Phenol, 4-(phenylamino)-, sulfurized, leuco deriv.

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Organisation of Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, March 1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

Deviations:

no

Test guideline 4

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

Test material information

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

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.
Lactation: Pups were kept with the dam until termination in Macrolon cages.
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 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%.

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating.
- Age at mating of the animals in the study: Approximately 14 weeks
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by the appearance of an intravaginal copulatory plug and/or by determination of the estrous stage. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).

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.

Pups were not dosed directly but could have potentially be exposed to the test substance in utero, via maternal milk or from exposure to maternal urine/faeces.

Frequency of treatment:

Once daily for 7 d/w.

Duration of test:

Males: 31 days
Females: 41-49 days

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.
- Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.
- Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.

Examinations

Maternal examinations:

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

GENERAL REPRODUCTION DATA
- Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.
- Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

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.
- The number of former implantation sites and corpora lutea were recorded for all paired females.
- Selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis: According to test guidelines
- All remaining animals and females which failed to deliver: 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

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.

SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5-7 zie rapport.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGHTS
No.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (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. (Ref. 1)
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution. (Ref. 3)
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data. (Ref. 2)
- The following additional methods of statistical analysis were used: Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 4) to determine intergroup differences followed by the Wilcoxon test (Ref. 5) 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. No statistical analysis was performed on histopathology findings.

References:
Ref. 1 Dunnett C.W., A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
Ref. 2 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Ref. 3 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).
Ref. 4 Kruskal W.H. and Wallis W.A.. Use of ranks in one-criterion variance analysis. Journal of the American Statistical Association 47 (260): 583-621, December (1952).
Ref. 5 Wilcoxon, F. Individual comparisons by ranking methods. Biometrics, 1, 80-83 (1945).

Indices:

Reproductive indices; For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring indices:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects. Remark: See remark on Bilirubin increase under Overall Remarks, Attachments.

Details on maternal toxic effects:
MORTALITY
No mortality occurred during the study period.

CLINICAL SIGNS
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.

FUNCTIONAL OBSERVATIONS
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.

BODY WEIGHTS
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
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 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
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.

MACROSCOPIC EXAMINATION
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.

ORGAN WEIGHTS
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.

MICROSCOPIC EXAMINATION
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.

REPRODUCTIVE DATA
No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. One control female (no. 49) did not mate, and one female at 300 mg/kg (no. 64) did not deliver offspring and was found to be non-pregnant based on absence of implantation sites. The incidence of these occurrences was considered unrelated to treatment.

DEVELOPMENTAL DATA
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early
postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

PARTURITION/MATERNAL CARE
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.

Details on embryotoxic / teratogenic effects:
There was no evidence of teratogenic effects based on the absence of relevant clinical signs and external macroscopic findings.

EARLY POSTNATAL PUP DEVELOPMENT
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

MORTALITY PUPS
One pup of the control group, two pups at 100 mg/kg, one pup at 300 mg/kg and two pups at 1000 mg/kg were found dead or missing during lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS PUPS
One pup of litter 68 (300 mg/kg) had a missing tail. The nature and incidence of this finding remained within the range considered normal for pups of this age, and was therefore considered to be of no toxicological relevance.

BODY WEIGHT PUPS
Body weights of pups were considered to have been unaffected by treatment.

MACROSCOPY PUPS
Absence of milk in the stomach noted for all pups from litter 80 (1000 mg/kg) was not considered to be treatment-related or toxicologically relevant since these pups survived until the scheduled necropsy, indicating they were all sufficiently fed during the first days of lactation. Also the incidence was confined to one litter. This finding is likely secondary to the time when the actual necropsies were performed, and is not attributable to treatment. The nature and incidence of a missing tail remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No toxicity was observed up to the highest dose level tested.

Fetal abnormalities

Key result

Abnormalities:

effects observed, non-treatment-related

Overall developmental toxicity

Key result

Developmental 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 an OECD 422 study with rats, no reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg). Based on these results, a reproduction No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg/day was derived (based on main component C.I. Leuco Sulphur Brown 96, corresponding to approximately 6098 mg test substance including C.I. Leuco Sulphur Brown 96/kg body weight).