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Toxicological information

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:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication

Data source

Reference
Reference Type:
publication
Title:
Screening of toxicological properties of 4-methoxybenzoic acid by oral adminstration to rats
Author:
Mariko Shirota, Takayuki Seki, Kazumi Tago, Hiroyasu Katoh, Hideki Marumo, Mami Furaya, Tomoko Shindo and Hiroshi Ono
Year:
2008
Bibliographic source:
The Journal of Toxicological Sciences Vol. 33, no. 4, 431-445, 2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
28 days repeated dose oral toxicity study was performed to determine the toxic nature of 4-methoxybenzoic acid
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: 4-methoxybenzoic acid
- Molecular formula: C8H8O2
- Molecular weight: 136.1492 g/mol
- Substance type: Organic
- Physical state: No data
Specific details on test material used for the study:
- Name of test material: 4-methoxybenzoic acid
- Molecular formula: C8H8O2
- Molecular weight: 136.1492 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: 98.95% pure

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
No data
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan (Atsugi Breeding Center, Atsugi, Kanagawa, Japan)
- Age at study initiation: 5 weeks approximately
- Weight at study initiation: No data
- Fasting period before study:
- Housing: The animals were kept individually in metallic cages with metal-meshed floors
- Diet (e.g. ad libitum): Solid rodent chow (CE-2, CLEA Japan) ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25˚C
- Humidity (%): 40-75%
- Air changes (per hr): 15 complete changes/hour
- Photoperiod (hrs dark / hrs light): 12hr (lights on 7 hrs)

IN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
No data
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% sodium carboxumethylcellulose solution
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test compound was finely ground in mortar and then suspended in 0.5% sodium carboxumethylcellulose solution to give dose levels of 0, 100, 300 or 1000 mg/kg bw/day

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% sodium carboxumethylcellulose solution
- Concentration in vehicle: 0, 100, 300 or 1000 mg/kg bw/day
- Amount of vehicle (if gavage): 5 mL/Kg
- Lot/batch no. (if required): No data
- Purity: No data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
0, 100, 300 or 1000 mg/kg bw/day
No. of animals per sex per dose:
0 mg/Kg bw/day: 10 males and 10 females
100 mg/kg bw/day: 5 males and 5 females
300 mg/kg bw/day: 5 males and 5 females
1000 mg/Kg bw/day: 10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 7 days preliminary repeated dose oral toxicity study was performed in which 1000 mg/Kg did not show any toxic effects. Based on this the main study doses were selected as 0, 100, 300 or 1000 mg/Kg bw/day

- Rationale for animal assignment (if not random): Stratified allocation based on body weight measured on the day before initial dosing
- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: No data
- Section schedule rationale (if not random): No data
Positive control:
No data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: prior to initial dosing and once a week until the end of recovery period
- Cage side observations checked in table [No.?] were included. Mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to initial dosing and once a week until the end of recovery period

BODY WEIGHT: Yes
- Time schedule for examinations: 3 times during the first week of the treatment and twice a week thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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 data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were No data examined: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to necropsy
- Anaesthetic used for blood collection: Yes, pentobarbital sodium
- Animals fasted: Yes
- How many animals: No data
- Parameters checked in table [No.?] were examined. Cell counts, RBCs, WBCs, platelets, hemoglobin concentration and differential WBC counts. Hematocrit, mean concentration of hemoglobin in the RBC (MCHC) and mean content of hemoglobin in the RBC were calculated. The Prothrombin time (PT) and activated partial thromboplastin time were measured.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to necropsy
- Animals fasted: Yes
- How many animals: No data
- Parameters checked in table [No.?] were examined. Plasma concentration of total protein, albumin, total cholesterol, glucose, blood urea nitrogen, creatinine, triglyceride, total bilirubin, inorganic P, Ca and activities of AST, ALT, ALP and GTP were determined. A/G ration and plama concentration of Na and K were measured.

URINALYSIS: Yes
- Time schedule for collection of urine: at final week of treatment and at final week of recovery study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined. pH, occult blood, protein, ketone bodies, urobilinogen and bilirubin, color, turbidity and sediments. The volume and weight of 24hrs urine was measured and the specific gravity of the urine was calculated

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

OTHER: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, necropsy was performed after 18-24 hts of fasting on the day following final treatment and on the day following recovery period. Organs such as brain, thymus, heart, liver, kidneys, spleen, testis, adrenals and epididymides were weighed

HISTOPATHOLOGY: Yes, brain, thymus, heart, liver, kidneys, spleen, testis, adrenals and epididymides along wth spinal cord, lungs, bronchi, stomach, ileum, colon, seminal vesicles, ovaries, uterus, vagina, urinary bladder, femoral marrow, mesentric lymphnodes, mandibular lymphnodes and ischiadic nerves were dissected out and fixed in buffered formalin for microscopic examination. The testes were fixed in Bouin;s solution, with post fixation in buffered formalin. These organs/tissues were processed for paraffin embedded block, sections cut from the blocks were stained with hematoxylin-eosin.
Other examinations:
No data
Statistics:
Graded findings in the histopathological examination were analysed using the Mann Whitney U test. Data from the urinanalysis usine quality test were analyzed using a chi-square test. The other data were analyzed with multiple comparisons when comparing the data from more than 2 groups with those from the control. When comparing the data from a single group with those from the control, Student’s t-test or Aspin-Welch’s test was applied, an analysis of variance (ANOVA) test or Kruskal Wallis rank test was applied, following examination of the uniformity of variatons by Barlett’s test. Significant differences from the control were determined by Dunnett’s test. A p value of less than % was judged a significant difference.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Some animals of the 1000 mg/kg bw/day group showed temporary salivation after dosing. No other clinical signs were observed.
Mortality:
no mortality observed
Description (incidence):
No mortality and murbidity was noted
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight of males and females in the compound treated groups changes similarly to those of the control group at any period
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption in females of 1000 mg/Kg bw/day was slightly higher than in the controls from day 7-8 of treatment. No difference in food intake was observed for male rats of any group.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Increase in water changes was observed, which was confined by an observation of water feeding bottles in the metabolic cages.
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of treatment period, an increase in AST activity and a slight decrease in the protein concentration were noted in the 1000 mg/kg bw treated females compared to the controls. No significant differences were observed between the compound related and control groups for males and those at the end of the recovery period from animals of both sexes.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urine volume measured on day 23 of treatment was larger in the males given 300 mg/Kg nw/day or more of the compound and in females given 1000 mg/Kg bw/day when compared to the control animals. Also, usine specific gravities were decreased in the males of these groups. Urinanalysis showed some minor changes and control groups and were suggestive of any toxic effects.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No abnormalities were noted in the neurobehavioral test at the last week of treatment
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant differences from the control were observed in absolute organ weights or in organ weights relative to body weight in any of the compound treated group of males or females.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No abnormality suggestive of any toxic effects were observed in any organs or tissues on gross examination at necropsy.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No abnormality suggestive of any toxic effects were observed in any organs or tissues on microscopic examination.
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
No data

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No significant effects were noted at the mentioned dose level

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table: Urianalysis of rats treated orally

Dose (mg/Kg)

On day 23 of treatment

On Day 9 of recovery

0

100

300

1000

0

1000

No. of animals

10/10

5/5

5/5

10/10

10/10

10/10

Urinary volume

 

 

 

 

 

 

Males

15.6±2.2

15.6±2.1

20.9±4.7*

23.8±4.7**

18.3±4.7

24.3±4.6

Females

11.7±3.5

11.9±3.4

13.0±4.3

22.1±7.5**

13.3±2.8

18.3±5.6

Specific gravity

 

 

 

 

 

 

Males

1.058±0.008

1.051±0.007

1.043±0.011**

1.045± 0.006**

1.056±0.009

1.038±0.006**

Females

1.045±0.012

1.046±0.008

1.043 0.007

1.038±0.009

1.041±0.010

1.044±0.011

* P<0.05

** P<0.01

 

Table: Hematology data

Dose (mg/Kg)

End of treatment

End of recovery

Males

Females

Males

Females

0

100

300

1000

0

100

300

1000

0

1000

0

1000

RBC

755±53

780±15

770±27

755±18

742±19

771±26

750±25

751±30

797±13

766±22**

749±25

766±24

Hemoglobin

14.9± 0.6

15.2±0.2

15.2±0.3

15.0±0.2

14.8±0.3

14.9±0.3

14.7±0.3

14.7±0.5

15.2±0.1

14.6±0.3**

14.5±0.4

14.4±0.7

Hematocrit

44.8±2.9

46.0±0.7

45.5 v1.1

45.2±0.4

44.2±1.0

44.9±1.3

44.4±0.7

43.6± 1.9

45.3±0.8

43.5±1.0**

42.6±1.3

55.8±2.0

MCV

59.4 2.2

58.9±0.8

59.0±1.3

60.0± 1.5

59.5± 1.1

58.3±1.2

59.2±1.4

58.0±1.1

56.8±1.4

56.8 0.8

56.9± 1.2

55.8±2.0

MCH

19.8±0.8

19.5±0.6

19.8±0.6

19.9± 0.6

20.0± 0.6

19.6± 0.3

19.8±0.6

19.6±0.3

19.1± 0.4

19.0± 0.3

19.4±0.4

18.8 v0.7

MCHC

33.3±0.1

33.0±0.7

33.6±0.3

33.2±0.2

33.6±0.4

3.7±0.3

33.5± 0.2

33.8±0.6

33.5±0.6

33.5±0.4

34.0±0.3

33.7±0.3

PlateletPT

102.9± 13.9

104.5± 16.3

106.6± 8.1

101.5±10.4

96.3± 7.0

100.5±9.4

93.3±83.9

83.9±3.1

96.1± 19.6

99.3±8.4

101.8± 12.7

102.7± 10.2

APTT

22.1±0.8

22.9±1.7

20.5±2.4

20.1±1.0

18.5±1.0

18.2±1.4

17.8±1.2

16.7±1.4

21.6±1.5

21.0±0.4

17.1±0.8

16.9±0.8

WBC

90.8±16.7

61.5±20.5

67.7±17.6

74.4± 21.7

61.0± 18.1

54.9±14.8

46.7± 12.4

59.8±7.8

69.4± 13.4

65.4±14.2

43.9± 2.9

58.8±14.5

Differential leucocyte counts

 

 

 

 

 

 

 

 

 

 

 

 

Neutrophils

10±5

11±2

10±2

11 3

7±2

6±3

8±4

8±4

13±5

15±3

10±4

10±3

Eosinophils

1±0

1±0

1±0

0±0

1±0

1±1

1±0

1±0

1±0

1±1

2±1

1±1

Basophils

0±0

0±0

0±0

0±0

0±0

0±0

0±0

0±0

0±0

0±0

0±0

0±0

Monocytye

3±1

4±1

5±1

4±1

3±1

4±1

3±1

3±1

4±1

4±2

4±1

4±2

Lymphocytes

85±5

85±3

85±3

85±3

89±3

88±3

88±4

88±4

81±4

79±4

85 5

85 5

* P<0.05

** P<0.01

 

Table: Clinical chemistry data

Dose (mg/Kg)

End of treatment

End of recovery

Males

Females

Males

Females

0

100

300

1000

0

100

300

1000

0

1000

0

1000

TP

5.0±0.2

5.2±0.2

5.1±0.1

5.0±0.0

5.4±0.3

5.3±0.3

5.2±0.3

4.8±0.3**

5.6±0.4

5.4±0.1

5.5±0.1

5.7±0.3

Albumin

3.0±0.2

3.0±0.3

3.0±0.1

3.0±0.1

3.2±0.2

3.2±0.2

3.2±0.2

2.9±0.2

3.0±0.2

3.0±0.1

3.2±0.2

3.3±0.4

A/G

1.48±0.15

1.46±0.27

1.38±0.27

1.44±0.08

1.51±0.17

1.48±0.17

1.64±0.11

1.56±0.09

1.14±0.08

1.28±0.14

1.41± 0.20

1.37±0.24

BUN

16±2

17±2

16±3

16±1

23±3

21±2

23±1

20±2

19±3

15±3

21±1

24±3

Creatinine

0.6±0.1

0.6±0.1

0.6±0.0

0.6±0.1

0.6±0.1

0.6±0.1

0.7±0.1

0.7±0.1

0.7±0.0

0.6±0.1

0.7±0.0

0.8±0.1

Glucose

130±13

137±32

143±13

155±24

109±13

113±21

118±7

108±10

137±10

139±16

125±12

128±10

T. Choleterol

34±8

33±4

37±5

40±5

41±8

48±9

45±10

31±8

41±15

38±10

48±9

54±4

Triglyceride

19±5

22±7

28±14

35±15

11±5

9±3

10±3

9±2

24±13

29±12

13±2

22±12

T.Bil

0.07±0.04

0.08±0.03

0.09± 0.05

0.07 v0.03

0.09±0.03

0.07±0.03

0.09±0.04

0.09± 0.03

0.08± 0.02

0.09±0.03

1.10±0.04

0.08±0.02

IOng. P

9.0±0.7

8.7±0.8

8.4±0.3

8.6±0.3

7.9±0.8

8.1±0.7

8.2±1.1

8.4±0.5

7.2±0.9

6.5±0.6

7.4±0.9

7.5±0.5

Ca

8.9±0.1

8.9±0.3

9.0±0.1

9.2±0.2

9.1±0.1

9.1±0.2

9.1±0.2

8.8±0.4

8.9±0.2

8.8±0.1

9.1±0.2

9.2±0.2

Na

145.8±0.6

145.1±0.9

145.8±0.3

145.5± 0.9

145.5± 0.8

144.7±0.7

145.6± 0.8

146.0±1.4

146.2±0.4

145.9± 0.6

143.1±0.8

143.4±0.2

K

4.48±0.53

4.33±0.36

4.08±0.26

4.29±0.24

4.18±0.39

4.31±0.20

4.20±0.28

4.05±0.38

4.03±0.26

3.96±0.12

4.11±0.28

3.98 0.32

Cl

109.0±1.7

108.3±1.7

108.1±1.2

107.8± 1.4

109.1± 1.2

107.5± 1.0

109.9±1.1

109.3± 2.3

108.2± 1.1

108.7±0.6

107.2±0.6

107.8±0.6

ALP

441± 143

451±58

511± 190

459±56

319± 65

261±62

299±75

266±46

366±80

374±29

219±36

231±39

ALT

31±6

27±4

29±3

37±15

25±2

22±2

22±5

29±5

35±5

32±4

24±3

28±5

AST

72±10

66±11

67±7

88±24

69±3

66±5

69±8

94±16*

83±14

66±2

62±3

66±5

GTP

0±1

0±1

1±0

1±1

1±0

1±0

1±1

1±0

0±1

1±1

1±1

1±1

 

Applicant's summary and conclusion

Conclusions:
The No observed adverse effects level (NOAEL) for 4-methoxybenzoic acid is considered to be 300 mg/Kg bw/day when male and female Sprague Dawley rats were treated with the test chemical for 28 days.
Executive summary:

28 days repeated dose oral toxicity study was performed to determine the toxic nature of 4-methoxybenzoic acid. The study was performed using male and female Sprague Dawley rats for 28 days. The test chemical wassuspended in 0.5% sodium carboxumethylcellulose solution to give dose levels of 0, 100, 300 or 1000 mg/kg bw/day. During the study period, the animals were observed for clinical signs, mortality, changes in body weight and food consumption, hematology, clinical chemistry, urinalaysis, neurobehavioral changes and were subjected to gross and histopathology.No mortality and murbidity was noted. Some animals of the 1000 mg/kg bw/day group showed temporary salivation after dosing. No other clinical signs were observed. Body weight of males and females in the compound treated groups changes similarly to those of the control group at any period. Food consumption in females of 1000 mg/Kg bw/day was slightly higher than in the controls from day 7-8 of treatment. No difference in food intake was observed for male rats of any group. Increase in water changes was observed, which was confined by an observation of water feeding bottles in the metabolic cages. At the end of treatment period, an increase in AST activity and a slight decrease in the protein concentration were noted in the 1000 mg/kg bw treated females compared to the controls. No significant differences were observed between the compound related and control groups for males and those at the end of the recovery period from animals of both sexes. Urine volume measured on day 23 of treatment was larger in the males given 300 mg/Kg nw/day or more of the compound and in females given 1000 mg/Kg bw/day when compared to the control animals. Also, usine specific gravities were decreased in the males of these groups. Urinanalysis showed some minor changes and control groups and were suggestive of any toxic effects. No abnormalities were noted in the neurobehavioral test at the last week of treatment. No significant differences from the control were observed in absolute organ weights or in organ weights relative to body weight in any of the compound treated group of males or females. No abnormality suggestive of any toxic effects were observed in any organs or tissues on gross and histopathological examination at necropsy. Based on the observations made, the No observed adverse effects level (NOAEL) for 4-methoxybenzoic acid is considered to be 300 mg/Kg bw/day when male and female Sprague Dawley rats were treated with the test chemical for 28 days.