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Carcinogenicity

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Description of key information

Not carcinogenic by oral route in mice and rats (NTP, 1992, Key, rel.1)

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 03, 1981 to November 04, 1983
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
GLP study conducted similarly to OECD Guideline 451 without deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: GAF Corporation (New York, NY) / Lot 600-BLO
- Physical state: Clear, colorless liquid
- Analytical purity: > 97%
- Impurities (identity and concentrations): 11 impurities were identified by GC method, there is no data on chemical identity of these impuries.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in the dark at 5 °C at the study laboratory.
- Stability under test conditions: Stability studies performed by the analytical chemistry laboratory using gas chromatography indicated that y-butyrolactone was stable as a bulk chemical for at least 2 weeks at temperatures to 60 °C. The stability of the bulk chemical was monitored by the study laboratory using gas chromatography and infrared absorption periodically during all phases of the studies. No change in the study material was detected.
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Frederick Cancer Research Facility (Frederick, MD)
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Males -55 days; Females -62 days
- Weight at study initiation: At week 1, approx. 23.7 g (males) / 18.5 g (females)
- Fasting period before study: No
- Housing: Animals were housed 5/cage (male mice were housed individually from February 9, 1983 and female mice were housed individually from July 1, 1983) in solid-bottom polycarbonate cages (Lab Products, Inc., Garfield, NJ)
- Diet: NIH-07 Rat and Mouse Ration, Open formula, pellets (Zeigler Bros., Inc., Gardners, PA), available ad libitum
- Water: Tap water (Birmingham Water Works) via outside-the-cage automatic watering system (Edstrom Industries, Inc., Waterford, WI), available, ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature: 16-29 °C
- Humidity: 25-79 %
- Air changes: Minimum of 15 per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: November 03, 1981 to November 04, 1983
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared by mixing appropriate amounts of test item and corn oil to give the required concentrations. Dose formulations were prepared weekly and discarded 2 weeks after the date of preparation. During the studies, the dose formulations were stored in sealed amber serum vials in the dark at 5 °C for no longer than 2 weeks.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw

STABILITY
Gas chromatography confirmed the stability of the dose formulations when stored 14 days in the dark at temperatures to 25 °C.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the test item dose formulations were performed using gas chromatography. The dose formulations were analyzed at approximately 8-week intervals; 96% (27/28) of the dose formulations for mice were within 10% of the target concentrations.
Duration of treatment / exposure:
103 weeks
Frequency of treatment:
5 days/week
Post exposure period:
1 week
Dose / conc.:
262 mg/kg bw/day (actual dose received)
Dose / conc.:
525 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
50
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses selected for 2 year study for mice were 262 and 525 mg/kg bw/day, which were based on the chemical related mortality observed in male mice (3/10) and in female mice (1/10) at 1050 mg/kg bw/day in a 13 week study.
Rationale for animal assignment (if not random): Animals were grouped by weight intervals, then groups were assigned to cages. A table of random numbers was used to assign cages to treatment groups.
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed at study initiation, weekly for 13 weeks, and monthly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed at study initiation, weekly for 13 weeks, and monthly thereafter.

FOOD CONSUMPTION: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
NECROPSY: Animals found moribund or surviving to the end of the 2-year studies were killed. Necropsy was performed on all animals.

GROSS PATHOLOGY: Yes; At necropsy, all organs and tissues were examined for gross lesions.

HISTOPATHOLOGY: Yes; all major tissues were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned, and stained with haematoxylin and eosin for microscopic examination. Complete histopathlogical examinations were performed on all control, all high-dose and low dose male mice. Selected tissues were examined from low-dose female mice. Histopathology examinations were performed on all grossly visible lesions in all dose groups.
Other examinations:
None
Statistics:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Animals found dead of other than natural causes or found missing were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided. The incidences of neoplasms or non-neoplastic lesions were given as the numbers of animals bearing such lesions at a specific anatomic site to the numbers of animals with that site examined microscopically. Analysis of tumor incidence was performed using logistics regression analysis. Other tests used were life table test (Cox; 1972; Tarone, 1975), Fisher exact test and the Chochran-Armitage trend test (Armitage, 1971; Gart et. al., 1979). For analysis of continuous variables tests performed include Dunnett (1955) and Williams (1971, 1972), and Jonckheere’s test (Jonckheere, 1954).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
High dose (525 mg/kg bw/day) males and females were slightly lethargic after dosing.
Mortality:
mortality observed, treatment-related
Description (incidence):
High dose (525 mg/kg bw/day) males and females were slightly lethargic after dosing.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
High dose (525 mg/kg bw/day) females had reduced final mean body weights at the end of the study.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Low dose (262 mg/kg bw/day) males had statistically significant increased incidence of proliferative lesions, primarily hyperplasia, of the adrenal medulla.
Other effects:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
High-dose male mice were partially sedated or lethargic and inactive shortly after dosing; There was a significantly lower survival of high-dose male mice. This was attributed partially to fighting during the first year of the study, when the animals were housed in groups of five (males were housed individually after approximately 66 weeks on study). Survival of low- and high-dose female mice was similar to controls.
Survival rate for male mice: 35/50 (control), 30/50 (262 mg/kg bw/day) and 12/50 (525 mg/kg bw/day)
Survival rate for female mice: 38/50 (control), 34/50 (262 mg/kg bw/day) and 38/50 (525 mg/kg bw/day)

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of low- and high-dose male mice followed a similar pattern throughout the study and were consistently lower than the mean body weights of the controls. The decrement in bodyweight gain was evident as early as week 3 and continued to increase until approximately week 66. Mean body weights of low- and high-dose males were within 10% of the mean body weight of the controls through week 27; from week 32 to week 66 the decrement increased to a maximum of 17%. During week 67, all male mice were housed individually; thereafter, the difference between the mean body weights of dosed males and control mice decreased. By the end of the study, the final mean body weights of low- and high-dose male mice were only 6% less than that of the controls.
In female mice, the mean body weights of both dose groups were within 10% of those of the controls through week 27. Thereafter, weight gains of low-and high-dose females steadily declined relative to controls, and the differences did not diminish after the females were housed individually at week 87. At the end of the study, the final mean body weights of low-and high-dose female groups were 17% and 14% lower than that of the controls.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no non-neoplastic degenerative lesions associated with the administration of test item. Decreases in a number of miscellaneous spontaneous legions in low- and high dose male mice were attributed to decreased survival and were not considered related to test item. The observed dose-related increases in several non-neoplastic lesions in male mice were considered to be associated with fighting or bite wounds.

HISTOPATHOLOGY: NEOPLASTIC
Adrenal Medulla: There was a statistically significant increase in the incidence of focal hyperplasia in low-dose male mice. Moreover, there was a marginal increase in the incidence of pheochromocytomas (benign or malignant combined) in low-dose male mice compared to controls, although neither the trend test nor the pairwise comparison was statistically significant. Because focal hyperplasia and pheochromocytomas constitute a morphological and biological continuum, the increased incidence of these lesions, principally hyperplasia, may be related to test item administration. The lack of a dose response may be related to the reduced survival in the high-dose group. In female mice, there was no apparent increase in the incidence of adrenal medulla proliferative lesions associated with the administration of test item (hyperplasia: control, 3/50; high-dose, 1/49; pheochromocytoma, benign or malignant: 0/50, 2/49).

Liver: Hepatocellular adenomas or carcinomas (combined) occurred with a statistically significant negative trend in male mice, and the incidences in low- and high dose groups were significantly lower than the incidence in the controls by survival-adjusted analyses (24/50, 8/50, 9/50). The overall incidence of hepatocellular neoplasms in NTP historical control males receiving corn oil by gavage is 210/599 (35.1%, range 14%-52%).

Harderian Gland: Adenomas in male mice occurred with a statistically significant negative trend, and the incidences in low-and high-dose groups were significantly less than controls by survival-adjusted analyses (8/50, 1/50, 0/50). The overall historical control incidence of this tumor in males is 38/600 (6.3%) with a range of 0% to 16%. Thus, the significance of the decrease may be due to the rather high incidence in controls in this study, rather than to the administration of test item. The incidence of harderian gland neoplasms in low- and high-dose female mice was not decreased (2/50, 2/50, 4/50).

Miscellaneous Non-neoplastic Lesions: Decreases in a number of miscellaneous spontaneous non-neoplastic lesions in low- and high-dose male mice were attributed to decreased survival and were not considered related to test item administration. The observed dose-related increases in several non-neoplastic lesions in male mice were considered to be associated with fighting or bite wounds. The skin lesions were primarily located around the genitalia and backs, and the lymphoid hyperplasia of the inguinal lymph node was considered to be an immunological response to superficial bacterial infections of the bite wounds. Prostatitis is frequently seen in group-housed male mice and is believed to be the result of ascending bacterial infections resulting from bite wounds on and around the genitalia. Depletion of lymphocytes from the thymus (also called thymic atrophy) often accompanies debilitation and stress and was usually seen in mice dying early from fight wounds. The leukocytosis, hemorrhage, and congestion of the lung were also seen principally in males dying early from fight wounds.
Key result
Dose descriptor:
NOAEL
Effect level:
525 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: neoplastic
Remarks on result:
other: carcinogenicity
Key result
Dose descriptor:
LOAEL
Effect level:
262 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
Remarks on result:
other: toxicity
Key result
Critical effects observed:
no

Table 7.7/1: Mean body weights and survival of male mice

Weeks on Study

 

Vehicle Control

262 mg/kg bw/day

525 mg/kg bw/day

Av. Wt. (g)

Number of Survivors

Av. Wt. (g)

Wt. (% of controls)

Number of Survivors

Av. Wt. (g)

Wt. (% of controls)

Number of Survivors

1

23.6

50

24.1

102

50

23.7

100

50

2

25.8

50

25.9

100

50

26.3

102

50

3

28.8

50

27.4

95

50

27.0

94

50

4

29.1

50

28.5

98

50

27.9

96

50

5

30.1

50

29.8

99

50

28.8

96

50

6

31.3

50

29.0

93

50

30.0

96

50

7

32.4

50

31.7

98

50

30.9

95

50

8

33.1

50

31.9

96

50

31.5

95

50

9

33.9

50

32.4

96

50

32.2

95

50

10

34.6

50

33.5

97

50

32.8

95

50

11

35.1

50

34.2

97

50

34.0

97

49

12

35.7

50

34.7

97

50

34.4

96

49

13

36.1

50

34.6

96

50

33.6

93

49

15

36.7

50

35.4

97

50

35.2

96

48

18

37.5

50

35.3

94

50

36.0

96

44

22

39.9

50

37.2

93

48

37.5

94

44

27

41.9

49

38.0

91

47

38.7

92

38

32

44.3

49

39.8

90

46

39.0

88

36

35

44.4

49

39.2

88

46

39.6

89

36

40

46.0

49

40.8

89

45

40.2

87

36

44

45.8

49

40.7

89

45

38.6

84

36

48

46.1

49

40.6

88

45

40.9

89

35

52

48.1

49

41.4

86

44

41.7

87

35

56

48.9

48

42.1

86

43

41.0

84

33

58

48.7

48

40.6

83

40

41.4

85

33

62

49.9

48

41.4

83

39

41.7

84

32

66

50.0

47

42.7

85

38

41.3

83

30

70

48.1

45

42.5

88

38

41.3

86

30a

74

46.9

45

41.9

89

38

40.2

86

28

78

47.5

44

42.6

90

36

41.2

87

28

82

46.5

44

43.2

93

36

42.9

92

24

86

47.4

41

43.0

91

34

40.9

86

20

90

47.4

39

43.8

92

32

41.8

88

19

94

45.3

38

42.4

94

32

41.8

92

17

98

43.9

38

42.4

97

30

42.0

96

14

102

44.2

36

41.6

94

30

41.7

94

12

Terminal sacrifice

35

-

-

30

-

-

12

Mean for weeks

1-13

31.5

-

30.6

97

-

30.2

96

-

14-52

43.1

-

38.8

90

-

38.7

90

-

53-102

47.3

-

42.3

89

-

42

88

-

a The number of animals weighed for this week is fewer than the number of animals surviving.

Table 7.7/2: Mean body weights and survival of female mice

Weeks on Study

Vehicle Control

262 mg/kg bw/day

525 mg/kg bw/day

Av. Wt. (g)

Number of Survivors

Av. Wt. (g)

Wt. (% of controls)

Number of Survivors

Av. Wt. (g)

Wt. (% of controls)

Number of Survivors

1

18.5

50

18.7

101

50

18.4

100

50

2

19.8

50

19.8

100

50

20.1

102

50

3

21.3

50

20.9

98

50

21.0

99

50

4

22.0

50

21.5

98

50

21.0

96

50

5

23.2

50

22.5

97

50

22.4

97

50

6

23.0

50

23.3

101

50

22.4

97

50

7

23.8

50

23.8

100

50

23.1

97

50

8

24.3

50

23.4

96

50

22.8

94

50

9

24.6

50

24.0

98

50

23.9

97

50

10

25.8

50

24.8

96

50

25.0

97

50

11

26.5

50

26.0

98

50

25.6

97

50

12

26.6

50

25.3

95

50

25.5

96

50

13

27.1

50

25.0

92

50

24.8

92

50

15

26.9

50

25.7

96

50

26.0

97

50

18

27.6

50

26.1

95

50

26.2

95

50

22

29.2

50

28.2

97

50

27.8

95

50

27

31.7

50

29.2

92

50

28.9

91

50

32

34.6

50

29.2

84

50

30.6

88

50

35

33.1

50

30.9

93

50

30.8

93

50

40

35.9

50

31.5

88

50

30.7

86

50

44

36.6

50

32.6

89

50

32.5

89

50

48

37.4

50

33.4

89

50

33.0

88

50

52

39.2

50

34.6

88

50

34.8

89

50

56

39.8

50

34.5

87

50

35.5

89

50

58

40.1

50

34.9

87

50

36.0

90

50

62

40.2

50

35.6

89

50

36.6

91

50

66

43.3

50

38.5

89

50

39.7

92

50

70

44.8

50

38.4

86

48

39.8

89

49

74

44.9

50

38.4

86

48

39.1

87

48

78

47.1

50

38.8

82

47

39.4

84

48

82

46.0

50

39.2

85

45

40.3

88

48

86

48.2

46

40.2

83

41

40.5

84

46

90

45.9

42

38.7

84

39

39.0

85

46

94

47.1

41

38.3

81

35

40.3

86

44

98

44.1

39

36.9

84

35

39.5

90

41

102

44.4

38

36.7

83

34

38.3

86

39

Terminal sacrifice

38

-

-

34

-

-

38

Mean for weeks

1-13

23.6

-

23.0

97

-

22.8

97

-

14-52

33.2

-

30.1

91

-

30.1

91

-

53-102

44.3

-

37.6

85

-

38.8

88

-

 

Table 7.7/3: Lesions of the adrenal medulla in male mice

 

Lesions

Vehicle Control

262 mg/kg

525 mg/kg

Hyperplasia

Overall ratesa

2/48 (4%)

9/50 (18%)

4/50 (8%)

Logistic regression testsb

P=0.071

P=0.011

P=0.191

Benign Pheochromocytoma

Overall rates

1/48 (2%)

5/50 (10%)

1/50 (2%)

Adjusted ratesc

2.3%

16.7%

5.3%

Terminal ratesd

0/34 (0%)

5/30 (17%)

0/12 (0%)

First incidence (days)

582

729 (T)

640

Logistic regression tests

P =0.352

P =0.073

P =0.760

Malignant Pheochromocytoma

Overall rates

1/48 (2%)

1/50 (2%)

0/50 (0%)

Benign or Malignant Pheochromocytomae

Overall rates

2/48 (4%)

6/50 (12%)

1/50 (2%)

Adjusted rates

4.90%

20.0%

5.3%

Terminal rates

0/34 (0%)

6/30 (20%)

0/12 (0%)

First incidence (days)

582

729 (T)

640

Logistic regression tests

P=0.472

P =0.092

P=0.592N

(T) Terminal sacrifice

aNumber of lesion-bearing animals/number of animals necropsied or examined microscopically for this lesion

bBeneath the control incidence are the P values associated with the trend test. Beneath the dosed group incidence are the P values corresponding to pairwise comparisons between the controls and that dosed group. The logistic regression tests regard tumors in animals dying prior to terminal kill as nonfatal. A lower incidence in a dose group is indicated by N.

cNumber of lesion-bearing animals/effective number of animals, i.e., number of animals alive at first occurrence of this tumor type in any of the groups

dObserved incidence at terminal kill

eHistorical incidence for 2-year NTP corn-oil gavage studies with vehicle control groups (mean ± standard deviation): 18/582 (3.1% ± 1.8%), range 0%-6%

Table 7.7/4: Selected non-neoplastic lesions in male mice

Lesions

Vehicle Control

262 mg/kg

525 mg/kg

Inguinal Lymph Node

Lymphoid hyperplasia

0/50

5/50*

5/49*

Lung

Congestion

1/50

1/50

4/50

Hemorrhage

0/50

1/50

7/50**

Leukocytosis

1/50

2/50

5/50

Prostate Gland

Inflammation, suppurative

1/49

5/48

8/48*

Skin

Acanthosis

12/50

36/50**

39/50**

Inflammation, chronic

4/50

17/50**

19/50**

Pigmentation

3/50

12/50**

19/50**

Ulcer

4/50

15/50**

17/50**

Hair follicle, atrophy

1/50

11/50**

16/50**

Thymus

Depletion

0/42

5/39*

6/38**

Epithelial hyperplasia

0/42

4/39*

4/38*

* Significantly different (P<0.05) from the control group by the logistic regression tests

** P<0.01

aNumber of lesion-bearing animals/number of tissues examined

Conclusions:
Under the conditions of this study, there was no evidence of carcinogenic activity of test item in female B6C3F1 mice. There was equivocal evidence of carcinogenicity of test item in male B6C3F1 mice at 262 mg/kg bw/day based on the marginally increased incidences of adrenal medulla pheochromocytomas and hyperplasia. Sensitivity of the study in male mice to detect a carcinogenic effect was reduced by the low survival of the high-dose group associated with fighting.
Executive summary:

In a carcinogenicity study performed similarly to OECD Guideline 451 and in compliance with GLP, B6C3F1 mice (50/sex/dose) were administered γ-butyrolactone at 0, 262 and 525 mg/kg bw/day, 5 days/week for 103 weeks. The following parameters were performed: clinical signs, mortality, body weight, gross pathology, histopathologic – neoplastic and non-neoplastic lesions.

High-dose mice were partially sedated or lethargic and inactive shortly after dosing. There was a significantly lower survival of high-dose male mice.  This was attributed partially to fighting during the first year of the study, when the animals were housed in groups of five. Survival of low- and high-dose female mice was similar to controls.

Survival rate for male mice: 35/50 (control), 30/50 (262 mg/kg bw/day) and 12/50 (525 mg/kg bw/day)

Survival rate for female mice: 38/50 (control), 34/50 (262 mg/kg bw/day) and 38/50 (525 mg/kg bw/day)

The mean body weights of dosed male mice were lower than those of the controls throughout the study, but the differences in mean bodyweights decreased when male mice were housed individually at week 67. The final mean body weights of dosed male mice were 6% lower than that of the controls. Mean body weights of dosed female mice were also lower than those of the controls throughout the study, and the final mean bodyweights were from14% to17% lower than that of the controls.

There were no non-neoplastic degenerative lesions associated with the administration of test item. Increased incidences of proliferative lesions, primarily hyperplasia, of the adrenal medulla in low-dose male mice were associated with test item administration (pheochromocytoma, benign or malignant: 2/48, 6/50, 1/50; hyperplasia: 2/48, 9/50, 4/50). The incidence of hepatocellular neoplasms in both dose groups of male mice was lower than the incidence in the controls (hepatocellular adenoma or carcinoma: 24/50, 8/50, 9/50). The incidences of harderian gland adenoma in the dosed groups of male mice were also significantly lower than the incidence in the controls.

Under the conditions of this study, there was no evidence of carcinogenic activity of test item in female B6C3F1 mice at 262 or 525 mg/kg bw/day. There was equivocal evidence of carcinogenicity of test item in male B6C3F1 mice at 262 mg/kg bw/day based on the marginally increased incidences of adrenal medulla pheochromocytoma and hyperplasia. Sensitivity of the study in male mice to detect a carcinogenic effect was reduced by the low survival of the high-dose group associated with fighting.

Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 10, 1981 to November 14, 1983
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
GLP study conducted similarly to OECD Guideline 451 without deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: GAF Corporation (New York, NY) / Lot 600-BLO
- Physical state: Clear, colorless liquid
- Analytical purity: > 97%
- Impurities (identity and concentrations): 11 impurities were identified by GC method, there is no data on chemical identity of these impuries.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in the dark at 5 °C at the study laboratory.
- Stability under test conditions: Stability studies performed by the analytical chemistry laboratory using gas chromatography indicated that y-butyrolactone was stable as a bulk chemical for at least 2 weeks at temperatures to 60 °C. The stability of the bulk chemical was monitored by the study laboratory using gas chromatography and infrared absorption periodically during all phases of the studies. No change in the study material was detected.
Species:
rat
Strain:
Fischer 344
Remarks:
F344/N
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Frederick Cancer Research Facility (Frederick, MD)
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 61 days
- Weight at study initiation: At week 1, approx. 190 g (males) / 138 g (females)
- Fasting period before study: No
- Housing: Animals were housed 5/cage throughout the study in solid-bottom polycarbonate cages (Lab Products, Inc., Garfield, NJ)
- Diet: NIH-07 Rat and Mouse Ration, Open formula, pellets (Zeigler Bros., Inc., Gardners, PA), available ad libitum
- Water: Tap water (Birmingham Water Works) via outside-the-cage automatic watering system (Edstrom Industries, Inc., Waterford, WI), available, ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature: 16-29 °C
- Humidity: 31-79 %
- Air changes: Minimum of 15 per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: November 10, 1981 to November 14, 1983
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared by mixing appropriate amounts of test item and corn oil to give the required concentrations. Dose formulations were prepared weekly and discarded 2 weeks after the date of preparation.
During the studies, the dose formulations were stored in sealed amber serum vials in the dark at 5 °C for no longer than 2 weeks.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw

STABILITY
Gas chromatography confirmed the stability of the dose formulations when stored 14 days in the dark at temperatures to 25 °C.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the test item dose formulations were performed using gas chromatography. The dose formulations were analyzed at approximately 8-week intervals; 98% (41/42) of the dose formulations were within 10% of the target concentrations.
Duration of treatment / exposure:
103 weeks
Frequency of treatment:
5 days/week
Post exposure period:
1 week
Dose / conc.:
112 mg/kg bw/day (actual dose received)
Remarks:
males
Dose / conc.:
225 mg/kg bw/day (actual dose received)
Remarks:
males and females
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Remarks:
females
No. of animals per sex per dose:
50
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses selected for the 2-year study in male rats were 0, 112, and 225 mg/kg bw/day. These doses were based on the mortality in males at 900 mg/kg bw/day and the depressed body gain in males at 450 mg/kg bw/day in the 13-week study. Because of the lower mortality in females at 900 mg/kg bw/day, the doses selected for the 2-year study in female rats were 0, 225, and 450 mg/kg bw/day.
- Rationale for animal assignment (if not random): Animals were grouped by weight intervals, then groups were assigned to cages. A table of random numbers was used to assign cages to treatment groups.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed at study initiation, weekly for 13 weeks, and monthly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed at study initiation, weekly for 13 weeks, and monthly thereafter.

FOOD CONSUMPTION: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
NECROPSY: Animals found moribund or surviving to the end of the 2-year studies were killed. Necropsy was performed on all animals.

GROSS PATHOLOGY: Yes; At necropsy, all organs and tissues were examined for gross lesions.

HISTOPATHOLOGY: Yes; all major tissues were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned, and stained with haematoxylin and eosin for microscopic examination. Complete histopathologic examinations were performed on rats that died or were killed moribund prior to day 637. Selected tissues were examined from all low-dose rats. Histopathology examinations were performed on all grossly visible lesions in all dose groups.
Other examinations:
None
Statistics:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Animals found dead of other than natural causes or found missing were censored from the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided. The incidences of neoplasms or non-neoplastic lesions were given as the numbers of animals bearing such lesions at a specific anatomic site to the numbers of animals with that site examined microscopically. Analysis of tumor incidence was performed using logistics regression analysis. Other tests used were life table test (Cox; 1972; Tarone, 1975), Fisher exact test and the Chochran-Armitage trend test (Armitage, 1971; Gart et. al., 1979). For analysis of continuous variables tests performed include Dunnett (1955) and Williams (1971, 1972), and Jonckheere’s test (Jonckheere, 1954).
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical findings attributed to test item administration.
Mortality:
mortality observed, treatment-related
Description (incidence):
There was no adverse effect of the test substance on survival in male or female.
There was a marginally significant increased survival of dosed males compared to controls; however, pairwise comparisons of survival between controls and low- or high dose groups showed no significant difference. The higher survival rates in the 225 mg/kg bw/day male dose group are due in part to the marginally decreased incidence of mononuclear cell leukemia (control, 16/50; low dose, 15/50; high dose, 9/50). Survival was similar in all female groups.
Survival rate for male rats: 24/50 (control), 27/50 (112 mg/kg bw/day) and 32/50 (225 mg/kg bw/day)
Survival rate for female rats: 28/50 (control), 27/50 (225 mg/kg bw/day) and 28/50 (450 mg/kg bw/day)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In male rats there was no body weight change associated with administration of 112 or 225 mg/kg bw/day test material. In female rats there was no body weight change associated with administration of the low dose (225 mg/kg bw/day). In female rats there was a reduction in body-weight gain associated with administration of the high-dose (450 mg/kg bw/day) from week 6 to end of the study. The mean body weight of high dose females was within 10% of the mean body weight of the controls until week 58; by the end of 2-year studies the mean body weight was 20% lower than that of the controls.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no significant gross pathological effects reported.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no significant non-neoplastic lesions reported.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Skin: Several morphological types of epithelial neoplasms, principally benign neoplasms, occurred more frequently in dosed male rats than in controls. The incidence of keratoacanthoma was marginally increased in low- and high-dose males, but pairwise comparisons were not significant (control, 1/50; low-dose, 4/50; high-dose, 6/50). The overall historical control incidence of keratoacanthomas in NTP corn oil gavage studies with F344/N male rats is 26/770 (3.4%) with a range of 0% to 12%. Because the incidences of keratoacanthoma in low-and high-dose male rats are not significantly greater than the incidence in the controls and because the incidences are within the range for historical controls, the marginally increased incidence of keratoacanthoma is not considered related to test item administration. Further, all keratoacanthomas occurred in animals killed at 2 years, and it is likely that the apparent increase in this neoplasm reflects in part the increased survival in the high-dose group relative to controls.
Basal cell adenomas occurred in four low-dose males; none were observed in high-dose or control males. One basal cell carcinoma occurred in a high-dose male rat. Although the incidence in the low-dose group was not significantly greater than the incidence in the control group, basal cell adenomas occur infrequently in male rats. The overall historical incidence of basal cell and related neoplasms in corn oil gavage controls is 13/770 (1.7%) with a range of 0% to 5%. The basal cell adenomas were not considered related to test item administration, because they did not occur at a significantly increased incidence in the low dose group and did not occur with an increased incidence in the high-dose group.

Mesothelium: Mesotheliomas occurred in four high-dose males and one low-dose male rat, but were not present in controls. The historical incidence of mesotheliomas in corn oil control male rats is 26/770 (3.4%) with a range of 0% to 10%. Thus, the apparent increased incidence reflects the low incidence in control males and is not considered to be related to test item administration.

Mammary Gland: The incidence of fibroadenomas in female rats occurred with a statistically significant (P<0.01) negative trend, and the incidence in the high-dose group was significantly lower than that of the controls (22/50, 14/50, 6/50). The overall historical control incidence for fibroadenomas in female rats is 298/770 (38.7%) with a range of 18% to 56%. The decreased incidence of fibro-adenomas in low-and high-dose female rats was considered related to test item administration. The incidence of mammary gland cysts (markedly dilated ducts or glands lined by a single layer of epithelium) also showed a statistically significant (P<0.01) negative trend (42/50, 35/50, 23/50).

Pituitary Gland: There was a statistically significant (P<0.01) decrease in the incidence of cysts in the pars distalis of high-dose female rats (25/49, 13/37, 11/48). Cysts of the pars distalis are cavities filled with serum proteins displacing the parenchyma and often occur within focal hyperplasia or adenoma. A decreased incidence of adenomas in high-dose females was not statistically significant (22/49, 24/37, 16/48).

Hematopoietic System: The incidence of mononuclear cell leukemia in male rats occurred with a significant negative trend, and the incidence in the high-dose males was significantly less than controls (16/50, 15/50, 9/50). Mononuclear cell leukemia is a common neoplasm in male F344/N rats with a overall historical control incidence of 164/770 (21.3%) and a range of 4% to 38%.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
225 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Remarks on result:
other: carcinogenicity
Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Remarks on result:
other: carcinogenicity
Key result
Dose descriptor:
NOAEL
Effect level:
225 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: toxicity
Key result
Critical effects observed:
no

Table 7.7/1: Mean body weights and survival of male rats

Weeks on study

Vehicle control

112 mg/kg bw/day

225 mg/kg bw/day

Av. Wt. (g)

Number of survivors

Av. Wt. (g)

Wt. (% of controls)

Number of survivors

Av. Wt. (g)

Wt. (% of controls)

Number of survivors

1

191

50

191

100

50

187

98

50

2

225

50

222

99

50

221

98

50

3

243

50

240

99

50

238

98

50

4

265

50

264

100

50

262

99

50

5

284

50

280

99

50

278

98

50

6

298

50

295

99

50

293

98

50

7

308

50

305

99

50

301

98

50

8

316

50

315

100

50

310

98

50

9

330

50

327

99

50

323

98

50

10

339

50

337

99

50

331

98

50

11

343

50

340

99

50

334

98

50

12

354

50

349

99

50

346

98

50

13

360

50

359

100

50

354

98

50

17

380

50

380

100

50

376

99

50

21

402

50

406

101

50

403

100

50

26

425

50

430

101

50

425

100

50

31

443

50

446

101

50

444

100

50

34

453

50

451

100

50

449

99

50

40

465

50

467

100

50

465

100

50

43

473

50

470

99

50

468

99

50

47

482

50

482

100

50

478

99

50

51

487

50

487

100

50

480

99

50

53

489

50

490

100

50

482

99

49

58

498

50

497

100

47

487

98

49

62

500

50

497

99

47

490

98

48

66

502

50

502

100

47

496

99

48

70

501

46

499

100

47

490

98

48

74

499

45

499

100

46

491

98

48

78

498

43

497

100

44

484

97

47

82

494

42

496

100

42

489

99

45

86

493

37

494

100

39

489

99

43

90

484

35

489

101

37

487

101

42

94

480

33

479

100

35

475

99

42

98

476

27

472

99

33

462

97

37

102

466

25

467

100

30

461

99

34

Terminal sacrifice

24

-

-

27

-

-

32

Mean for weeks

1-13

297

-

294

99

-

291

98

-

14-52

446

-

447

100

-

443

99

-

53-102

491

-

491

100

-

483

98

-

 

Table 7.7/2: Mean body weights and survival of female rats

Weeks on study

 

Vehicle Control

225 mg/kg bw/day

450 mg/kg bw/day

Av. Wt. (g)

Number of survivors

Av. Wt. (g)

Wt. (% of controls)

Number of survivors

Av. Wt. (g)

Wt. (% of controls)

Number of survivors

1

139

50

139

100

50

137

98

50

2

155

50

155

101

50

151

98

50

3

162

50

162

100

50

156

96

50

4

173

50

171

99

50

166

96

50

5

180

50

181

100

50

173

96

50

6

187

50

185

99

50

177

95

50

7

190

50

187

99

50

180

95

50

8

194

50

192

99

50

182

94

50

9

196

50

195

100

50

184

94

50

10

199

50

199

100

50

189

95

50

11

202

50

199

99

50

189

94

50

12

203

50

200

99

50

189

93

50

13

206

50

204

99

50

192

93

50

17

214

50

209

98

50

200

93

50

21

220

50

216

98

50

206

94

49

26

230

50

226

98

50

215

93

48

31

238

50

235

99

50

222

93

48

34

243

50

237

98

50

223

92

46

40

252

50

245

97

50

234

93

46

43

255

50

252

99

49

236

92

46

47

268

49

260

97

49

241

90

45

51

271

49

268

99

48

246

91

45

53

276

49

274

99

47

248

90

45

58

289

49

283

98

46

254

88

45

62

300

49

294

98

45

261

87

45

66

306

49

301

98

45

268

87

44

70

313

49

305

98

45

270

86

44

74

323

46

313

97

43

275

85

44

78

326

45

316

97

43

276

85

44

82

328

42

322

98

42

277

85

43

86

331

41

319

96

42

272

82

37a

90

334

41

323

97

42

278

83

37

94

331

40

325

98

40

277

84

36

98

333

34

321

96

37

273

82

31

102

339

30

323

95

28

272

80

30

Terminal sacrifice

 -

28

 -

 -

27

 -

 -

28

Mean for weeks

1-13

184

-

182

99

-

174

95

-

14-52

243

-

239

98

-

225

93

-

53-102

318

-

309

97

-

269

85

-

a The number of animals weighed for this week is fewer than the number of animals surviving.

Conclusions:
Under the test conditions, there was no evidence of carcinogenic activity of test item in male or female rats. There was a decreased incidence of mammary gland fibroadenomas and cysts and pituitary cysts in female rats which were associated with the administration of test item.
Executive summary:

In a carcinogenicity study performed similarly to OECD Guideline 451 and in compliance with GLP, Fischer 344 rats (50/sex/dose) were administered γ-butyrolactone at 0, 112 and 225 mg/kg bw/day to male rats and 0, 225 and 450 mg/kg bw/day to female rats, 5 days/week for 103 weeks. The following parameters were performed: clinical signs, mortality, body weight, gross pathology, histopathologic – neoplastic and non-neoplastic lesions.

 

There were no clinical findings or adverse effect on survival in male or female rats attributed to test item. The mean body weights of male rats administered test item were similar to those of the controls throughout the study. The mean body weight of high-dose females was lower than that of the controls after week 5 and was 10% to 20% lower than that of the controls throughout the second year. The survival of high-dose male rats was slightly higher than that of the controls (control, 24/50; low-dose, 27/50, high-dose, 32/50) due primarily to a lower incidence of mononuclear cell leukemia in the high-dose group (16/50, 15/50, 9/50). The survival of dosed females was similar to that of the controls (28/50, 27/50, 28/50). No increased incidences of neoplasms or non-neoplastic lesions in male rats were related to the administration of test item for 2 years. In female rats, negative trends were observed in the incidences of cysts (42/50, 35/50, 23/50) and fibroadenomas of the mammary gland (22/50, 14/50, 6/50) and in cysts of the pituitary pars distalis (25/49, 13/37, 11/48). These decreases were considered to be related to test item administration. There was no evidence of carcinogenic activity of test item in male and female rats.

 

Under the test conditions, there was no evidence of carcinogenic activity of test item in male or female rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
225 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
GLP study of high quality (Klim. 1)

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Only an old subcutaneous study with γ-undecalactone was available.

In this carcinogenic study (Swern, 1970), γ-undecalactone was administered via subcutaneous injections to a group of 16 female Swiss-Webster mice at a dose level of 25 mg/injection; 3 times a week for 4 weeks. Untreated and vehicle (tricaprylin) controls were also included in the study. All mice were weighed at the start of the experiments and at regular intervals throughout the ensuing period of observation of 18 to 24 months.

Total alive mice at 6 months were recorded to be 97/100, 171/203 and 4/16 in vehicle control, untreated and treatment groups, respectively. No excessive weight losses or gains were found as compared with untreated control groups. Subcutaneous sarcomas, breast cancers pulmonary and other tumors were identified in control groups. No tumor was observed in any of the mice in treatment group. 

Under the test conditions, there was no evidence of carcinogenic activity of γ-undecalactone in female Swiss-Webster mice.

More recent studies were performed by oral route on the read-across substance γ-butyrolactone, a lactone considered adequate for read-across purpose (see § "Toxicokinetics").

In these carcinogenicity studies performed similarly to OECD Guideline 451 and in compliance with GLP, B6C3F1 mice (50/sex/dose) were administered γ-butyrolactone at 0, 262 and 525 mg/kg bw/day, 5 days/week for 103 weeks and Fischer 344 rats (50/sex/dose) were administered γ-butyrolactone at 0, 112 and 225 mg/kg bw/day to male rats and 0, 225 and 450 mg/kg bw/day to female rats, 5 days/week for 103 weeks (NTP, 1992). The following parameters were performed: clinical signs, mortality, body weight, gross pathology, histopathologic – neoplastic and non-neoplastic lesions.

Survival rate for male mice: 35/50 (control), 30/50 (262 mg/kg bw/day) and 12/50 (525 mg/kg bw/day)

Survival rate for female mice: 38/50 (control), 34/50 (262 mg/kg bw/day) and 38/50 (525 mg/kg bw/day)

There was no evidence of carcinogenic activity of test item in female B6C3F1 mice at 262 or 525 mg/kg bw/day. There was equivocal evidence of carcinogenicity of the source substance in male B6C3F1 mice at 262 mg/kg bw/day based on the marginally increased incidences of adrenal medulla pheochromocytoma and hyperplasia. Sensitivity of the study in male mice to detect a carcinogenic effect was reduced by the low survival of the high-dose group associated with fighting.

  

The survival of high-dose male rats was slightly higher than that of the controls (control, 24/50; low-dose, 27/50, high-dose, 32/50) due primarily to a lower incidence of mononuclear cell leukemia in the high-dose group (16/50, 15/50, 9/50). The survival of dosed females was similar to that of the controls (28/50, 27/50, 28/50). No increased incidences of neoplasms or non-neoplastic lesions in male rats were related to the administration of test item for 2 years. In female rats, negative trends were observed in the incidences of cysts (42/50, 35/50, 23/50) and fibroadenomas of the mammary gland (22/50, 14/50, 6/50) and in cysts of the pituitary pars distalis (25/49, 13/37, 11/48). These decreases were considered to be related to test item administration. There was no evidence of carcinogenic activity of the source substance in male and female rats.

 

Under the test conditions, there was no evidence of carcinogenic activity of γ-butyrolactone in B6C3F1 mice and Fischer 344 rats.

There is evidence suggesting lack of carcinogenicity of γ-butyrolactone in experimental animals (IARC, 1999).

γ-butyrolactone is not classifiable as to its carcinogenicity to humans (group 3) in the overall evaluation of IARC (1999)

IARC (1999) IARC Monograph on the Evaluation of carcinogenic Risks to humans.Vol. 71, Re-evaluation of Some Organic Chemicals, hydrazine and hydrogen peroxide.

Finally, γ-Undecalactone is not classifiable as to its carcinogenicity to human based on the read-across approach with γ-Butyrolactone

Justification for classification or non-classification

Harmonized classification

γ-Undecalactone has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self classification

Based on the available data, no additional self-classification is proposed regarding carcinogenicity according to the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and the GHS.