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Diss Factsheets

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:
25 May - 13 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
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
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
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
Guideline:
other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.
Deviations:
no
Qualifier:
according to guideline
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

Constituent 1
Reference substance name:
phenol, 4-(phenylamino)-, sulfurised, leuco deriv.
Molecular formula:
not applicable
IUPAC Name:
phenol, 4-(phenylamino)-, sulfurised, leuco deriv.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system 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 12 weeks.
- Weight at study initiation: mean weight at start of treatment was 339 gr (males) or 215 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.
- 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: 25 May - 13 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 approximately 5 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. Correction was made for the purity of the test substance (16.4%).
- Storage conditions of formulations: At ambient temperature.
- Justification for use and choice of vehicle: Based on trial formulations performed at WIL Research Europe B.V. 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 13 (06 June 2012) and at the end of treatment (09 July 2012), using gravimetry. Samples of formulations of Day 13 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). 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 41-49 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). Two females of Group 2 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 499625; Based on the results of this range finding study, dose levels selected for the main study (28 days toxicity study) were 100, 300 and 1000 mg/kg body weight. No clinical signs indicative of toxicity 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, immediately (0-15 minutes) 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 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

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 20 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 approximately 26.5 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals 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 (manyto-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.

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
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period. Black staining of the tail or back, and black faeces as seen at 100, 300 and/or 1000 mg/kg were considered to be related to staining properties of the test substance (a black paste). The black staining of the tail correlated microscopically with external black-brown material on the surface of the tail skin, and was most likely due to the presence of remnants of the test substance in the faeces. Laboured respiration was shown by one female at 1000 mg/kg on two days of the post coitum phase only. The incidence of scabs, alopecia and rales among the dose groups 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. These were therefore considered signs of no toxicological relevance.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in body weights and body weight gain were noted. The slightly lower body weight gain of males at 1000 mg/kg throughout the treatment period (achieving statistical significance on all occasions, as well as for body weights on Day 15) was very minor in nature. The statistically significant lower body weight gain of males at 100 mg/kg occurred in the absence of a dose-related trend, and was also minor in nature. No toxicological relevance was therefore ascribed to these changes.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control animals.
Food efficiency:
no effects observed
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
Description (incidence and severity):
No toxicologically relevant changes occurred in haematological parameters of treated rats. The statistically significant lower prothrombin time (PT) of males at 100 and 300 mg/kg occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats
of this age and strain. The higher reticulocyte count of females at 1000 mg/kg also remained within the range considered normal for rats of this age and strain, and occurred in the absence of any supportive changes in red blood cell parameters. These changes in haematological parameters were therefore considered to be of no toxicological relevance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant higher bilirubin levels were noted in males and females at 1000 mg/kg (for females due to one higher value for animal no. 72), and in males also at 100 and 300 mg/kg (not statistically significant for males at 100 mg/kg). Any other statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and
remained within the range considered normal for rats of this age and strain. These changes consisted of lower glucose levels in males at 1000 mg/kg, higher aspartate aminotransferase activity (ASAT) in males at 300 mg/kg, lower sodium and chloride levels in males at 300 mg/kg and lower inorganic phosphate levels in males at 100 mg/kg.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals. Motor activity was similar across the dose groups. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes were noted in organ weights and organ to body weight ratios. The statistically significant higher testes and epididymides to body weight ratio at 1000 mg/kg was attributed to the lower terminal body weights. The statistically significant higher testes to body weight ratio at 300 mg/kg, the lower brain weight of males at 300 mg/kg, and higher kidney weights of females at 1000 mg/kg were minor in nature and occurred in the absence of a dose-related trend (for testes weight when uncorrected for body weight). The statistically significant higher uterus weight at 1000 mg/kg remained within the range considered normal for rats of this age and strain, and was not supported by any morphological lesions. No toxicological relevance was therefore ascribed to these changes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy did not reveal any toxicologically relevant alterations. Black discolouration of the tail noted for all males at 1000 mg/kg was attributed to staining properties of the test substance (see Clinical Signs), and to be of no toxicological relevance. Other incidental findings among control and treated animals were within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and included pelvic dilation of the kidneys, red discolouration of the thymus or mandibular lymph nodes, scab formation on the skin, alopecia, enlarged clitoral glands, gray-white foci on the liver, and a watery-clear cyst on the ovaries.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic findings noted. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis. There were no morphological findings in the reproductive organs of either sex which could be attributed to treatment.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
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

Key result
Critical effects observed:
no

Any other information on results incl. tables

Analysis of dose preparations

The mean concentrations analysed in the formulations of Group 2, Group 3 and Group 4 prepared for use on Day 13 were 83, 83 and 99% of target concentration 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 68, 52 and 83% of target concentration respectively. Results for Group 2, Group 3 and Group 4 were corrected for the mean of Group 1 and their sample weight.

 

Homogeneity(gravimetry) 

The coefficient of variation for the formulations of Group 2, Group 3 and Group 4 prepared for use on Day 4 were 19, 6.3 and 1.1% respectively.

 

Stability (IR spectrophotometry)

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:
In a combined repeated dose study with reproduction/developmental toxicity screening (OECD 422) in rats the derived NOAEL for the parental animals was >= 1000 mg/kg bw.
Executive summary:

In a combined repeated dose and reproduction/developmental study according to OECD 422 five rats/sex/group were administered once daily at dose levels of 0, 100, 300 and 1000 mg/kg bw/d by oral gavage. 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 41-49 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 s cheduled necropsy). Two females of Group 2 were not dosed during littering.

No mortality occurred and no relevant clinical signs of toxicity were noted during the observation period. Black staining of the tail or back, and black faeces as seen at 100, 300 and/or 1000 mg/kg were considered to be related to staining properties of the test substance (a black paste). There were no relevant differences in funtional observations, body weight and food consumption when compared to the control group.

Changes in haematological parameters and clinical biochemistry were considered to be of no toxicological relevance. Necropsy did not reveal any toxicologically relevant alterations. The changes in organ weights and organ to body weight ratios were not considered to be of toxicological relevance.No treatment related microscopic findings were noted.

Based on the results of this OECD 422 study in rats, where no parental toxicity was observed, the NOAEL was determined to be the highest dose level applied (1000 mg/kg bw/d).