Histidine

Carcinogenicity

Description of key information

Read-across was done from  L-histidine monohydrochloride (HMHC). Based on this result, and on the structural, chemical

and toxicological similarities between L-histidine monohydrochloride (HMHC) and L-histidine, it is concluded that L-histidine is not carcinogenic in F344 rats of either sex.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

not specified

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

male/female

Details on test animals or test system 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

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on 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

Post exposure period:

3 weeks

Remarks:

Doses / Concentrations:
1.25%
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
2.5%
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.

Positive control:

no

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.

Clinical signs:

no effects observed

Description (incidence and severity):

No statistical significant differences between control and tested animals.

Mortality:

no mortality observed

Description (incidence):

No statistical significant differences between control and tested animals.

Body weight and weight changes:

no effects observed

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 sites, histological types and incidences of tumours in each group of both sexes are summarized in Table 2.
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.

Relevance of carcinogenic effects / potential:

In the present study, although a variety of tumours was noted in all groups, including the control group, the organ distribution and histological types were essentially the same as those of spontaneous tumours described previously in F344 rats.

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

Table 2: Sites and types of tumours in F344 rats given HMHC in the diet for 104 weeks

Sites	Types of tumours	Number of rats with tumours
		Males			Females
		0%	1.25%	2.5%	0%	1.25%	2.5%
Effective n° of rats		50	49	50	50	50	50
Brain	Astrocytoma	0	1	0	1	1	0
Pituitary gland	Adenoma	7	3	8	7	10	12
Thyroid gland	C-cell adenoma	3	1	2	0	1	0
	C-cell carcinoma	2	1	1	0	1	1
Adrenal gland	Cortical adenoma	0	0	1	0	0	0
	Pheochromocytoma	9	8	5	1	2	1
	Malignant Pheochromocytoma	1	1	2	1	0	0
Pancreas	Islet cell adenoma	0	0	2	0	1	1
Liver	Adenoma	0	0	1	0	0	0
Oral cavity	Squamous cell carcinoma	0	0	1	0	0	0
Tongue	Squamous cell papilloma	0	2	0	0	0	0
	Squamous cell carcinoma	0	0	0	0	1	0
Large intestine	Lipoma	0	0	1	0	0	0
Haematopoietic organs	Mononuclear cell leukemia	9	7	4	4	6	6
	Malignant lymphoma	0	0	1	1	0	0
Spleen	Fibroma	1	1	0	0	0	0
Testis	Interstitial cell tumour	45	49	45	-	-	-
Mammary gland	Fibroma	1	0	0	0	1	0
	Fibroadenoma	2	0	0	3	2	3
	Adenolipoma	0	0	0	1	0	0
	Adenocarcinoma	0	0	0	0	1	1
Ovary	Scirrhous adenocarcinoma	-	-	-	1	0	0
Uterus	Adenoma	-	-	-	0	2	2
	Endometrial stromal polyp	-	-	-	9	6	6
	Endometrial stromal sarcoma	-	-	-	1	0	1
Preputial clitoral gland	Adenoma	2	3	1	0	0	1
	Squamous cell carcinoma	0	0	1	1	1	0
Lung	Adenoma	3	4	1	0	1	0
	Adenocarcinoma	0	0	0	1	0	0
Kidney	Lipoma	1	0	0	0	0	0
	Transitional cell papilloma	0	1	0	0	0	0
Skin/subcutis	Trichoepithelioma	0	0	0	1	0	0
	Keruloacanthoma	1	0	0	0	0	0
	Squamous cell papilloma	1	1	2	0	0	0
	Fibroma	1	1	3	0	0	1
	Lipoma	0	1	0	1	0	0
	Malignant schwannoma	0	0	1	1	0	0
Zymbal’s gland	Squamous cell papilloma	0	2	2	0	1	0
	Squamous cell carcinoma	0	0	0	0	0	1
Harderian gland	Adenoma	1	0	0	0	0	0
Abdominal cavity	Lymphocytic sarcoma	0	0	0	1	0	0
	Mesothelioma	3	3	2	0	0	0

Conclusions:

Therefore it is concluded that HMHC is not carcinogenic in F344 rats of either sex.

Executive summary:

In this study the long-term toxicity 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 or 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.

A variety of tumours occured in all groups, including the control group. However, the organ distribution and histological types of tumours were the same as those spontaneously occuring and described previously in F344 rats. No statistically significant increase in the incidence of any tumour was found in the treated groups of either sex.

Therefore, it was concluded that under the present experimental conditions, HMHC is not carcinogenic in F344 rats.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Read-across from L-histidine monohydrochloride (HMHC) to L-histidine is considered justified based on strong similarities with regard to chemical structure and metabolic pathways. Orally administered L-histidine will be protonated in the stomach under the influence of the hydrochloric acid present in the gastric juice. Hence, the systemic behaviour of both substances is identical.

Reason / purpose for cross-reference:

read-across source

Conclusions:

Read-across was done from L-histidine monohydrochloride (HMHC). Based on this result, and on the structural, chemical
and toxicological similarities between L-histidine monohydrochloride (HMHC) and L-histidine, it is concluded that L-histidine is not carcinogenic in F344 rats of either sex.

Executive summary:

Read-across was done from L-histidine monohydrochloride (HMHC).

In this study the long-term toxicity 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 monohydrochloride (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 or 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.

A variety of tumours occured in all groups, including the control group. However, the organ distribution and histological types of tumours were the same as those spontaneously occuring and described previously in F344 rats. No statistically significant increase in the incidence of any tumour was found in the treated groups of either sex.

Therefore it is concluded that HMHC is not carcinogenic in F344 rats of either sex.

Based on this result, and on the structural, chemical and toxicological similarities between L-histidine monohydrochloride (HMHC) and L-histidine, L-histidine is under the present experimental conditions considered as to be not carcinogenic in F344 rats of either sex.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

chronic

Species:

rat

Justification for classification or non-classification

The criteria used for the classification of carcinogenicity are depicted in Table 3.6.1 of the CLP regulation No 1272/2008.

As there was no evidence obtained from the carcinogenicity study in rats, HMHC cannot be placed in either category for carcinogens and thus the substance does not require classification acoording to the CLP regulation.

Based on the structural, chemical and toxicological similarities between L-histidine monohydrochloride (HMHC) and L-histidine, the same conclusion in regards to the classification according the CLP regulation applies to L-histidine and L-histidine can therefore not be placed in either category for carcinogens.

Additional information

Read-across was done from L-histidine monohydrochloride (HMHC).

In the study of Ikezaki (1996) rats were dosed with L-histidine monohydrochloridc (HMHC) for 104 weeks, similar to OECD 453. The concentrations were 0 [control], 1.25 and 2.5% in the diet for 104 weeks and correspond to 0, 472.9 and 955 mg/kg bw for male rats and 0, 558.4, 1105.2 mg/kg bw for females.

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 or male rats were similar in both treated and control groups.

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.

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

A variety of tumours occured in all groups, including the control group. However, the organ distribution and histological types of tumours were the same as those spontaneously occuring and described previously in F344 rats. No statistically significant increase in the incidence of any tumour was found in the treated groups of either sex.

Therefore, it was concluded that under the present experimental conditions, HMHC is not carcinogenic in F344 rats.

Based on this result, and on the structural, chemical and toxicological similarities between L-histidine monohydrochloride (HMHC) and L-histidine, L-histidine is under the present experimental conditions considered as to be not carcinogenic in F344 rats of either sex.

Justification for selection of carcinogenicity via oral route endpoint:
The study describes a chronic cancerogenicity test performed on rats which was carried out according to OECD 453.