mean survival time are summarized in Table 1.
Final survival rate of female rats in the 2.5% group was 74 % and slightly lower than those (88-90%) in the other two groups. Animals in the 2.5% groups of both sexes showed slightly shorter mean survival although these were not statistically significant. The survival rate of female rats after wk 75 in the 2.5% group was slightly lower than those in the other two groups, whereas the survival curves or male rats were similar in both treated and control groups.

FOOD CONSUMPTION AND COMPOUND INTAKE
Data for total intake of the test chemical, estimated from the food consumption data are summarized in Table 1.

HAEMATOLOGY
Statistically significant increases in red blood cell count (P < 0.05), haemoglobin value (P < 0.05), haematocrit (P < 0.01) and platelet count (P < 0.01)
were observed in male rats given 2.5% HMHC, although no significant changes were found in females.

ORGAN WEIGHTS
In females, the relative weights of the brain in the 2.5 % group and of the adrenals in the 1.25 and 2.5% groups were significantly higher (p < 0.05 and 0.01) than the respective control values whereas the absolute weights were comparable in all groups. The absolute lung weights in male rats were significantly lower (P < 0.05) in the 1.25 and 2.5% groups than in the controls.

GROSS PATHOLOGY
The first autopsy was performed at week 47, when a male rat in the 2.5% group died of a lipoma arising in the submucosa of the rectum. Therefore, all rats surviving beyond the 47-week time point were included in the effective numbers, except for one malein the 1.25% group that died at week 98, for which organs were not available for histopathological examination because of autolysis. The overall incidences of tumours in each group were 100% in males and 52-64% in females. There were no significant differences between the control and treated groups of both sexes in overall tumour incidences. Tumours were found in many organs or tissues in all groups of both sexes, including the control group. In all male groups, tumours of the testes were most frequent, followed by lesions in the adrenals, heamatopoietic organs and pituitary. All testicular tumours were benign interstitial cell tumours, the most frequently encountered spontaneous tumours in F344 rats. In females, tumours of the pituitary, uterus, heamatopoietic organs and mammary glands were common. Tumours were also detected in other organs/tissues of rats of both sexes of all groups, but at low incidences. The incidence of pituitary adenoma in females were relatively high in the treated groups as compared with the control group, but there were no significant differences.
Therefore, the organ distribution and histological types of tumours were essentially the same as those spontaneous tumours described previously in F344 rats.

NON-NEOPLASTIC LESIONS
Non-neoplastic changes were observed frequently in all groups, including control groups, without any significant differences between the groups. Sperm granulomas in the epididymis, which were observed in male rats fed with 5.0% HMHC in the previous subchronic study, were not found in any group in the present study. Indicating that no damage to the tubule epithelium occured with 2.5% HMHC feeding.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
472.9 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
organ weights and organ / body weight ratios
gross pathology
Dose descriptor:
NOEL
Effect level:
558.45 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 1: Data food consumption, intake HMHC, final survival rate and mean survival time

Dose (%)

Food consumption (g/animal/day)

HMHC intake

Final survival rate (%)

Mean survival time (week) (mean±SD)

Daily intake (mg/kg bw/day)

Total intake (g/kg bw/104 weeks)

Males

0

13.4

0.0

0.0

80

104.5± 7.8

1.25

13.3

472.9

345.2

76

104.1± 9.3

2.5

13.1

955.0

697.2

76

102.4± 12.8

Females

0

9.3

0.0

0.0

88

105.5± 7.2

1.25

9.2

558.4

407.7

90

106.1± 5.4

2.5

8.9

1105.2

806.8

74

103.3± 9.8

Applicant's summary and conclusion

Conclusions:
It was concluded that HMHC is not toxic to F344 rats of either sex.
Even though a NOAEL was not derived in this study, we can say that the NOAEL for male is 472.9 mg/kg bw/day and for females 558.45 mg/kg bw/day, as no adverse effects occured in the 1.25% test group for both sexes.
Executive summary:

In this study the long-term toxicity (104 weeks) and carcinogenicity effects of histidine were examined in Fischer 344 (FJ44) rats. It is not stated that the study is GLP, however, the methodology is similar to the OECD 453.

Groups of 50 males and 50 females were fed L-histidine monohydrochloridc (HMHC) in their diet at concentrations of 0 [control], 1.25 and 2.5% for 104 weeks. The dose levels were selected based on the results of a previously performed subchronic toxicity study (Ikezaki et al, 1994, Bulletin of the National Institute of Health Sciences 112, 57 -63). In the previous study the body weights were depressed and formation of sperm granulomas in the epididymis was histologically evident in males fed with 5.0% HMHC.

The real HMHC intake was verified and calculated and are indicated in the table below:

Dose (%)

HMHC intake

Daily intake

(mg/kg bw/day)

Total intake

(g/kg bw/104 weeks)

Males

0

0.0

0.0

1.25

472.9

345.2

2.5

955.0

697.2

Females

0

0.0

0.0

1.25

558.4

407.7

2.5

1105.2

806.8

After 104 weeks exposure, the surviving rats were observed for an additional 3 weeks wereafter they were sacrificed.

Only one male rat died during the exposure period. Final survival rate of female rats in the highest dose group was 74 % and thus slightly lower than those (88-90%) in the other two groups. Animals in the 2.5% groups of both sexes showed slightly shorter mean survival although these were not statistically significant. Also, the survival curves of male rats were similar in both treated and control groups.

The growth curves indicated that the 2.5% group had a significant depression of body weight gain (males: 76-104 weeks; females: 84-100 weeks). The mean body weights of both sexes of the 1.25% group were comparable with the controls, although significant differences were sporadically noted at various time points.

In females, the relative weights of the brain and adrenals in the tested groups were significantly higher than the controls, whereas the absolute weights were comparable in all groups. The absolute lung weights in male rats were significantly lower in the tested groups than in the controls.

Statistically significant increases in red blood cell count, haemoglobin value, haematocrit and platelet count were observed in male rats fed with 2.5% HMHC, while no significant changes were found in females.

Non-neoplastic changes were observed frequently in all groups, including control groups, without any significant differences between the groups.

Even though no NOAEL was derived in this study, no adverse effects were observed at the dose of 472.9 mg/kg bw/day for males and 558.45 mg/kg bw/day for females (1.25% test group for both sexes).