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Description of key information

LOAEL (male/female): 1561 mg/kg bw/d

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
only two dose levels, limited documentation (clinical signs, gross pathology)
Reason / purpose:
reference to same study
Principles of method if other than guideline:
The carcinogenicity of glycine was assessed in rats by application in the drinking water.
GLP compliance:
no
Species:
rat
Strain:
Fischer 344/DuCrj
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Co. Japan, Inc. Atsugi
- Age at study initiation: 6 wks
- Housing: in plastic cages (3/sex/cage)
- Diet (ad libitum): pellet diet (CRF-1, Charles River, Japan)
- Water (ad libitum)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 2
- Humidity (%): 55 ± 5
Route of administration:
oral: drinking water
Vehicle:
unchanged (no vehicle)
Duration of treatment / exposure:
108 wks
Frequency of treatment:
continuously via drinking water
Dose / conc.:
2.5 other: % (nominal in water)
Remarks:
equivalent to 1534 mg/kg bw/day for males and 1587 mg/kg bw/day for females (actual ingested)
Dose / conc.:
5 other: % (nominal in water)
Remarks:
equivalent to 3280 mg/kg bw/day for males and 3082 mg/kg bw/day for females (actual ingested)
No. of animals per sex per dose:
50
Control animals:
other: drinking water without test substance
Details on study design:
- Dose selection rationale: on the basis of a preliminary subacute experiment (not further specified)
Observations and examinations performed and frequency:
BODY WEIGHT: Yes
- Time schedule for examinations: every one or two weeks

FOOD CONSUMPTION: Yes
- Time schedule for examinations: every four weeks

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at week 108
- How many animals: all survivors
- Parameters checked: white/red blood cells, heamoglobin (Hb), haematocrit (Ht)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at week 108
- How many animals: all survivors
- Parameters checked: glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), creatine phosphokinase (CPK), blood urea nitrogen (BUN), creatinine (CRE), Urea (UA), total protein (TP), albumin/globulin rate (A/G rate), blood glucose (BG), phospholipid (PL), total cholesterol (Tch), triglyceride (TG), natrium, potassium, calcium, chloride

URINALYSIS: Yes
- Parameters checked: leukocytes, nitrite, urobilinogen, protein, pH, ketone, glucose
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all animals, including those that died or were sacrificed upon becoming moribund during the experiment)
HISTOPATHOLOGY: Yes (all organs were examined and samples were fixed in 10% buffered neutral formalin, embedded in paraffin, and routinely stained with hematoxylin and eosin; histological analysis of tumors in renal pelvis)
Statistics:
Wilcoxon t-test or chi-squared test
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
5% dose group: slightly increased mortality in females compared to controls
Mortality:
mortality observed, treatment-related
Description (incidence):
5% dose group: slightly increased mortality in females compared to controls
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
2.5 and 5% dose group: slight dose-dependent inhibition of mean body weight gain in both sexes
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
5% dose group: significant changes of Hb and HT in males and females
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
2.5 and 5% dose group, males and females: significantly changes in CPK, BUN, TP, Tch and TG were observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
2.5 and 5% dose group: papillary necrosis and transitional hyperplasia was observed in male and female animals (no cases seen in the controls)
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
2.5 and 5% dose group, females: kidney papillomas were seen, which were not observed in controls
Details on results:
CLINICAL SIGNS AND MORTALITY
Mortality of females of the high dose group was slightly increased when compared to controls (number of surviving females and males: 31, 46, 41 and 40, 45, 40 for the high, low, control group).

BODY WEIGHT AND WEIGHT GAIN
2.5 and 5% dose group, males and females: Mean body-weights were slightly but dose-dependently inhibited when compared to controls over the hole study period (only growth curves were given).

HAEMATOLOGY
5% dose group, males: A slight but not significant increase in white blood cells was observed. The Hb was significantly increased when compared to controls.
5% dose group, females: A slight but not significant increase in white blood cells and a slight but not significant decrease in red blood cells was observed. The Hb and HT were significantly decreased when compared to controls.
2.5% dose group, females: A slight but not significant increase in white blood cells and a slight but not significant decrease in red blood cells was observed.

CLINICAL CHEMISTRY
2.5 and 5% dose group, males and females: The CPK values were significantly decreased when compared to controls in both sexes and both dose groups. BUN was significantly increased in both sexes of the 5% dose group.
2.5 and 5% dose group, females: A significant and dose-dependent decrease of TP and Tch and a significant and dose-dependent increase in TG was observed.
Some sporadic effects on isolated parameters were observed which were considered as not treatement-related (see Table 1 "Any other information on results incl. tables").

URINALYSIS
No significant differences in urinalysis data between treated and control male and female animals were observed. The only change was increased numbers of erythrocytes in glycine-treated male and female rats. This was expected from haemorrhage in necrotic renal papillae (see histopathology).

ORGAN WEIGHTS
No significant effects on the relative organ weights were observed in the treated groups when compared to controls.

HISTOPATHOLOGY: NON-NEOPLASTIC
The incidences of papillary necrosis in the kidney of male rats fed 2.5 and 5% glycine were 4 and 7%, while the corresponding figures for females were 30 and 32%. Since necrosis of the renal papillae was limited to glycine-treated rats it was very likely linked to the induction of papillomas. Transitional hyperplasia in renal pelvis was observed in 2 female animals of the 2.5% dose group. Calcification indices in kidney were 39, 32 and 42% of the 0, 2.5 and 5% groups. Endometrial polyps in the uterus were increased in both treatment group females (see Table 2/3 "Any other information on results incl. tables").
Overall, for the males, tumors were found in 90% of the high dose group, 97% of the low dose group and in 90% of the controls, while the corresponding figures for females were 59, 65 and 39%. These tumors were considered to be spontaneous in nature (see Table 3 "Any other information on results incl. tables").

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
One renal cell tumor was seen in one male of the high dose group. Renal pelvis papillomas were found in 8% of the low dose group and in 6% of the high dose group females, but not in the controls. Since spontaneous tumors in this tissues are very rare, they were considered as treatment-related (see Table 3 "Any other information on results incl. tables").
Dose descriptor:
LOAEL
Remarks:
carcinogenicity
Effect level:
1 561 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: histopathology
Dose descriptor:
NOAEL
Remarks:
carcinogenicity
Effect level:
< 1 561 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: histopathology
Critical effects observed:
not specified

Table 1: Clinical chemistry data for rats treated with the test substance (mean values ± standard deviation)

 

Males

Females

Group (%)

0

2.5

5

0

2.5

5

No. of rats

40

45

40

41

46

31

GOT (K-U)

122 ± 72

120 ± 57

114 ± 27

130 ± 53

117 ± 40

136 ± 68

GPT (K-U)

51 ± 34

55 ± 37

53 ± 12

59 ± 22

56 ± 12

66 ± 16

LDH (K-U)

2879 ± 772

2710 ± 972

2514 ± 819

2572 ± 1369

2666 ± 836

2556 ± 952

ALP (KA-U)

16 ± 6

15 ± 5

17 ± 5

19 ± 9

19 ± 5

25 ± 7*

CPK (IU/L)

754 ± 321

563 ± 217*

509 ± 197*

706 ± 392

545 ± 160*

446 ± 158*

BUN (mg/dL)

18 ± 3

17 ± 2

21 ± 3*

18 ± 2

17 ± 2

21 ± 3*

CRE (mg/dL)

0.6 ± 0.07

0.5 ± 0.10*

0.5 ± 0.11*

0.5 ± 0.06

0.5 ± 0.06

0.5 ± 0.07

UA (mg/dL)

2.3 ± 1.5

1.9 ± 0.6

2.5 ± 1.3

2.8 ± 1.8

2.0 ± 0.9*

2.5 ± 1.7

TP (g/dL)

6.9 ± 0.5

6.6 ± 0.3*

6.8 ± 0.4

7.5 ± 0.7

7.1 ± 0.5*

6.8 ± 0.7*

A/G (rate)

0.6 ± 0.09

0.7 ± 0.06*

0.7 ± 0.06*

0.8 ± 0.07

0.8 ± 0.06

0.8 ± 0.09

BG (mg/dL)

131 ± 46

127 ± 30

140 ± 51

136 ± 35

127 ± 15

125± 27

PL (mg/dL)

216 ± 62

205 ± 54

214 ± 61

211 ± 37

205 ± 36

202 ± 34

Tch (mg/dL)

150 ± 51

130 ± 39

129 ± 42

117 ± 22

104 ± 19*

98 ± 17*

TG (mg/dL)

175 ± 82

162 ± 59

198 ± 78

154 ± 73

209 ± 102*

226 ± 105*

Na (mEq/L)

144 ± 2

141 ± 2*

143 ± 2

141 ± 3

139 ± 1*

141 ± 4

K (mEq/L)

5.3 ± 0.7

4.6 ± 0.8*

5.3 ± 1.0

5.0 ± 0.9

4.4 ± 0.5*

4.7 ± 0.8

Ca (mg/dL)

11.1 ± 0.9

10.3 ± 0.5*

11.2 ± 1.1

11.2 ± 1.4

10.6 ± 0.6*

10.7 ± 1.1

Cl (mEq/L)

99 ± 1.9

101 ± 2.3*

99 ± 3.5

99 ± 2.0

98 ± 2.3

97± 4.3

*p<0.05, compared to control

Table 2: Histological findings for the urinary system for rats treated with the test substance

No. of affected rats (%)

 

Males

Females

Test substance concentration (%)

0

2.5

5

0

2.5

5

Effective No. of rats

40

45

40

41

46

31

Urinary system

 

 

 

 

 

 

Kidney

 

 

renal cell tumor

0

0

1(2)

0

0

0

papillary necrosis

0

2(4)

3(7)

0

14(30)

10(32)

calcification

0

0

0

16(39)

15(32)

13(42)

Pelvis

 

 

transitional hyperplasia

0

0

0

0

2(4)

0

transitional cell tumor

1(2)

0

0

0

0

0

papilloma

0

0

0

0

4(8)

2(6)

Table 3: Organ distribution and histological diagnosis for tumors in rats treated with the test substance

 

No. of affected rats (%)

 

Males

Females

Test substance concentration (%)

0

2.5

5

0

2.5

5

Effective No. of rats

40

45

40

41

46

31

Tumor development

 36(90)

44(97) 

36(90) 

 27(59)

30(65) 

12(39) 

Genital system

Testis

interstitial cell tumor

35(87)

44(97)

36(90)

-

-

-

Prostate

adenoma

1(2)

2(4)

2(4)

-

-

-

calcification

-

-

-

-

-

-

Uterus

endometrial polyp

-

-

-

5(12)

18(39)

12(38)

endometrial sarcoma

-

-

-

0

1(2)

0

adenocarcinoma

-

-

-

1(2)

0

0

Mammary gland

firboma

0

0

0

1(2)

0

0

adenoma

0

0

0

0

1(2)

1(3)

Endocrine system

Pituitary gland

adenoma

9(22)

6(13)

7(17)

13(31)

12(26)

9(29)

Thyroid gland

C-cell adenoma

4(10)

1(2)

3(7)

4(9)

3(6)

2(6)

C-cell carcinoma

0

2(4)

1(2)

1(2)

1(2)

0

papillary adenoma

1(2)

0

1(2)

0

2(4)

0

papillary adenocarcinoma

0

1(2)

0

0

0

0

Adrenal gland

pheochromocytoma

5(13)

7(15)

5(13)

0

1(2)

0

Pancreas

islet cell tumor

6(15)

1(2)

2(4)

0

1(2)

0

Digestive system

Liver

neoplastic nodule

1(2)

0

0

0

0

1(3)

Respiratory system

Lung

adenoma

0

2(4)

0

0

1(2)

0

Urinary system

Kidney

renal cell tumor

0

0

1(2)

0

0

0

transitional cell tumor

1(2)

0

0

0

0

0

papilloma

0

0

0

0

4(8)

2(6)

Urinary bladder

papilloma

0

0

0

0

0

0

Body cavity

Peritoneum

mesothelioma

2(5)

2(4)

1(2)

0

0

0

Hematopoietic system

Leukemia

 

4(10)

6(13)

3(7)

6(14)

9(17)

3(9)

Integument, musculoskeletal system

Subcutis

fibroma

3(8)

0

0

4(10)

2(4)

0

 

lipoma

0

1(2)

0

0

0

1(2)

 

preputial/clitoral gland tumor

3(6)

2(4)

2(4)

0

0

1(2)

 

fibrosarcoma

0

0

1(2)

0

0

0

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 2), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.9.1, of Regulation (EC) No 1907/2006.

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Carcinogenicity of glycine after oral administration to male and female Fischer 344/DuCrj rats was investigated by Kitahori et al. (1994). The unchanged test substance was applied continuously via drinking water at 2.5 and 5% for 108 weeks to 50 animals per sex. The concentrations used correspond to 1534 and 3280 mg/kg bw/day for males and to 1587 and 3082 mg/kg bw/d for females, respectively, based on the body weights and water consumption data given in the publication. Control animals received drinking water without the test substance. Dose concentrations were chosen on the basis of a preliminary subacute experiment (unspecified). Observations and examinations comprised body weight development, food and water consumption, haematology, clinical chemistry, urinalysis, gross pathology and histopathology.

Mortality of the high dose group females was slightly increased when compared to controls (31 vs. 41 surviving animals). Mean body weights of both sexes were slightly and dose-dependently inhibited over the whole study period when compared to controls. In the high dose group a slight, but not statistically significant increase in white blood cells was observed in both sexes when compared to controls. In addition, a slight decrease in red blood cells and a statistically significant decrease of the haemoglobin and haematocrit levels was observed in females. In the low dose group females showed a slight, but not statistically significant increase in white blood cells and decrease in red blood cells was observed when compared to controls. In both sexes of both dose groups, the creatine phosphokinase values were significantly lower when compared to controls. Blood urea nitrogen was significantly increased in both sexes of the high dose group. In females of both dose groups, a significant and dose-dependent decrease of total protein and total cholesterol and a significant and dose-dependent increase in triglycerides was observed. Some sporadic effects on isolated parameters were even more observed with regard to clinical chemistry which were considered as not treatement-related.

The incidences of papillary necrosis in male rats fed 2.5 and 5% glycine were 4 and 7%, while the corresponding figures for females were 30 and 32%. Since necrosis of the renal papillae was limited to glycine-treated rats it was very likely linked to the induction of papillomas. Transitional hyperplasia in renal pelvis was observed in 2 female animals of the low dose group. Calcification indices in kidney were 39, 32 and 42% in females of the control, 2.5 and 5% groups, respectively. These effects were considered as non-neoplastic histopathology findings. Overall, tumors in males were found in 90% of the high dose group, 97% of the low dose group and in 90% of the controls, while the corresponding figures for females were 39, 65 and 59%. These tumors were considered to be spontaneous in nature.

With regard to neoplastic histopathology in males, one renal cell tumor was seen in one animal of the high dose group. Renal pelvis papillomas were found in 8% of the low dose group and in 6% of the high dose group females, but not in the controls. Since spontaneous tumors in this tissues are very rare, they were considered as treatment-related. Necrosis of the renal papillae was also limited to glycine-treated rats and were therefore very likely to be linked to the induction of papillomas. Besides necrosis, calcification was observed in female rats. Since untreated and treated animals were affected, calcification in the kidney of females can rather not be linked to the formation of renal pelvis papillomas. Probably, calcification is a common effect in aged female rats. Yasukata et al. (1985) smilarly reported phytic-acid induced calcification in the kidneys. In their study, calcification was also present in the untreated controls. Subchronic intraperitoneal glycine administration was reported by Vasudevan et al. (1987) to result in a 44% incidence of hyperplastic, preneoplastic, and neo-plastic lesions in the urinary bladder (cancer 1/9, papillomas 3/9, and hyperplasia 1/9). The urinary bladder of glycine-treated rats within this study was exposed to high levels of orotic acid as a result of an enhanced urea cycle due to the high glycine levels. High levels of orotic acid were considered to be involved in the tumor formation within this study. The crystalline structure of orotic acid and the subsequent permanent irritating properties to tissues, might be a reason for tumor formation, especially in the renal pelvis, which serves as collecting funnel for the urine where transit time might be high. Oxalic acid is a metabolite after glycine degration. It exists in crystalline shape in aqueous solutions and might therefore be irritating to the renal-bladder-tract, too, leading to the overall formation of renal pelvis papillomas. However, it remains unexplained why the effect is restricted to female animals.

In summary, the hypothesized mechanim is a result of a unrealistic, high glycine uptake. Renal pelvis tumors were observed at administered doses of >1000 mg/kg bw/d, but daily glycine uptake in adult humans is 3 - 5 g, corresponding to 40 - 70 mg/kg bw (body weight of 75 kg). Therefore, renal pelvis papilloma formation is not assumed to be relevant for humans.

References

Yasukata J., Shigihara S., Ichikawa M. and Tomita H. (1985) The influence of food additives on zinc concentration in organs of rats. Biryou Kinziku Taisha 133: 13 -22

Vasudevan S., Laconi E., Abanobi S.E., Rao P.M., Rajalakshmi S. and Sarma D.S. (1987) Effect of glycine on the induction of orotic aciduria and urinary bladder tumorigenesis in the rat. Toxicol Pathol 15: 194 -197

Justification for classification or non-classification

The available data on carcinogenicity do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.