Adenine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1979

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

only males, 12 males/group, administration period was 36 days

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Adenine

EC Number:

200-796-1

EC Name:

Adenine

Cas Number:

73-24-5

Molecular formula:

C5H5N5

IUPAC Name:

adenine

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: ARS Sprague Dawley, Madison, Wisconsin, USA
- Age at study initiation: adult
- Weight at study initiation: 150 - 200 g
- Housing: individually
- Diet: Purina Chow, ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- not specified (constant temperature-humidity environment)

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

36 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

35 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

70 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

130 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

196 mg/kg bw/day (nominal)

Doses / concentrations 6

Dose / conc.:

286 mg/kg bw/day (nominal)

Doses / concentrations 7

Dose / conc.:

367 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 male/group

Control animals:

yes, concurrent no treatment

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 and 36

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the experimental period
- Anaesthetic used for blood collection: No
- How many animals: all surviving animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the experimental period
- How many animals: all surviving animals

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Other examinations:

The systolic blood pressure of the rat was measured.

Statistics:

The Student’s t-test was used.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

High doses of adenine produced gradual ataxia, muscle weakness, and dyspnea. Most animals experienced convulsions prior to death. Several animals succumbed as the result of respiratory failure.

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortality was observed up to 70 mg/kg bw/d dose group. In the 130 mg/kg/d dose group two animals died between day 21 and 36. In the 196 mg/kg/d dose group four animals died between day 11 and 20. In the 286 mg/kg/d dose group eight animals died. In the 367 mg/kg/d dose group all animals died during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Control animals gained an average of 27% in total body weight during the 36-day period. Adenine was shown to have a marked and significant effect upon growth as measured by body weight increase. At the lowest level of adenine there was approximately 30% inhibition of normal weight increase. In the 70 and 130 mg/kg/d dose group, ca. 50% inhibition of weight gain was observed. In the two highest dose groups, a loss of weight was observed.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A reduced food intake was observed from the 196 mg/kg bw/d dose group upwards.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Adenine levels less than 0.1% failed to change the erythrocyte count, hemoglobin, hematocrit, and mean corpuscular volume. The erythrocyte count, hemoglobin, and hematocrit increased 37, 21, and 23 %, respectively at 0.8% dietary level. A decrease of approximately 10% was noted in the mean corpuscular volume. The number of leukocytes in the peripheral blood was increased at all levels of dietary adenine. At 0.4 and 0.8 % adenine the number of leukocytes in the peripheral blood was more than double that of controls.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

All parameters showed a significant change when the level of dietary adenine exceeded 0.4%. It is interesting to note that the blood urea nitrogen was essentially normal at low dietary levels of adenine but significantly elevated at levels greater than 0.1%.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Kidneys gradually increased in weight and more than doubled the weight of control kidneys when 0.8 % adenine was present in the diet. At the 1.6% adenine level the weight of the kidney was only slightly increased and appeared hemorrhagic.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopically, the kidneys at the 0.05 % level appeared normal. At the 0.1 % level most kidneys were normal in size and weight. The kidneys of three animals appeared slightly pale. At 0.2% adenine gross pathological changes were clearly obvious. All kidneys examined were larger and weighed more than normal kidneys. Large grey spots were noted on the cortical surface. When dietary adenine exceeded 0.2% the most striking pathological kidney change was its enormous size. The kidneys appeared mottled, grey, granular, edematous, and softer than normal.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

On cross-section radiating white bands appeared throughout the medulla and extended to the papilla. The cortical layer was greatly thickened and discolored.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Rats treated with greater than 0.2% dietary adenine developed a substantial increase in systolic blood pressure but no significant EKG changes after 2 weeks of feeding.

Effect levels

1

Key result

Dose descriptor:

other: LD50

Effect level:

227 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

mortality

2

Dose descriptor:

LOAEL

Effect level:

35 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

130 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Table 1: The Mortality of Rats on Various Levels of Dietary Adenine Base

Adenine Base		Mortality	Days of Death
%	mg/kg bw/d		1 – 10	11 – 20	21 - 36
0.05	35 ± 4.1	0/12	-	-	-
0.1	70 ± 5.4	0/12	-	-	-
0.2	130 ± 17.0	2/12	-	-	2
0.4	196 ± 76.0	4/12	-	4	-
0.8	286 ± 133.0	8/12	4	2	2
1.6	367 ± 220.0	12/12	6	6	-
Values represent the mean ± SE.

Table 2: Relative Body Weight Gain and Food Intake in Rats on Dietary Adenine Base

Adenine base (% in diet)	Consumption per day (average)		Weight (g) (average)		Change“ (%)
	Water (mL)	Food (g)	Initial	Final
Control	27.2 ± 10.2	11.5 ± 3.1	161 ± 18.4	219 ± 9.7	27 ± 7.0
0.05	26.5 ± 2.5	12.3 ± 1.4	173 ± 5.4	215 ± 21.7	19 ± 7.0*
0.1	28.3 ± 3.9	12.3 ± 0.9	174 ± 2.9	211 ± 11.7	17 ± 5.0**
0.2	40.0 ± 4.9**	11.3 ± 1.6	175 ± 11.0	203 ± 25.0	13 ± 10.0**
0.4	40.0 ± 14.0**	9.6 ± 2.0*	170 ± 3.5	156 ± 35.0	9 ± 19.0**
0.8	50.0 ± 10 0**	8.0 ± 1.1**	175 ± 15.0	148 ± 9.0	-16 ± 10.0**
1.6	31.0 ± 10.0*	5.3 ± 1.1**	169 ± 6.0	137 ± 10.1	-19 ± 6.0
Values represent mean percentage change in body weight with reference to respective control animals +SE. Twelve animals were used at each dose level. * Significantly different from control values, p <0.05. ** Significantly different from control values, p <0.01.

Table 3: Body and Relative Kidney Weights of Rats on Dietary Adenine Base

Adenine base (% in diet)	Body weight (g)	Kidney weight (g)	% of Bodyweight
Control	220 ± 9.8	2.4 ± 0.12	1.1 ± 0.04
0.05	215 ± 12.7	2.6 ± 0.10	1.2 ± 0.06
0.1	211 ± 11.7	2.7 ± 0.20	1.3 ± 0.04*
0.2	212 ± 14.1	3.7 ± 0.22	1.6 ± 0.11*
0.4	149 ± 35.0	4.2 ± 0.36	2.9 ± 0.38**
0.8	154 ± 9.0	5.6 ± 0.41	3.6 ± 0.15**
1.6	133 ± 10.0	2.7 ± 0.13	2.0 ± 0.16**
Values represent mean percentage change in body weight with reference to respective control animals +SE. Twelve animals were used at each dose level. * Significantly different from control values, p <0.05. ** Significantly different from control values, p <0.001.

Table 4: Hematological Values for Rat Plasma on Dietary Adenine Base

No of animals	Adenine Base (%)	WBC (x10E3)	RBC (10E6)	HGB (g/100 mL)	HCT (%)	MCV (m³)
12	Control	4.4 ± 0.87	6.2 ± 0.3	13.2 ± 0.3	32.9 ± 2.0	52.5 ± 1.2
12	0.05	6.6 ± 1.2**	6.87 ± 0.7	14.2 ± 0.3	34.5 ± 3.0	49.8 ± 2.4
12	0.1	8.3 ± 1.2***	6.4 ± 0.3	13.9 ± 0.4	33.0 ± 1.5	49.7 ± 1.5
12	0.2	7.6 ± 1.6***	5.7 ± 0.5*	11.9 ± 0.5*	29.0 ± 2.5*	50.8 ± 2.1
7	0.4	10.3 ± 0.8***	5.7 ± 0.9*	12.0 ± 0.6*	28.6 ± 3.6*	49.0 ± 1.4
6	0.8	11.2 ± 1.0***	8.5 ± 2.8***	16.0 ± 5.2***	40.7 ± 15.8***	47.0 ± 2.2**
Values represent the mean ± SE. * Significantly different (p<0.05) from control values ** Significantly different (p<0.01) from control values *** Significantly different (p<0.001) from control values

Table 5: Clinical Chemistry Values of Rat Plasma on Dietary Adenine Base

No of animals	Adenine Base (%)	Ca (mg/100 mL)	P (mg/100 mL)	Glucose (mg/100 mL)	Uric acid (mg/100 mL)	Alb (mg/100 mL)	Alk phos(mU/mL)	BUN (mg/100 mL)
12	Control	9.7 ± 0.8	5.2 ± 0.8	160 ± 10	2.2 ± 0.7	3.5 ± 1.0	210 ± 26	24.5 ± 3.1
12	0.05	10.1 ± 1.3	5.5 ± 0.9	165 ± 12	1.8 ± 0.6	4.1 ± 1.1	220 ± 31	24.5 ± 2.8
12	0.1	9.7 ± 1.1	5.7 ± 0.9	165 ± 12	2.5 ± 0.8	3.8 ± 0.8	225 ± 46	28.0 ± 3.8
12	0.2	10.0 ± 2.0	5.6 ± 2.0	160 ± 12	2.5 ± 0.5	34 ± 0.8	210 ± 21	37.0 ± 4.2
7	0.4	10.5 ± 2.1	5.9 ± 1.0	140 ± 16 *	2.3 ± 0.4	2.4 ± 0.6	200 ± 30	94.0 ± 10.6
6	0.8	4.7 ± 1.2**	4.6 ± 0.8	120 ± 18**	2.0 ± 0.8	1.7 ± 0.8**	80 ± 12**	10.0 ± 16.1**
Values represent the mean ± SE. * Significantly different (p<0.01) from control values ** Significantly different (p<0.001) from control values

Applicant's summary and conclusion

Conclusions:

In an in vivo repated dose toxicity study in rat similar to OECD 407, an LD50 oral of 227 mg/kg bw was determined.

Executive summary:

The subacute toxic effects (36 days) of the test item (0.05, 0.10, 0.20, 0.40 0.80 and 1.6% of the diet, resembling 35, 70, 130, 196, 286, and 367 mg/kg bw/d) was investigated in young male SD rats. Animals consumed normal amounts of food and water at low test item levels (0.05-0.10%). Higher levels (>0.20%) significantly reduced food consumption and increased water intake. Evidence of gross pathology in soft tissues was absent at 0.05 to 0.10% dose group. Levels greater than 0.10% produced definite pathological changes in the kidney. The kidney was greatly enlarged and its absolute weight increased more than 2x normal. The enlarged kidney were granular, edematous, greyish, and soft. A significant change in the blood urea nitrogen occurred when the level of the test item approached 0.20%. No significant changes were found in blood uric acid at all dietary levels. Low levels did not change the hematological indices with the exception of the leukocyte count. A significant leukocytosis occurs at all levels (0.05-1.6%) of adenine. An increase in systolic blood pressure but no change in EKG was noted at levels >0.20% adenine. An oral LD50 of 227 mg/kg bw/d was determined.