2-ethylhexyl (4-chloro-2-methylphenoxy)acetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15.12.2004 - 31.05.2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

read-across source

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(4-chloro-2-methylphenoxy)acetic acid

EC Number:

202-360-6

EC Name:

(4-chloro-2-methylphenoxy)acetic acid

Cas Number:

94-74-6

Molecular formula:

C9H9ClO3

IUPAC Name:

(4-chloro-2-methylphenoxy)acetic acid

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

impurity 2

Reference substance name:

4-chloro-o-cresol

EC Number:

216-381-3

EC Name:

4-chloro-o-cresol

Cas Number:

1570-64-5

Molecular formula:

C7H7ClO

IUPAC Name:

4-chloro-2-methylphenol

Test material form:

solid

Test animals

Species:

dog

Strain:

Beagle

Details on species / strain selection:

Beagle dogs, 24 males and 24 females from WOBE Ltd. Hungary (Protocol No. 31/2004) were used. Before the study the animals were quarantined for 38 days in the conditions identical to the conditions during the experiment. Mean weight of males at the start of experiment was 9.6 kg males and of females 9.3 kg. The animals were marked by serial numbers 1 - 48. These numbers were placed on the cages together with the marking of sex and group.

Sex:

male/female

Administration / exposure

Route of administration:

oral: capsule

Vehicle:

other: gelatine capsules

Details on oral exposure:

The test substance was administered per os in gelatine capsules every day at 9 a.m. for 90 days. Doses were calculated on the current body weight. The animals were weighed once a two weeks. The gelatine capsules without test substance were administered to animals of control group.

Analytical verification of doses or concentrations:

no

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

For control group

Doses / concentrations 2

Dose / conc.:

1 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

15 mg/kg bw/day (nominal)

Results and discussion

Results of examinations

Mortality:

no mortality observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

< 1 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

other: • inflammatory changes in liver and focal adverse effect on development of spermiogenesis

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

1.      Clinical Observation and Body Weight

All the animals during observation period were without any visible signs of intoxication. There were no treatment-related deaths of animals in the study. Occurrence of diarrhoea (exist only 1 day) in all animals (from all groups) between 37^th– 72^ndday of test substance application was observed. This observation was not related to test substance MCPA.

Body weight of all animals moderate increased. After statistical evaluation of the body weight, significant differences between control group and dose groups were not detected.

2.      Ophtalmoscopic Examinations

ophtalmoscopic examinations were performed prior to test substance administration and at study termination.

Eye surround — clear, without oedema, scars, mattery matrix’s, border eyelashes into correct position, activity is correct, tear-duct are standard. Basic position of eyeball and activity are sans pathology. Eye — adequate large, calm, position physiological, activity unlimited, color without pathological changes. Cornea — safe, silky, sans oedema and undulation. Sclera - light, sans bulg and scars. Camera anterior — accordingly deep, clear, iris with saved desing and color. Pupilla — adequate respond to light, black, central stored, round. Lens — transparent, without fog. Reflex of camera posterior is normal, target of optical nerve limited in niveau of the retina, rose — white. Retina is rose, vessels adequate, reflect well, peripheral of the retina without focus changes, section of the macula lutea sans dystrophic changes, reflex of foveolar adequate, green — blue.

The finding in frontal and in background segment of eye in all animals under to Ophtalmoscopic examination consider to physiological.

No changes in Ophtalmoscopic examination before application of the MCPA and at study termination were registered.

3.      Hematological Results and Discussion

The statistical evaluation of results shows slight (but statistical significant) decrease of hemoglobin and eosinophils in males dose group 15 mg*kg^-1in sample time 4 against control group (in this case hemoglobin value of control group was increased against starting value). In females of dose groups 1 and 5 mg*kg^-1in sample time 2 was registered statistical significant decrease of eosinophils (increased value was also in control group). Hemoglobin concentrations in females dose group 15 mg*kg^-1in sample time 3 were significant decreased in comparison with control group. The increased MCV was registered in males (dose group 1 mg*kg^-1) in all sampling times against starting values.. After 60 days of MCPA application significant decrease of MCHC against starting values was registered in all groups (include control groups). Statistical significant decrease of lymphocytes against starting values was registered in males of control group and dose group 15 mg*kg^-1in sample time 3. In females, dose group 1 mg*kg^-1significant increase of RBC against starting value was registered after 90 days of application the test substance. In females of the highest dose group significant decrease MCHC against starting value from 60 day of application to 90 day and in satellite animals (sample time 4) was recorded too. Significant decrease of eosinophils number against starting values in sample time 3 was registered in females dose group 1 mg*kg^-1.

Described changes have not direct context with MCPA substance. According experiences from other studies performed in testing facility, similar variance in values of hematological parameters are periodically meet. It has been probably a consequence of nonspecific reactions of animals and only with statistical but not physiological significance.

4.      Clinical Chemistry Results and Discussion

The results showed the significant increased activities of enzyme ALT. The elevation of ALT against control group has been noticed in males dose group 5 mg*kg^-1after 30 and 90 day of MCPA application. The elevation of ALT activities against control group and starting values was registered in males dose group 15 mg*kg^-1after 30 and 60 day, too. The increased activities of ALT in females dose group 15 mg*kg^-1after 30 and 60 day against starting values and after 60 days of application against control and starting value were determined. Reduced activity of ALP was registered in males dose groups 5 and 15 mg*kg^-1from sample time 1. Decrease of activity ALP was registered in dose group 1 mg*kg^-1 in sample times 2 and 3. Females have reduced activity of ALP in all dose groups, too (Table 33-34). In males of dose groups 5 and 15 mg.kg-' was registered significant increased concentration of urea against control group and starting value from 30 day of treatment. After 90 day was observed significant increase of urea only against control group. Significant increased concentration of urea against control group and starting value was registered in females dose group 15 mg*kg^-1from sample time 1 to sample time 3. The urea values of females dose group 5 mg*kg^-1were significant increased against control group after 60 and 90 day of MCPA application. In all doses of MCPA highly increased concentration of creatinine was observed. In all mentioned cases the changes were reversible. At the end of 28-day recovery period animals of the highest dose satellite group have shown biochemical parameters on normal level. After 60 day of test substances application males and females dose group 15 mg.kg-1 showed decreased glucose level. This decrease was temporal and mild, but statistical significant.

Other monitored biochemical parameters ( AST, Chol, TAG, TBil, TP, Alb, Ca, Phos, Na, K, Cl) had no changes or singular changes. These changes have no direct connection with test substance application. They are results of intra-individual and inter-individual variability, nonspecific reaction of animals, eventually have only statistical character.

The test substance MCPA applied to Beagle dog already after 30 days caused increase of ALT activities, decreasing of parameter ALP and elevation of urea and creatinine. Present trend continued till the end of MCPA application. Dependence on dose is evident. In the case of ALP and creatinine changes were recorded in all dose groups. In the cases of ALT and urea in dose groups 5 and 15 mg*kg^-1. Decrease in glucose concentration was observed only in animals of dose group 15 mg*kg^-1.

Changes were reversible, mentioned biochemical parameters in all animals of satellite groups were on normal level. Different response to administration of MCPA in males and females was registered. Males responded more sensitive. Obtained results indicated that target organs of MCPA toxicity are liver and kidney.

5.      Pathology Results and Disscusion

5.1 Statistical Analysis

The relative weights of the right and left testis were significant decreased in the males from highest dose group treated at 15 mg/kg/day in comparison with controls. Relative weights of other organs in the treated animals were not significantly different from those in the control group.

5. 2. Gross Pathology

The ovaries cysts were found in females No 31 (Control group) and No 36 (low dose group). The samples of ovaries with cysts were prepared for microscopically examination.

5. 3. Histopathology

Liver

Focal hepatocellular necrosis, inflammatory changes, mononuclear nodules were observed predominantly in treatment animals. The inflammatory changes were found in the parenchyma of liver and in the portal tract too. They were observed commonly in male from high dose group (15 mg/kg/day MCPA). In the control group they were less common.

Lungs

In female No 28 from control group inflammatory lesions were found markedly. The alveolar septa were infiltrated with degenerative mononuclear, multinucleate cells and macrophages contained hemosiderin.

Kidneys

The calcium deposits in the lumen of medulla tubules were observed mostly in females No 26 (control group), 35 (low dose group), 41 (high dose group) and in male No 20 (high dose group). These foci were small, individual tubules only. The surrounding tissue of kidney was microscopically normal. Sporadic glomerular atrophy was found in female No 28 (control group), No 35 (low dose group) and No 41 (high dose group) .

Testes

Testicular atrophy and occurrence of the multinucleate giant cells were observed in male No 17. In this dog there was absence of spermatids in the epididymal tubules. In male No 18 there was observed absence of spermatogenic cells in several tubules. Both animals were from the highest dose group. Sertoli cells and Leydig cells were not affected. Small focal absence of spermatogenic cells in tubules of testis was found in males No 11 (low dose group), No 16 (medium dose group) and No 21 (satellite high dose group).

Ovary

In females No 25, 31 (control group) and No 36 (low dose group) cysts were found. They had variable sizes. The cysts wall of ovaries (females No 25 and 36) was consisted of inner thin layer of fibrous connective tissue, a central wide zone of granulosa-luteal cells and a capsule of ovarial stroma, which was compressed. The cysts in female No 31 were lined by a single layer of flattened epithelial cells. The ovarial cortex with follicles was found.

Esophagus

Small inflammatory focus in the mucosa in female No 27 (control group) was observed.

Stomach

In female No 26 (control group) focal necrosis of epithelial cells in mucosa was found.

Spinal cord

Small hemorrhages in the anterior horn in female No 25 and in female No 26 in the pars intermedia was observed. Both females were from control group.

Skin

In female No 41 sporadic inflammatory change in the sweat gland was observed.

Spleen

The findings of granular haemosiderin in spleen were less frequently in males than in females. We did not find any difference between controls and high dose groups. In the remain organs and tissues no histopathologic changes were observed.

 

5. 4. Discussion of Results

This study indicate, that MCPA produce adverse effect in liver and testes. Occurrence of inflammatory changes in liver was most common in males from high dose group. They were smaller at high dose group in female dogs. The incidence of liver lesions was decreased in lower dose groups. In the control group they were minimal frequency. Similar histopathologic changes were observed in liver dogs treated with 2,4-D. Decrease testes weights at high dose level in testis right minus 17% and testis left minus 23% versus control were registered. This fact correlated with the focal testicular atrophy, loss of spermatogenic cells and fusion of spermatids to form multinucleate giant cells in testes. Small focal absence of spermatogenic cells was observed in male of lower dose group and in satellite high dose group, too. MCPA had adverse effect on development of spermiogenesis. Ovarian cysts were observed in two females from control group and in one female from low dose group. Lutein cysts or cystic corpora lutea are occasionally found in the ovaries of several species, especially the dog. They are most often found in nulliparous bitches. The pathogenic mechanism involved in the production of lutein cysts is not known. Inclusion cysts arise from ovarian surface epithelium. Microscopically they are variable in size and consist of a single loculated fluid-filled space usually lined by a single layer of flattened epithelial cells. Inclusion cysts are more commonly found in older individuals. The finding of focal calcification in the kidneys in animals of the control group were found as well as in the treated animals. The nature and frequency of these lesions in the females from the highest dose group were similar to control group. Renal calcifications in the dogs may be seen in animals in which there is no other concurrent renal changes. Focal mineralization in the medulla is a frequent finding in the rat, dog and cat and may occasionally be seen in other species of laboratory animals. The pathogenesis of their development and their relationship to renal calculi, however, has not been clearly defined. They were not related to the effect of test substances. In the pathomorfologic examination of other organs and tissues from dogs non extensive changes were stated. These changes appeared in the organs of dogs from control groups most often.

Applicant's summary and conclusion

Executive summary:

CONCLUSION

For a consideration acquired from this study, it may be stated:

·        there were no deaths during the study

·        mean body weight of doses groups were similar to those of control

·        the test substance MCPA had no effect on hematological parameters

·        obtained results from biochemical examinations indicated that target organs are liver and kidney

·        inflammatory changes in liver and focal adverse effect on development of spermiogenesis

·        the NOAEL (no-observed-adverse-effect-level) of MCPA in oral administration to Beagle dogs for 90 days is under1 mg*kg^-1/day.