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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007
Reference Type:
publication
Title:
Pathology of Laboratory Rats and Mice
Author:
Cotchin, E., and Roe, F.J.C.
Year:
1967
Bibliographic source:
Blackwell Scientific Publications, pp. 440
Reference Type:
publication
Title:
Handbook of Toxicology Pathology
Author:
Haschek, W.M., and Rousseaux, C.G. (eds)
Year:
1991
Bibliographic source:
Academic Press, CA, p. 444

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Housing and Husbandry: F0 generation rats assigned to the main satellite/recovery mated portions of the study were individually housed in stainless steel, wire-bottomed cages, except during the cohabitation and postpartum periods. During cohabitation, each pair of male and female rats was housed in the male rat's cage. Beginning no later than gestation day (DG) 20, F0 generation female rats assigned to natural delivery were individually housed in nesting boxes. The study room was maintained under conditions of positive airflow, temperature and humidity were monitored and maintained. Animals were given standard diet and water ad libitum under a 12 hour light and 12 hour dark photoperiod.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Stock Preparation: A blended stock suspension was prepared once at the testing facility and stored at room temperature, protected from light. Suspensions of the blended test substance and/or vehicle for dosage administration were prepared daily and also stored at room temperature, protected from light. Prepared formulations were stirred continuously during dosage administration.

Dosage Selection: Doses were selected on the basis of results from a range-finding study. In that study there were no mortalities at doses as high as 2000 mg/kg/day. Body weight gains of the males only were reduced in the 1000 and 2000 mg/kg/day groups during the first week, and although they subsequently rebounded, the early reductions resulted in overall reduction at the end of the test period. Therefore, doses of 0, 250, 500 and 1000 mg/kg/day were selected for the definitive test.
Duration of treatment / exposure:
at least 39 days (males); through lactation day 4 (females)
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (corn oil vehicle), 250, 500 and 1000 mg/kg/day
Basis:
other: nominal
Control animals:
yes, concurrent vehicle
Details on study design:
Method Overview: Sixty male and 80 female rats were randomly assigned to four dosage groups, 15 male rates and 20 female rats per dosage group. The test material was administered orally (via gavage) on a daily basis beginning 14 days before a 14 day cohabitation period. Dosage continued through the day prior to sacrifice, after completion of the cohabitation period (minimum of 39 days of administration). In female rats, dosage continued up to and including day 4 of lactation. Male and female rats assigned to the satellite recovery portion of the study were not administered the test substance and/or the vehicle for 14 days prior to sacrifice starting when the first main study female rat assigned to the main study reached day 4 of lactation.

Examinations

Observations and examinations performed and frequency:
Parameters Evaluated: All of the main and recovery rats were examined for viability, clinical observations, detailed clinical observations (main study rats only), body weight and body weight changes and feed consumption values. Maternal behavior was observed at various times for specific purposes. In addition, each litter was evaluated for viability at least twice daily and clinical observations once daily. Each litter was counted once daily. Pup weights were recorded on lactation days (DL) 1 and 4 for litters of dams assigned to the main study hematology and clinical chemistry portion of the study. Pup body weights were also recorded periodically for various purposes. Urine and blood samples were collected for evaluation periodically and a functional observational battery (FOB) and motor activity assessment was conducted on study day (DS) 40 (male rats) or DL5 (female rats). All surviving main study rats were sacrificed on the day following the last administration of the test substance and/or the vehicle, after a minimum of 39 days of dosage. Satellite/recovery mated female rats were sacrificed on DL18. Satellite/recovery non-mated male and female rats were Sacrificed 4 days after the first main study rat reached DL4.
Sacrifice and pathology:
Necropsy: The following tissues were weighed and/or retained from all rats: liver, kidneys, testes, epididymides, seminal vesicles with coagulation gland and prostate (male only), and ovaries, vagina, a mammary gland and uterus with cervix. The following tissues were retained from the five male and female rats (main study) assigned to hematology, clinical biochemistry and histological evaluations: small and large intestines (Peyer's patches), lungs, trachea, esophagus, mandibular and mesenteric lymph nodes, peripheral nerve (sciatic), stomach, seminal vesicles with coagulating gland, spinal cord, thyroid, urinary bladder, prostate, vagina and a mammary gland, bone marrow and gross lesions. Additionally, the brain, adrenals, spleen, thymus and heart were weighed and retained for possible histological evaluation from the five male and female rats (main study) assigned to hematology, clinical biochemistry and histological evaluations. All gross lesions were examined histologically. Histological examination was performed on all (main study rats assigned to clinical chemistry evaluation only) control and high test substance dose group rats, and on the thymus and thyroids from all in the 250, 500 and 1000 mg/kg/day dosage groups. Pups that died were examined and/or preserved for future analysis. On DL4, pups from litters of dams assigned to the clinical chemistry and hematology portion of the study were sacrificed, as were pups from litters of dams assigned
to the FOB portion of the study on DL5. Necropsies were performed.

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:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
Mortality: No treatment-related mortalities were observed. One early sacrifice occurred in one male rat as the result of an injury. No treatment-related clinical signs were observed in the F0 male and female rats and F1 generation rats (main and recovery portions of the study).

Body Weight: Body weight gains of the male rats assigned to the main study were significantly reduced for the entire study (DSs 1 to 39) in the 1000 mg/kg/day dosage group. No reductions in body weight or body weight gain was observed in the male rats assigned to the recovery portion as high as 1000 mg/kg/day. No effects on body weight or body weight gain were observed in the female rats during any stage of the study at doses up to and including 1000 mg/kg/day. Summary data for the male rats is shown in the table below; full data are in the study report.

Feed Consumption: No effects were observed on the absolute or relative feed consumption for either sex a high as 1000 mg/kg/day.

Reproduction: No effects were observed on estrous, mating, fertility, natural delivery or litter observations at any dose.

Behavior and Motor Activity: No effects on any of the parameters examined in the FOB, motor activity or urinalysis were observed.

Clinical Chemistry: There were no statistically significant or biologically important differences in the urinalysis, hematology or clinical chemistry parameters.

Organ Weights: No statistically significant differences were observed in organ weights, the ratio (%) of organ weights to terminal body weights, or the ratio (%) of organ weights to brain weights in either sex at any dose.

Microscopic Effects: Treatment-related microscopic changes were observed in the thyroid of male and female rats at all dosage levels and in the thymus of the 1000 mg/kg/day dosage group female rats. No other treatment-related microscopic changes were observed. The changes in the thyroid consisted of hyperplasia and hypertrophy of the follicular epithelial cells. These microscopic changes occurred in a dose-responsive manner. According to various sources, the thyroid of rats is susceptible to environmental variations that are reversible and considered of minimal toxicological significance. Thyroid changes may be a response to stress, and therefore, a combination of dosing and stress may have contributed to the effects seen in this study (e.g., Cotchin and Roe 1967; Haschek and Rousseaux 1991). Microscopically, the thymus was reduced in size due to atrophy of the cortical and medullary lymphoid lobules in the 1000 mg/kg/day dosage group (females only). No atrophy was observed in the males of any dosage group or the females in the 250 and 500 mg/kg/day groups. Thymic atrophy or decreased cellularity is a relatively nonspecific finding in young rats, but is often associated with stress and can occur in young rats with significant decreased weight gain or loss. Total incidence of thymus and thyroid observations are shown below.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Sex:
male
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: reduced body weight gain
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Dose Group

I

II

III

IV

I

II

III

IV

Sex

Males

Females

No. Animals

5

5

5

5

5

4

5

5

Thymus

No. Examined

5

5

5

5

5

3

5

5

No. Normal

5

5

5

5

5

3

5

0

- atrophy, focal [total]

[0]

[0]

[0]

[0]

[0]

[0]

[0]

[5]

   - minimal

0

0

0

0

0

0

0

2

   - mild

0

0

0

0

0

0

0

3

Thyroid

No. Examined

5

5

5

5

4

4

5

5

No. Normal

5

4

2

1

3

2

1

- cyst(s), ultimobrancial

0

0

0

0

1

0

0

1

- hypertrophy/hyperplasia, follicular [total]

   - epithelium

[0]

[1]

[3]

[4]

[0]

[2]

[3]

[4]

   - minimal

0

0

0

1

0

2

2

2

   - mild

0

1

2

1

0

0

1

2

   - moderate

0

0

1

2

0

0

0

0

Dosage Group

I

II

III

IV

Dosage (mg/kg/d)

0

250

500

1000

Body Weight (g)

   - Day 1

345.9 ± 8.6

346.4 ± 8.9

346.9 ± 8.4

345.6 ± 6.5

   - Day 39

525.6 ± 45.0

489.0 ± 37.9

501.5 ± 31.7

478.8 ± 20.6

Applicant's summary and conclusion

Conclusions:
Based on the body weight reductions in male rats, the NOAEL was considered to be 500 mg/kg/day in male rats and 1000 mg/kg/day in female rats. The overall NOAEL is 500 mg/kg-bw/day.
Executive summary:

This study determined the sub-chronic toxicity of the test substance to rats. Groups of rats were exposed by oral gavage to concentrations of 0, 250, 500 or 1000 mg/kg/day of test substance. Males were exposed daily for 39 days, and females were exposed daily through lactation day 4. Animals were observed regularly for body weight, mortality, clinical signs, food consumption, and behaviour. At the end of the study, the animals were sacrificed and examined for gross pathology, organ weights, histopathology, clinical chemistry, hematology, and urinalysis parameters. Adverse effects were seen in the thymus at all dose levels, however, these changes are most likely stress related and are considered to be of minimal toxicological significance. Body weight reduction was seen in male rats at the 1000 mg/kg/day dose level. The LOAEL for male rats is 1000 mg/kg/day, and the NOAEL is 500 mg/kg/day. No adverse effects were seen in female rats. The NOAEL for female rats is 1000 mg/kg/day.