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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 May 2012 - 10 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.
Deviations:
no
Qualifier:
according to
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
Principles of method if other than guideline:
In addition, the procedures described in the report essentially conform to the following guidelines:
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
semi-solid (amorphous): gel
Remarks:
migrated information: paste
Details on test material:
- Name of test material (as cited in study report): C.I. Leuco Sulphur Brown 37
- Substance type: Dark brown paste
- Physical state: Paste
- Lot/batch No.: ESA 0010188
- Expiration date of the lot/batch: 01 July 2014
- Storage condition of test material: At room temperature in the dark
- Volatile: No
- Reactivity: Reactive to oxygen, risk of oxidation in contact with air
- Test substance handling: Avoid long exposure to air
- pH (1% in water, indicative range): 8.8 - 8.4

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: mean weight at start of treatment was 328 gr (males) or 208 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages. Pups were kept with the dam until termination
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES
From: 18 May 2012 - 10 July 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
- Method of formulation: Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. A correction was made for the purity of the test substance (13.5%).
- Storage conditions of formulations: At ambient temperature.
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe and on information provided by the Sponsor.
- Dose volume: 20 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on Day 4 (21 May 2012) and at the end of treatment (02 July 2012), using gravimetry. Samples of formulations of Day 4 were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Samples at end of treatment were analyzed for accuracy of preparation only. All samples were collected and analysed in duplicate. Stability in vehicle over 4 hours at room temperature could not be determined analytically (IR spectrophotometry; see Results). The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Duration of treatment / exposure:
Males were exposed for 31 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 46-53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). ( 3, 1, 2, 2 females of group 1, 2, 3, 4, respectively were not dosed during littering).
Frequency of treatment:
Once daily, 7 d/w
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of a 10-day dose range finding study (Project 499623: Based on the results of this range finding study, dose levels for the main study (28 days toxicity study) were 100, 300 and 1000 mg/kg body weight. No clinical signs were observed. Therefore, clinical observations in the main study were conducted immediately after dosing, and functional observation tests were conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals.)
- Selection of animals for selected measurements: 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology. Only females with live offspring were selected.
Positive control:
Not required.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: Daily, detailed clinical observations were made in all animals, at least immediately (0-15 min) after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.

BODY WEIGHT
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION
- Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY
Yes

WATER CONSUMPTION
No. Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION
No

HAEMATOLOGY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines, and including Met-haemoglobin.

CLINICAL CHEMISTRY
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS
No

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: According to test guidelines
Sacrifice and pathology:
GROSS PATHOLOGY
- All animals were fasted overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy were deeply anaesthetised and subsequently exsanguinated.
- Selected 5 animals/sex/group: According to test guidelines
- All remaining females which failed to deliver and the remaining males: According to test guidelines

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines
- All remaining males: Epididymides and Testes

HISTOPATHOLOGY
- According to test guidelines
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Based on subjective appraisal.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
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:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY
No mortality occurred during the study period.

CLINICAL SIGNS
No clinical signs of toxicity were noted during the observation period. Scabs and alopecia occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period.

BODY WEIGHTS
No toxicologically relevant changes in body weights and body weight gain were noted. The statistically significant lower body weight gain of males at 1000 mg/kg (whole mating period) and 300 mg/kg (Day 8 of the mating period) was minor in nature, and did not show a clear dose-related trend. No toxicological relevance was therefore ascribed to this change.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats. The statistically significant increase in relative Met-haemoglobin content of males at 1000 mg/kg (and an apparent increase among females of this dose group) was slight in nature. Also, since other red blood cell parameters were unaffected by treatment, this change was considered to be of no toxicological relevance. The statistically significant lower white blood cell count of females at 100 mg/kg was considered to be of no toxicological relevance as this change occurred in the absence of a dose-related trend and remained within the range considered normal for rats of this age and strain.

CLINICAL BIOCHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats. The statistically significant higher chloride level of males at 100 mg/kg was considered to be of no toxicological relevance as this change occurred in the absence of a dose-related trend and remained within the range considered normal for rats of this age and strain.

MACROSCOPIC EXAMINATION
Necropsy did not reveal any toxicologically relevant alterations. The incidence macroscopic findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a doserelated incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and consisted of a diaphragmatic hernia of the liver, a yellowish soft nodule on the epididymides, pelvic dilation of the kidneys, reduced size of the preputial glands, alopecia, thickened left horn of the uterus, tan or yellowish foci on the clitoral glands, tan discolouration of the clitoral glands, red foci on the glandular mucosa of the stomach and red foci on the thymus.

ORGAN WEIGHTS
No toxicologically relevant changes were noted in organ weights and organ to body weight ratios. All statistically significant changes in organ weight and organ to body weights remained within the range considered normal for rats of this age and strain, occurred in the absence of a dose-related trend, and/or had no histopathological correlates. No toxicological relevance was therefore ascribed to these changes, which consisted of a higher relative kidney and seminal vesicle weight of males at 1000 mg/kg, and higher absolute and relative ovary weight at 100 and 300 mg/kg.

MICROSCOPIC EXAMINATION
There were no microscopic findings in any of the animals suspected of infertility which could explain their lack of reproductive performance. All recorded microscopic findings were within the range of background pathology encountered in Wistar (Han) rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats. The spermatogenic staging profiles were normal for evaluated control males and males at 1000 mg/kg.

Effect levels

Dose descriptor:
NOAEL
Remarks:
Parental generation
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicity was observed up to the highest dose level tested.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The mean concentrations analysed in the formulations of Group 2, Group 3 and Group 4 prepared for use on Day 4 were 60, 87 and 88% respectively. The mean concentrations analysed in the formulations of Group 2, Group 3 and Group 4 prepared for use at the end of treatment were 84, 76 and 86% respectively. Results for Group 2, Group 3 and Group 4 were corrected for the mean of Group 1 and their sample weight. The coefficient of variation for the formulations of Group 2, Group 3 and Group 4 prepared for use on Day 4 were 26, 7.9 and 3.3% respectively. Major peaks in the IR spectra of the formulations derived from the vehicle (water and carboxymethylcellulose). The only peak that derived from the test substance was observed at 1120 cm-1. Given the minor size of this peak, no conclusion could be drawn on stability of the formulations.

Applicant's summary and conclusion

Conclusions:
Based on this OECD 422 study in rats, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg/day was derived (based on main component C.I. Leuco Sulphur Brown 37, corresponding to approximately 7407 mg test substance including C.I. Leuco Sulphur Brown 37/kg body weight).