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Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
Combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Study report from National Cancer Institute

Data source

Reference
Reference Type:
secondary source
Title:
Evaluation of Carcinogenicity, Teratogenecity &Mutagenic Activities of Selected Pesticides and Industrial Chemicals.
Author:
NTRL report
Year:
1968
Bibliographic source:
NTRL report, PB223159,1968

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
18 months combined repeated dose- carcinogenicity study was performed to evaluate the toxic effects of 4 methoxy phenyl acetic acid on mice.
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4-methoxyphenylacetic acid
EC Number:
203-166-4
EC Name:
4-methoxyphenylacetic acid
Cas Number:
104-01-8
Molecular formula:
C9H10O3
IUPAC Name:
(4-methoxyphenyl)acetic acid
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Benzeneacetic acid, 4-methoxy-
- Molecular formula : C9-H10-O3
- Molecular weight : 166.175
- Smiles notation : c1(ccc(OC)cc1)CC(O)=O
- InChl : 1S/C9H10O3/c1-12-8-4-2-7(3-5-8)6-9(10)11/h2-5H, 6H2, 1H3, (H,10,11)
- Substance type: Organic
- Physical state: Solid
Specific details on test material used for the study:
- Name of the test material: 4-methoxyphenylacetic acid
- EC name: 4-methoxyphenylacetic acid
- Molecular formula: C9H10O3
- Molecular weight: 166.175 g/mol
- Substance type: Organic
- Purity: No data

Test animals

Species:
mouse
Strain:
other: B6AKF1
Details on species / strain selection:
The animals were selected in expectation of a high susceptibility to carcinogenic stimuli coupled with the hardiness and longevity characteristic.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Cumberland View Farms, Clinton, Tennessee
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: Weanling (7 days of age)
- Weight at study initiation: At the time of dietary feeding: 10-19 gm
- Fasting period before study: No data
- Housing: Housed 6 mice/cage
- Diet (e.g. ad libitum): baked diet produced by D &G Co., Frederick, Maryland ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: No data

DETAILS OF FOOD AND WATER QUALITY: Routine tests to determine absence of Coliform, Salmonella and Pseudomonas spp. were made at regular intervals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:
other: Oral administration by stomach tube from 7th day of age to 28th weanling day following which the compound was mixed with the ground feed.
Details on route of administration:
Continuous daily oral administration by stomach tube was begun on the 7th day of age until the mice were at weanling age (28 days) following which the compound was mixed with the ground feed. The potential hazard for man associated with the vast majority of the chemicals studied is by the oral route and, in many cases, admixed with food. Hence, 4-methoxyphenylacetic acid was given by both gavage route and admixed with food.
Vehicle:
other: 0.5% gelatin (gavage) and baked diet (feed)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: 4-methoxyphenylacetic acid was suspended in 0.5% gelatin to give a dose range of 0 or 215 mg/Kg. The concentration of the final solution or suspension was such that the intended dose for a single animal was contained in 0.05 ml of the preparation. For feeding protocol, the chemical was mixed with feed in two Patterson-Kelly Twin Shell Liquids Solids Blenders.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food): baked diet
- Storage temperature of food: The portions of chemical feed were packaged separately in waxed paper bags, sealed in polyethylene bags and refrigerated.

VEHICLE
- Justification for use and choice of vehicle (if other than water): For gavage exposue: 0.5 % gelatin and For feed exposure: Baked diet
- Concentration in vehicle: 0.5% (215 mg/Kg)
- Amount of vehicle (if gavage): 0.05 mL
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
By Gas chromatographic analyses
Duration of treatment / exposure:
18 months
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
0.0 or 215.0 mg/Kg
No. of animals per sex per dose:
Total: 108
0 mg/Kg: 18 Male and 18 Female (Untreated)
0 mg/Kg: 18 Male and 18 Female (Vehicle)
215 mg/Kg: 18 Male and 18 Female
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The basic concept of the dosage choice was the use of a maximum tolerated dose. The calculated dose was not adjusted to the changing body weight during the three weeks of stomach tubing but a single adjustment was made at the time of conversion from stomach tube to mixture in the feed. The following preliminary toxicity studies were conducted prior to initiation of main study. The study was carried out on random-bred mice beginning at seven to eight days of
age for oral studies. All dosages used were selected from a list made up of the values 1.0, 2.15, and 4.64 multiplied by appropriate powers of 10.

Phase 1- Single Dose: Toxicity of single doses was determined by both oral and subcutaneous administration using groups of four animals per do!;e for each route. Initial range finding was accomplished by using dOHes differing by factors of 10. After 24 hours of observation, intermediatE! doses were given to additional groups of animals. From these data the maximum tolerated dose (defined as the largest dose which produced zero mortality), and the next higher and the next lower doses in the series, were identified. The maximum tolerated dose for subcutaneous administration, was identified in this phase of the study, was the dose to be used for single subcutaneous administration to weanling hybrids.

Phase II - Six Dose This phase was carried out by means of oral administration only and consisted of giving the MTD identified in Phase I, as well as the next higher and next lower doses, to groups of six animals every other day, for a total of six doses. As this study proceeded, indications of a need for higher or lower doses were available and new groups of animals using these additional doses were started as soon as indicated. From the acc
Phase III - Nineteen Dose This phase of the study was carried out by oral administration and only utilizing groups of six animals each. The maximum tolerated dose identified in Phase II plus the next higher and lower doses were administered daily for a total of nineteen doses. As in Phase II, new groups of animals, using higher or lower doses, were introduced into the study as indicated. At the end of Phase III, a maximum tolerated dose (still defined as above) was identified and this dose was then used to start hybrid animals for the definitive studies.

During the preliminary toxicity studies all cages were checked daily and animals weighed weekly. Weights were taken by groups rather than by individuals. Individual mice were not identified until such time as there was a specific reason for this, such as death. Moribund animals were killed and subjected to a gross autopsy. All surviving animals were held for at least 7 days following compound administration, then killed and subjected to a complete gross and/or microscopic examination.

- Rationale for animal assignment (if not random): No data
- 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: Daily
- Cage side observations checked in table [No.?] were included. mortaliity and morbundity

DETAILED CLINICAL OBSERVATIONS: Yes, careful observations were made like palpated for enlargement of liver and spleen
- Time schedule: weekly early in the study and less frequently thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: weekly early in the study and less frequently thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data
- 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: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Not specified
- Time schedule for examinations: Not specified
- Dose groups that were examined: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day before killing
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: All animals but only those smears from mice which showed splenomegaly, liver enlargement or lymph-adenopathy were stained and examined
- Parameters checked in table [No.?] were examined. Not specified

CLINICAL CHEMISTRY: Not specified
- Time schedule for collection of blood: Not specified
- Animals fasted: Not specified
- How many animals: Not specified
- Parameters checked in table [No.?] were examined. Not specified

URINALYSIS: Not specified
- Time schedule for collection of urine: Not specified
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
n table [No.?] were examined. Not specified

NEUROBEHAVIOURAL EXAMINATION: Not specified
- Time schedule for examinations: Not specified
- Dose groups that were examined: Not specified
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Not specified

IMMUNOLOGY: Not specified
- Time schedule for examinations: Not specified
- How many animals: Not specified
- Dose groups that were examined: Not specified
- Parameters checked in table [No.?] were examined. Not specified

OTHER: Not specified
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, An incision was made from the neck to the pubis and the skin reflected; another cut was made to expose the neck structures and various nodes and then the abdomen and thorax were opened. The animals were observed for signs of carcinogenic action of compounds in any organ. Incidental diseases (lesions) were noted. The total chest contents, liver, spleen, kidneys with adrenal glands, stomach (fixed whole), intestines, genital organs of male and female mice were obtained and placed in formalin saline. The head was severed from the body and all remains placed in fixative.

HISTOPATHOLOGY: Yes , total chest contents, liver, spleen, kidneys with adrenal glands, stomach (fixed whole), intestines, genital organs of male and female mice were obtained and placed in formalin saline and microscopic study was performed.
Other examinations:
No data
Statistics:
Chi square test (Fourfold method) & Yates Correction was used for specific tumor types

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
Clinical signs and mortality:
Clinical signs: Liver and spleen enlargement was not observed upon palpation.
Mortality: One mice of each sex died during the 18 months experimental study period

Body weight and weight gain: Significant weight gain was observed with the increase in duration from 4, 26, 52 weeks to 18 months

Food consumption and compound intake: No data available

Food efficiency: No data available

Water consumption and compound intake: No data available

Opthalmoscopic examination: No data available

Haematology: No data available

Clinical chemistry: No data available

Urinanalysis: No data available

Neurobehaviour: No data available

Organ weights: No data available

Gross pathology: Yes, Incidence of Type A Reticulum cell carcinoma observed in 1 female mice

Histopathology: Yes, details not available

Effect levels

Dose descriptor:
LOAEL
Effect level:
215 other: mg/kg
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Changes in body weight and Incidence of Type A Reticulum cell carcinoma observed in 1 female mice

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table: Statistical evaluation of total tumors and the three most prevelant tumors in animals receiving experimental compounds

 

Total tumors

Reticulum cell carcinoma

Pulmonary adema

Hepatoma

X

-

X

-

X: 0.05% probability

Table: Mortaliity and the related parameters

 

Male

Female

No. of mice at start

18

18

No. of mice 18 months

17

17

No. mice missing

1

0

No. mice died during experiment

1

1

No. mice negative

11

10

No. mice died with tumors

0

1

No. mice killed with tumors

0

0

No. mice killed or died, other diseases

6

7

Table: Body Weight changes during oral administration

Time (Weeks)

Start (4)

26

52

Term (weeks)

Sex

M

F

M

F

M

F

M

F

Weight gain

13

13

41

35

41

39

46

44

Table: Incidence of tumors

Tumors

Male

Female

Lymphatic luekamia

0

0

Type A Reticulum cell carcinoma

0

1

Type B Reticulum cell carcinoma

0

0

Pulmonary adenoma

0

0

Pulmonary carcinoma

0

0

Hepatoma

0

0

Hepatic carcinoma with kidney metastasis

0

0

Mammary carcinoma

0

0

Skin carcinoma

0

0

Applicant's summary and conclusion

Conclusions:
The Low Observed Adverse Effect Level (LOAEL) for 4-methoxyphenylacetic acid on B6AKF1 strain mice is 215 mg/Kg based on the changes in body weight and incidence of Type A Reticulum cell carcinoma observed in 1 female mice.
Executive summary:

Combined repeated dose- carcinogenicity study was conducted on male and female mice of strain B6AKF1 for 18 months. The chemical was used at dose levels of 0 or 215 mg/Kg and given daily for 18 months. Oral administration by stomach tube was initiated from 7th day of age to 28th weanling day following which the compound was mixed with the ground feed (baked diet). Animals were observed daily for any abnormalities and palpated weekly at time of weighing for enlargement of liver and spleen. Animals which appeared moribund were killed for necropsy. One mice of each sex died during the 18 months experimental study period. Significant weight gain was observed with the increase in duration from 4, 26, 52 weeks to 18 months. Incidence of Type A Reticulum cell carcinoma was observed in 1 female mice. Based on the observations made, Low Observed Adverse Effect Level (LOAEL) for 4-methoxyphenylacetic acid is 215 mg/Kg.