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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Already evaluated by the Competent Authorities for Biocides and Existing Substance Regulations.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
. Method developed by the US NTP specifically for the surposes of this study.
Deviations:
yes
Remarks:
. See below.
Principles of method if other than guideline:
The study deviated from 'Directive 88/302/EEC B.26 Subchronic 90-Day Oral Toxicity Study in Rodents' as follows;

No additional top dose group or control animals group were included in the study for observation of recovery from toxic effects after the treatment period.

Ophthalomological examinations were only carried out where the eyes showed clinical signs of gross abnormalities.

General eye examinations of the control and high dose group were not carried out.

Sensory activity and signs of neurotoxicity were not determined towards the end of the study. The study was conducted prior to this requirement being included in the guidelines. However, signs of reproductive toxicity were included in the test methodology.

Haematological, clinical chemistry and urinalysis parameters were not investigated.

Histopathological examinations did not include the aorta.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
Lot/Batch number: 533344
Description: Blue, crystalline solid
Purity: Not reported
Stability: Stable at room temperature

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
Species: Mouse
Strain: B6C3F1
Source: Simonsen Laboratories, Gilroy, California, USA
Sex: Male and Female
Age/weight at study initiation: Test animals were approximately 6 weeks old at study initiation. Male mean bodyweights ranged from 20.9-21.6 g, mean female bodyweights ranged from 17.1-18.6 g.
Number of animals per group: In the study, groups of 10
animals per sex were tested at each dose level.
Control animals: Yes (10 males and 10 females).

Administration / exposure

Route of administration:
oral: feed
Details on oral exposure:
Feed mix was available ad libitum throughout the study period. Doses were based on a preliminary 2-week feed study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Preparation of active ingredient in feed: Copper sulphate was mixed with NIH-07 Open Formula Diet in meal form.
Homogeneity analysis were conducted on the copper sulphate feed mixture using inductively coupled plasma-atomic emission spectroscopy. Samples taken prior to study initiation and twice during the study, confirmed homogeneity between feed mixtures.
Duration of treatment / exposure:
92 days
Frequency of treatment:
7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1000, 2000, 4000, 8000 or 16000 ppm in the feed (providing estimated intakes of 0, 44, 97, 187, 398 and 815 mg Cu/kg bw/day in males and 0, 52, 126, 267, 536 and 1058 mg Cu/kg bw/day in females).
Basis:
nominal in diet
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: none

Examinations

Observations and examinations performed and frequency:
Clinical signs:

Yes - test animals were observed weekly for clinical signs

Mortality:

Yes - test animals were observed twice daily for mortality/morbidity.


Body weight:

Yes - Individual bodyweights were recorded prior to the start of the study, on Day 1 and weekly thereafter.


Food consumption:

Yes - test animals were observed once weekly for food consumption.


Water consumption:

Not reported.


Ophthalmoscopic examination:

See histological examinations.


Haematology

No haematology parameters were investigated


Clinical Chemisty

No clinical chemistry parameters were investigated


Urinalysis

No urinalysis investigation was carried out
Sacrifice and pathology:
Organ Weights:

liver, kidneys, adrenals, testes, epididymides, uterus, ovaries, thymus, spleen, brain, heart

Gross and histopathology:

Number of animals: Complete necropsies were performed on all animals in the control and high dose groups and on all other animals that died early
Time point: See above
Parameters: adrenal glands, brain (three sections), esophagus, eyes (if grossly abnormal) femur with marrow, gross lesions, heart, intestines (large: cecum, colon, rectum: small: duodenum, jejunum. Ileum), kidneys, liver, lung/mainstream bronchi, lymph nodes (mandibular, mesenteric) mammary gland, nasal cavity and turbinates (three sections), ovaries, pancreas, parathyroid glands, pharynx (if grossly abnormal), pituitary gland, preputial or
clitoral glands, prostate gland, salivary glands, spinal cord/sciatic nerve (if neurological signs were present), spleen, stomach (forestomach, glandular stomach), testes (with epididymis) thymus, thyroid gland, trachea, urinary bladder and uterus.Other examinations

Supplemental histological examination:

To characterise the distribution of copper in the liver and kidney, sections of both organs from selected males and females were stained for copper using the rhodanine method.

In order to determine the nature of the proteinaceous droplets (see in previous study on rats) sections from selected animals were stained for carbohydrate (PAS method), protein (Mallory-Heidenhain method), lipofuscin (AFIP method) and a-2-microglobulin (immunochemistry). Perl's stain for iron was used to stain sections of spleen from mice in all groups .

Other examinations:
Sperm morphology and vaginal cytology:

Sperm morphology and vaginal cytology evaluations were performed on rats from the 0, 500, 200 and 4000 ppm groups (10 animals per sex and dose group). The method employed was as follows:

National Toxicology Program (NTP) 1987. Technical Protocol for Sperm Morphology and Vaginal Cytology Evaluations in Toxicity Testing for Rats and Mice, 10/31/82 version. Research Triangle Park, N.C.

Females: 12 days prior to sacrifice, the vaginal vaults of 10 individuals per dose group were lavaged and the aspirated lavage fluid and cells stained with Toluidine Blue. Relative numbers of leukocytes, nucleated epithelial cells and large squamous epithelial cells were determined and used to ascertain estrous cycle stage.

Males: Sperm motility was evaluated at necropsy. The left testis and epididymis were weighed, the tail of the epididymis was removed from the epididymis body and weighed. Test yolk was applied to slides and a small incision made in the cauda. The sperm effluxing from the incision were dispersed in the buffer on the slides, and the number of motile and non-motile spermatozoa counted for five microscopic fields per slide. Following motility determination, each left cauda were placed in phosphate buffered saline solution for sperm density determination with a hemacytometer.
Statistics:
The following statistical procedures were followed:

Dunnet, C.W. 1955. A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50, 1095-1121

Williams, D. A. 1971. Biometrics, 27, 103-117

Williams, D.A. 1972. The comparison of several dose levels with a zero dose control. Biometrics 28, 519-531

Shirley, E. 1977. A nonparametric equivalent of William's test for contrasting increasing dose levels of a treatment. Biometrics 33, 386-389

Dun, O.J. 1964. Multiple comparisons using rank sums. Technometrics 6, 241-252

Jonckheere, A.R. 1954. A distribution free k-sample test against ordered alternatives. Biometrika, 41, 133-145

Dixon & Massay 1951 Introduction to Statistical Analysis, McGraw-Hill Book Co.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
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
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Clinical signs:

No clinical signs of toxicity, considered to be substance related, were observed in male or female mice during the course of the study.

Mortality:

No mice in any of the dose groups died or were killed before the end of the 13-week study.

Body weight gain:

Mice exhibited a dose-related growth depression which resulted in more severe body weight depression at higher dose levels. Final mean bodyweights and bodyweight gains were slightly lower than those of the control group for males in the 4000 ppm group and were significantly lower for males and females in the 8000 and 16,000 ppm groups. See attached Table 1.

Food consumption and compound intake:

For both sexes in all dose groups, the average daily feed consumption was similar to, or exceeded that of the controls. The average daily compound consumption increased proportionally with increasing concentrations of copper sulphate pentahydrate in the feed. See attached Table 1.


BLOOD ANALYSIS

Haematology: Not applicable

Clinical chemistry: Not applicable

Urinalysis: Not applicable


SACRIFICE AND PATHOLOGY

Organ weights:

Significant decreases in organ weights were noted for the heart and kidney of high dose (16,000 ppm) male mice and the thymus and kidney of the high does females. In addition, dose related decreases in absolute liver weights were noted for males and females, with significant decreases occurring
in both sexes in the 8000 and 16,000 ppm groups and the 4000 ppm group in males. Generally relative organ weights for males and females in all dosed groups were greater than that of the controls, and many of these increases were significant for the higher dose groups. These changes in
absolute and relative organ weights could be attributed to the lower final mean weights of mice in the higher doses. See attached Table 1.

Gross and histopathology:

Chemical related gross lesions were limited to the forestomach of seven male and four female mice in the 16,000 ppm groups. This lesion was characterised as a focal white discolouration of the squamous mucosa in the area of the limiting ridge where it forms a junction with the glandular gastric mucosa. Histopathological findings included minimal to mild squamous cell hyperplasia with hyperkeratosis of the forestomach mucosa at the site of the limiting ridge. This lesion was present in male and female mice receiving 4000 ppm test substance or greater. There was no evidence of inflammation or erosion/ulceration in the forestomach, and there was no increase in hyperplasia or hyperkeratosis in other portions of the forestomach mucosa. See attached Table 2.

Supplemental histological examination:

The livers and kidneys of male mice in all groups and female mice in the control group and 16,000 ppm were stained for the presence of copper. Positive staining was limited to the livers of high-dose male and female mice. Staining was extremely minimal and consisted of only a few positive
staining hepatocytes in the entire liver section. Hepatocytes staining positive for copper contained a maximum of approximately 10 red granules per cell. Due to limited number of cells stained, no distribution of copper was apparent. There was no staining of livers in the lower doses or in the controls, and no staining was present in the kidneys of any mice.

Because of the reduction in iron in the spleen of rats (see Rat Repeat Dose Toxicity), additional sections of spleen from four mice in each dosed and control group were stained for iron. There was no difference between dosed and control mice in the amount of iron-positive granules in the spleen.

Sperm Morphology and Vaginal Cytology:

No significant findings were noted in males or females in any dose group. See attached Table 3.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Sex:
male/female
Basis for effect level:
other: Absence of hyperplasia and hyperkeratosis of the forestomach
Dose descriptor:
LOAEL
Effect level:
2 000 ppm
Sex:
male/female
Basis for effect level:
other: Presence of hyperplasia and hyperkeratosis of the forestomach

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
LOAEL: 2000 ppm for males and females

NOAEL: 1000 ppm for males and females
Executive summary:

Materials and Methods

The aim of the study was to examine the effect of copper sulphate (0,1000, 2000, 4000, 8000 or 16,000 ppm) administered to male and female B6C3F1mice in feed for 13 weeks.  The test organisms were observed throughout the study for signs of clinical toxicity, mortality, bodyweight changes and food consumption.  At the end of the study period all animals were sacrificed and subject to pathological examinations, sperm morphology and vaginal cytology. The study was conducted to a methodology developed by the US National Toxicology Programme specifically for the test.  The study was conducted in accordance with GLP.

 

Results and Discussion

There were no mortalities or signs of clinical toxicity observed in any of the test species during the duration of the study.  Opthalmoscopic examinations revealed no abnormalities at any dose level tested.  At gross pathology, significant decreases in heart and kidney weight were noted in the high dose males in the thymus and kidneys of high dose females.  There was also a significant decrease in liver weights in both sexes in the 8000 and 16000 ppm dose groups.  Chemical related gross lesions were limited to the forestomach of 7 male and 4 females in the 16000 ppm dose group. Histopathological findings included minimal to mild squamous cell hyperplasia with hyperkeratosis of the forestomach mucosa at the site of the limiting ridge.  Minimal positive staining for copper was present in the liver and was limited to the high-dose male and female mice.  No significant findings were noted following examination of the sperm morphology and vaginal cytology.