C-M5

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 15 March 2007 and 01 September 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: approximately five to eight weeks old
- Weight at study initiation: At the start of treatment the males weighed 151 to 196g, the females weighed 128 to 156g
- Fasting period before study: No
- Housing: The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Diet (e.g. ad libitum): A pelleted diet (Rodent 5LF2 (Certified) was used
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study
- Acclimation period: The animals were acclimatised for seven days during which time their health status was assessed


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Distilled water

The test material was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of C-M5 in the test material formulations was determined spectrophotometrically.
Samples:
The test material formulations were diluted with water to give a final, theoretical test material concentration of approximately 0.01 mg/ml.
Standards:
Standard solutions of test material were prepared in water at a nominal concentration of 0.01 mg/ml.
Procedure:
The standard and sample solutions were analysed spectrophotometrically using the following conditions:
Spectrophotometer : Camspec M550
Wavelength : : lamda max at ~ 530 nm
Cell path length : 1 cm
Reference medium : Water
Homogeneity Determinations:
The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.
Stability Determinations:
The test material formulations were sampled and analysed initially and then after storage at approximately +4ºC in the dark for fourteen days.
Verification of Test Material Formulation Concentrations:
The test material formulations were sampled and analysed within two days of preparation

The results indicate that the prepared formulations were within ± 8% of the nominal concentrations.

Duration of treatment / exposure:

28 days.

Frequency of treatment:

The test material was administered daily

Doses / concentrations

Remarks:

Doses / Concentrations:
dose levels of 25, 150, 300 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5 male and 5 female per dose.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on results of preliminary repeat dose study.
- Rationale for animal assignment (if not random): The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Positive control:

None.

Examinations

Observations and examinations performed and frequency:

Clinical signs, bodyweight development and food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period.

Sacrifice and pathology:

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Other examinations:

functional/behavioural toxicity

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.
All data were summarised in tabular form. Where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

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:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
Mortality: There were no unscheduled deaths during the study

Clinical signs: Pink/red staining on the cage tray liners and pink/red faeces was evident throughout the treatment period for animals of either sex treated with 1000, 300 and 150 mg/kg/day. Associated episodes of generalised pink fur staining and pink stained tails were also evident in animals of either sex treated with 1000 and 300 mg/kg/day.
Episodes of pink/red faeces was evident throughout the treatment period for animals of either sex treated with 25 mg/kg/day.
Staining of faeces and on cage tray liners remained evident in recovery animals of either sex treated with 1000 mg/kg/day throughout the treatment free period.


BODY WEIGHT AND WEIGHT GAIN
Males treated with 1000 mg/kg/day showed a reduction in bodyweight gain during the first week of treatment. No such effects were detected in females from this treatment group, animals of either treated with 300, 150 and 25 mg/kg/day or recovery animals following fourteen days without treatment.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No adverse effect on dietary intake or food efficiency was detected.



FOOD EFFICIENCY
No adverse effects

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
No intergroup differences were detected


HAEMATOLOGY
No toxicologically significant effects were detected.

CLINICAL CHEMISTRY
Animals of either sex treated with 1000 mg/kg/day showed increases in plasma albumin, aspartate aminotransferase and creatinine. Females from this treatment group also showed increases in total protein, albumin/globulin ratio and calcium concentration whilst males also showed an increase in plasma cholesterol.
The effect on cholesterol continued in recovery 1000 mg/kg/day males and at this time females were similarly affected.


URINALYSIS
No toxicologically significant effects were detected.

NEUROBEHAVIOUR
Behavioural Assessment. There were no toxicologically significant changes in the behavioural assessments observed.
Functional Performance Tests. There were no toxicologically significant changes in the functional performance parameters measured.
Sensory Reactivity Assessments. There were no treatment-related changes in sensory reactivity


ORGAN WEIGHTS
Females treated with 1000 mg/kg/day showed an increase in absolute and relative liver weight compared to controls. Recovery 1000 mg/kg/day females continued to show an increase in relative liver weight however absolute liver weight was statistically reduced. Recovery 1000 mg/kg/day females also showed a reduction in absolute kidney weight and an increase in relative kidney weight.

GROSS PATHOLOGY
Discolouration was detected in the majority of tissues in animals of either sex treated with 1000, 300 and 150 mg/kg/day. A red stained right eye was also evident in one female treated with 25 mg/kg/day. Discolouration of tissues continued to be evident for recovery 1000 mg/kg/day animals following cessation of treatment

HISTOPATHOLOGY:
The following treatment-related changes were detected:
KIDNEY: Tubular basophilia/degeneration and karyomegaly of proximal tubular epithelial nuclei were seen in relation to treatment for animals of either sex treated with 1000 mg/kg/day. In addition, vacuolation of the proximal tubular epithelium was also seen for three females at this dose level. Females treated with 300 mg/kg/day were similarly affected by all conditions.
There was some indication of regression of the conditions among Recovery 1000 mg/kg/day animals of either sex following an additional fourteen days without treatment, although karyomegaly remained prevalent for both sexes and tubular basophilia was still evident for females. Such changes are degenerative in nature although removal of treatment indicated that eventual reversibility of the lesions was likely.
STOMACH: Agglomeration of secretion, mucous cell hyperplasia, and mucosal basophilia/atrophy were observed in relation to treatment in the glandular stomach of animals of either sex treated with 1000 mg/kg/day. Acanthosis and hyperkeratosis of the limiting ridge was also seen as an effect of treatment in animals of either sex at this treatment level. Agglomeration of secretion and mucous cell hyperplasia were also seen in the gastric mucosa of animals treated with 300 mg/kg/day, with agglomeration of secretion only related to treatment for males treated with 150 mg/kg/day. Acanthosis and hyperkeratosis of the limiting ridge were also observed occasionally among animals from the low and intermediate dose levels, but were not convincingly related to treatment at other than the high dose level. Each of these observed conditions is occasionally seen as a spontaneous change among control rats.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: clinical signs; mortality; body weight; food consumption; food efficiency; water consumption and compound intake; haematology; clinical chemistry; urinalysis; gross pathology; organ weights; histopathology;

2

Dose descriptor:

NOEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: See results section

3

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: See results section

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

JUSTIFICATION OF NO OBSERVED EFFECT LEVEL

Oral administration of THE TEST MATERIAL to rats for a period of twenty-eight days at dose levels of up to 1000 mg/kg/day resulted in treatment-related effects at 1000, 300 mg/kg/day and in males treated with 150 mg/kg/day. These included microscopic changes identified as tubular basophilia/degeneration, karyomegaly of proximal tubular epithelial nucleiandvacuolation of the proximal tubular epitheliumin the kidneys, agglomeration of secretion, mucous cell hyperplasia, and mucosal basophilia/atrophy in the stomach, acanthosis and hyperkeratosis of the limiting ridge and vacuolation of histiocytes in the cervical and mesenteric lymph nodes.

The “No Observed Effect Level” (NOEL) for males was therefore considered to be 25 mg/kg/day and the ‘No Observed Effect Level’ (NOEL) for females was considered to be 150 mg/kg/day.

The gastric change that was observed in 150 mg/kg/day males was generally regarded as adaptive in nature and since there was no evidence of mucosal erosion, ulceration or associated inflammatory cell infiltrates the effect was generally regarded as not being adverse. For this reason the histopathological change was considered not to represent an adverse health effect and the “No Observed Adverse Effect Level” (NOAEL) for males was, therefore, considered to be 150 mg/kg/day.

Applicant's summary and conclusion

Conclusions:

The ‘No Observed Effect Level’ (NOEL) for females was considered to be 150 mg/kg/day and the ‘No Observed Effect Level’ (NOEL) for males was considered to be 25 mg/kg/day.
The “No Observed Adverse Effect Level” (NOAEL) for males was considered to be 150 mg/kg/day.

Executive summary:

Introduction. 

The study was designed to investigate the systemic toxicity of the test material and complies with the following regulatory guidelines:

i)                   Commission Directive 96/54/EC (Method B7).

ii)                  The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003 for a twenty-eight day repeat dose oral toxicity study as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973 of Ministry of International Trade and Industry (MITI) amended 2004.

iii)                The OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted).

iv)                USAEnvironmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.

Methods.

The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD^®(SD) IGS BR strain rats, for twenty-eight consecutive days, at dose levels of 25, 150, 300 and 1000 mg/kg/day (incorporating a correction factor for 93.4% purity). A control group of five males and five females was dosed with vehicle alone (Distilled water). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, bodyweight development and food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Conclusion

The oral administration of C-M5 at dose levels of 25, 150, 300 and 1000 mg/kg/day for a period of twenty-eight consecutive days resulted in treatment related effects in animals of either sex treated with 1000 and 300 mg/kg/day and in males treated with 150 mg/kg/day. No such changes were detected in females treated with 150 mg/kg/day or in males treated with 25 mg/kg/day. The ‘No Observed Effect Level’ (NOEL) for females was, therefore, considered to be 150 mg/kg/day and the ‘No Observed Effect Level’ (NOEL) for males was considered to be 25 mg/kg/day.

The gastric change that was observed in 150 mg/kg/day males was generally regarded as adaptive in nature and since there was no evidence of mucosal erosion, ulceration or associated inflammatory cell infiltrates the effect was generally regarded as not being adverse. For this reason the histopathological change was considered not to represent an adverse health effect and the “No Observed Adverse Effect Level” (NOAEL) for males was, therefore, considered to be 150 mg/kg/day.