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Repeated dose toxicity: oral

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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:
other: Non-GLP, guideline-equivalent study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1992

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:
some clinical chemistry parameters and pathology parameters
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
But-2-yne-1,4-diol
EC Number:
203-788-6
EC Name:
But-2-yne-1,4-diol
Cas Number:
110-65-6
Molecular formula:
C4H6O2
IUPAC Name:
but-2-yne-1,4-diol
Details on test material:
Purity of test substance was approximately 99%.

Test animals

Species:
rat
Strain:
other: Wistar Imp:DAK
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test animals were 6-8 weeks old at the start of dosing, with initial body weights of 188 +/- 20g (males) and 145 +/- 15g (females). Animals were randomly assigned either to control or test groups. They were kept in a room equipped with automatic light-cylcle timers and were housed in polypropylene bedding, four animals in each. Relative humidity and temperature were maintained at 40-60% and 20-21C, respectively. Food (Murigan pelled rodent chow, Wytwornia Pasz, Motycz, Poland) and tap water were provided ad libitum.

Administration / exposure

Route of administration:
oral: gavage
Details on oral exposure:
Distilled or diluted test material was administered with a syringe and a stainless-steel ball-tipped feeding needle in a volume of 1 ml/kg via oral gavage. Treated animals received the test material at doses of 1 mg/kg (low dose), 10 mg/kg (mid-dose), and 50 mg/kg (high dose). Control animals recieved equal volume of distilled water.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily, reported as "consecutive days" and interpreted as 7 days per week for 4 weeks.
Doses / concentrations
Remarks:
Doses / Concentrations:
1; 10; 50 mg/kg
Basis:

No. of animals per sex per dose:
8 males and 8 females per dose
Control animals:
yes
Details on study design:
Post-exposure period: none
Positive control:
none

Examinations

Observations and examinations performed and frequency:
Animals were observed daily for signs of overt toxicity. Body weights were recorded before the first dosing and twice a week thereafter during the test period. Food consumption was measured weekly.
Sacrifice and pathology:
At the end of the treatment period, rats were fasted overnight. Just before necropsy, blood samples were drawn from the tail vein for haemotological measurements (see Table 1). Animals were subsequently anesthetized with ethyl ether, sacrificed by exsanguination from the abdominal aorta and subjected to gross necropsy. Blood samples were collected for clinical chemistry measurements (see Table 2). The tissue and organs listed in Table 3 were fixed in 10% neutral-buffered formalin, processed and embedded in paraffin wax, sectioned, stained with hemotoxylin and eosin and evaluated microscopically. Evaluation of rats which died during the study was performed as described above except that the organs were not weighed and blood samples were not taken.
Statistics:
Except for body weight data, the differences between experimental and control groups were analysed using the following tests: Bartlett's test of homogeneity of variance was used at the P < 0.01 level. When variances were not significantly different, groups were compared with thte standard one-way variance (ANOVA). In the next step, Dunnet's test was used to determine which treatment groups differed from controls. In the absence of homogeneous variances, the Kruskal-Wallis test was used to assess differences and Dunn's summed rank test to determine which treatment group differed significantly from control. Body weight data were evaluated by analysis of covariance (ANCOVA), where day 1 was treated as a covariate, followed by Dunnett's test for comparison to controls. All these procedures were carried out with the TOXSTAT program.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
3 of 8 males and 3 of 8 females rats died from the high dose group
Mortality:
mortality observed, treatment-related
Description (incidence):
3 of 8 males and 3 of 8 females rats died from the high dose group
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain was lower in males from the high dose group compared to controls
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
slightly reduced in males from high-dose group
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):
See Table 4.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See Table 5.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute liver weights in the high dose male and female animals were significantly higher than in the controls. When expressed on a relative basis, the liver weights of females in the mid-dose group and of both sexes in the high dose group were significa
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
hepatic and splenic lesions
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Congested internal organs, pulmonary oedema and severe changes in liver and kidneys.
Histopathological findings: neoplastic:
not specified

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Dose descriptor:
LOAEL
Effect level:
10 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
haematology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Three male and three females in the highest group of doses died within the duration of the test. In the other experimental groups, there was no mortality. With the section of the animals, those that died in duration of the test had congested internal organs, pulmonary edemas and severe changes in liver and kidneys, which included diffuse hepatic parenchymal necrosis, accompanied by reactive mononuclear cells and granulocytes, fatty changes, as well as renal tubular degeneration and interstitial mononuclear cell infiltration in the kidney, (degeneration of the tubules among other things) were determined.

The body weight gain of the male animals in the highest group of doses was reduced, in the other groups of doses and with the female animals this effect was not significant. Food consumption was slightly reduced with the male rats in the highest group of doses. The absolute and relative liver weights and relative kidney weights of both sexes in the highest group of doses was significantly increased. The relative liver weight was increased with the female animals of the middle group of doses. The absolute kidney weights were increased in the female animals at the highest dose. The weights of spleen, suprarenal bodies and testicles were not changed.

 

Results of hematology and serum chemistry investigations are shown in Tables 4 and 5, respectively.

 

In the survivor animals in the high and middle doses, histopathological investigations showed dose-dependent liver damages and spleen changes. The authors state in summary that the notable features of the subacute poisoning are chronic liver inflammation and liver hypertrophy.

Table 4. Haematological Parameters

Parameter

Control

1 mg/kg

10 mg/kg

50 mg/kg

RBC (x 106mm-3) males

10.9±0.6

11.5±1.3

10.4±0.7

11.1±1

RBC (x 106mm-3) females

11.1±1.0

10.9±0.9

10±0.8*

8.8±1**

Haematocrit (%) males

52.0±1.7

51.6±1.4

51.9±1.2

50.6±2.3

Haematocrit (%) females

47.9±1.8

49.7±1.5

48.0±1.9

42.6±3.4**

Haemoglobin (g dl-1) males

14.3±1.1

15.6±0.8

16.0±1.1

14.7±1.2

Haemoglobin (g dl-1) females

13.5±0.7

13.6±1.2

13.5±0.4

12.3±0.6

Mean corpuscular haemoglobin (pg) males

13.2±0.8

13.8±1.8

15.5±1.5

13.3±1.9

Mean corpuscular haemoglobin (pg) females

12.2±0.9

12.5±1.6

13.6±1.3

14.1±2

Reticulocytes (‰) males

5.0±2.9

5.5±2.0

5.4±1.4

12.4±2.9**

Reticulocytes (‰) females

5.0±2.0

7.5±1.5

4.9±0.8

10.0±3.7**

WBC (x 106mm-3) males

16.1±3.1

17.5±3.1

15.8±3.5

24.1±2.9**

WBC (x 106mm-3) females

13.7±4.5

13.6±1.8

10.2±3.0

23.6±6.9**

Differential Neutrophils (x 106mm-3) males

4.4±2.0

3.8±1.3

3.7±1.7

7.4±2.3*

Differential Neutrophils (x 106mm-3) females

3.3±1.4

2.2±0.9

2.3±0.8

8.4±5.6**

Differential Lymphocytes (x 106mm-3) males

11.5±2.4

13.1±2.5

11.2±2.6

15.8±3.5*

Differential Lymphocytes (x 106mm-3) females

9.7±3.6

11.2±1.8

7.1±1.9

14.8±3.6*

* Significantly different from control (P < 0.05).

** Significantly different from control (P < 0.01).

Table 5. Serum Chemistry Parameters

Parameter

Control

1 mg/kg

10 mg/kg

50 mg/kg

Alanine aminotransferase (U/l) males

42.0±13.7

56.0±19.8

33.3±4.4

42.5±14.8

Alanine aminotransferase (U/l) females

32.5±5.3

34.4±12.3

34.5±10.8

43.7±8.2

Sorbital dehydrogenase (U/l) males

1.64±0.41

1.44±0.29

1.68±0.42

3.74±2.32**

Sorbital dehydrogenase (U/l) females

1.26±0.47

0.94±0.29

1.37±0.21

5.06±2.90**

Total protein (g/dl) males

6.16±0.28

6.38±0.19

6.14±0.41

6.56±0.27

Total protein (g/dl) females

6.85±0.21

6.86±0.29

6.79±0.23

7.33±0.32*

Albumin (g/dl) males

3.16±0.29

3.26±0.16

3.28±0.14

3.31±0.22

Albumin (g/dl) females

3.55±0.23

3.69±0.17

3.6±0.19

3.79±0.31

Glucose (mg/dl) males

109±17

98±25

112±12

142±24*

Glucose (mg/dl) females

124±21

128±29

99±26

122±26

* Significantly different from control (P < 0.05).

** Significantly different from control (P < 0.01).

Applicant's summary and conclusion

Executive summary:

2 -Butyne-1,4 -diol was given to male and female Wistar Imp:DAK rats by oral gavage for 28 consecutive days in daily doses of 1, 10, or 50 mg/kg/day. After 28 days all animals are necropsied. Blood samples were obtained and selected organs were weighed and prepared for histological examination. Treatment-related effects in the high-dose group consisted of: fatal cases in both sexes; decreased body weight gain in males; increase of absolute and/or relative weights of liver and kidneys in both sexes; depressed red blood cell count; haematocrit value and haemoglobin concentration in female rats ande elevated reticulocyte count and leukocyte count in both sexes; increased total serum protein content in females, elevated glucose concentration in males and higher activity of sorbital dehydrogenase in both sexes; and histopathological evidence of hepatotoxicity and nephrotoxicity in decedents, and hepatic and splenic changes in survivors. Minor hepatic, splenic, and erythrocyte changes were also found in some females given the middle dose. The dose of 1 mg/kg/day was considered to the NOAEL, and 10 mg/kg/day the LOAEL.