Registration Dossier

Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
02 December 2011 - 07 May 2012
Reliability:
2 (reliable with restrictions)
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 for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other:
Deviations:
no
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
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
Qualifier:
according to guideline
Guideline:
other: See other Guidelines under "Principles of method if other than guideline"
Deviations:
no
Principles of method if other than guideline:
In addition, the procedures in this study essentially conformed 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 (incl. QA statement)
Remarks:
issued 19th May 2011
Limit test:
no

Test material

Constituent 1
Reference substance name:
40766-30-1
Cas Number:
40766-30-1
IUPAC Name:
40766-30-1
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Phenyl-tolyl-ethane
- Substance type: clear, colourless liquid
- Purity: ≥ 95%
- Lot/batch No.: PTE-01
- Expiration date of the lot/batch: 17 October 2012
- Storage condition of test material: at room temperature in the dark under nitrogen
- Cas No.: 40766-30-1
- Specific gravity / density: 0.987 – 1.00 g/mL (20°C)
- Stability at higher temperatures: Yes, maximum temperature: 130°C, maximum duration: 24 hours under nitrogen seal. Stability in polyethylene glycol At least 24 hours (information provided by the Sponsor). As part of this study, stability for at least 6 hours at room temperature was confirmed over the used concentration range of 2 to 20 mg/mL (NOTOX Project 498194).

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
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 11 weeks.
- Weight at study initiation: mean weight range at start of treatment was 276-323 gr (males) or 182-214 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.
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
- Temperature (°C): 18.6 – 23.1
- Humidity (%): 27 - 88
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12
Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity (with a maximum of 4 hours). Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of pupillary reflex tests in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

IN-LIFE DATES: From 02 December 2011 to 07 May 2012

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level.
The container with formulation was flushed with nitrogen. Adjustment was made for specific gravity of the vehicle (factor: 1.125), but not for the relative density of the test substance which is very close to 1.00 g/mL (range: 0.987-1.00 g/mL) at 20°C.

Storage conditions of formulations: At ambient temperature

Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.

Dose volume: 5 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:
Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level.
The container with formulation was flushed with nitrogen. Adjustment was made for specific gravity of the vehicle (factor: 1.125), but not for the relative density of the test substance which is very close to 1.00 g/mL (range: 0.987-1.00 g/mL) at 20°C.

Storage conditions of formulations: At ambient temperature

Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
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.
- 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 staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
Duration of treatment / exposure:
Main males were exposed for 29 days, i.e. 2 weeks prior to mating, during the mating period and up to the day prior to necropsy.
Main females were exposed for 43-46 days, i.e prior to mating, during mating, during the post-coitum and lactation periods, and up to the day prior to necropsy.
Recovery males were exposed during the same period as Main males (i.e. for 29 days), followed by a 14-days treatment-free recovery period.
There were two cohorts Recovery females in this study. Treatment of the 1st cohort Recovery females was erroneously stopped together with that of the Recovery males (i.e. after 29 days), followed by a 15-days recovery period. Therefore, a 2nd cohort Recovery females was added to this study. This 2nd cohort Recovery females was exposed for a period comparable to that of the Main females (i.e. for 47 days), followed by a 14-days recovery period.
Three females (Group 1, 3 and 4 resp.) were not dosed during littering.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Once daily for 7 d/w.
Duration of test:
Males: 29 days
Females: 43-46 days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected in consultation with the sponsor based on the results of the dose range finding study.
Three females per group were dosed with 100, 300 or 1000 mg/kg bw daily for 14 days (5 mL/kg body weight/day). No mortality occurred at 100 mg/ kg bw/day, but females in other groups died or were killed in extremis in the first eight days of the experiment. In the group dosed with 100 mg/kg bw/day several clinical signs were noted. These clinical signs were noted at least immediately after dosing. Therefore, clinical observations in the main study were conducted immediately after dosing. Also, deviations in body weight gain were noted for two females in this group. No further macroscopic changes, changes in organ weights of livers and kidneys or changes in food or water consumption in this group were found.

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 (see also respective paragraphs). Only females with live offspring were selected.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule: Daily, detailed clinical observations were made in all animals. Based on the lack of a clear peak effect seen for animals in the dose range finding
study, these clinical observations were at least conducted within 30 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.

BODY WEIGHT:
Yes
- 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, except for Main males and Main females which were housed together for mating and for Main females without evidence of mating. Food consumption of mated Main 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 (iso-flurane)
- 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.
- 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:
- Yes, at end of treatment urinalysis was performed for selected Main males/group, Recovery males, and 2nd cohort Recovery females; At end of recovery, urinalysis was done for Recovery males, 1st cohort and 2nd cohort Recovery females.
- Urine samples were collected overnight (approximately 15-20 hours). During the sampling period animals were deprived of food but water was available. Urine was collected into a specimen vial, using a metabolism cage.
- Parameters checked were: According to test guidelines

FUNCTIONAL OBSERVATIONS:
Yes
- Time schedule for examinations: The selected males and females were tested at the end of treatment/ recovery.
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: 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:
SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5-7.
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 WEIGTHS
No.
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 (Ref. 1; 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 (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 3) was applied to frequency data.

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. 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.

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 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).
Ref. 3 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
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

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Four main females and One Recovery female (1st cohort) treated at 100 mg/kg bw/day showed hunched posture and/or piloerection on several days of treatment. There were no clinical signs of toxicological relevance for females up to 30 mg/kg bw/day.
Several animals showed increased salivation after dosing, which was not considered to be a sign of systemic toxicity.
Several incidents were noted, but they were found not to be related to exposure to the test substance:
(1) one Recovery female (2nd cohort) at 100 mg/kg bw/day had a single adenoma in the female mammary gland area.
(2) one Recovery female (2nd cohort) at 100 mg/kg bw/day suffered from an eye abnormality, most probably caused by damage during orbital blood sampling at the end of the treatment period.
Mortality:
no mortality observed
Description (incidence):
No substance related mortality occurred
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in body weights up to 100 mg/kg bw/day that were considered toxicological relevant. Reduced terminal body weights for Main females at 100 mg/kg bw/day were slight and were not considered to be toxicologically relevant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted.
Food efficiency:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related changes of haematology parameters were noted for (paired) Main females only at end of treatment. These changes included:
- Slightly higher red blood cells at 10 mg/kg bw/day (not statistically significant), 30 and 100 mg/kg bw/day.
- Lower reticulocytes at 10 and 30 mg/kg bw/day (not statistically significant), and 100 mg/kg bw/day.
- Lower red blood cell distribution width (RDW) at 30 and 100 mg/kg bw/day.
- Slightly higher haemoglobin at 10, 30 and 100 mg/kg bw/day.
- Lower platelets for females at 100 mg/kg bw/day.
No comparable changes were seen for (nulliparous) Recovery females. All other statistically significant changes recorded during this study were considered to be of no toxicological relevance as they were only very slight, remained within the range considered normal for rats of this age and strain and/or were noted at the end of the recovery period only. These changes included higher eosinophils and lower activated partial thromboplastin time (APTT) for 2nd cohort Recovery females at 100 mg/kg bw/day (all at end of treatment), and lower mean corpuscular haemoglobin (MCH) for 1st cohort Recovery females at 100 mg/kg bw/day (all at end of recovery). The changes in blood parameters noted for Main females at the lower dose levels of 10 and 30 mg/kg bw/day were not considered adverse due to the absence of any corroborative changes at the organ level.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
End of treatment
- Lower glucose for Main females at 100 mg/kg bw/day.
- Lower potassium for Main females at 30 and 100 mg/kg bw/day.
- Lower calcium for Main females at 100 mg/kg bw/day.
In the absence of any correlating organ weight changes and/or microscopic findings, these clinical biochemistry alterations are not considered adverse.
Endocrine findings:
not examined
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis did not reveal any toxicologically relevant change in females up to 100 mg/kg bw/day.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
Changes in motor activity were not dose related, consisted of increase in females, and were not accompanied by clinical signs and were thus considered not to be toxicologically significant.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The following (statistically significant) changes in organ weights distinguished treated animals from control animals at end of treatment:
- Increased thyroid weights (absolute and relative to body weight) for females at 10, 30 and 100 mg/kg bw/day. At the end of the two weeks recovery period, all organ weights for treated animals were in the normal range again. No microscopic findings correlating to the changes in organ weights were recorded, therefore the increased thyroid weights (absolute and relative to body weight) for Main females at 10, 30 and 100 mg/kg bw/day at end of treatment were not regarded as toxicologically relevant. In addition, the thyroid weight values of all groups were within normal limits with the concurrent control values at the lower end.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic examination at necropsy did not reveal any toxicologically relevant alterations that were related to treatment up to 100 mg/kg bw/day.
A thickened uterus and nodular yellowish contents in the cervix were noted for all four females with total litter loss at necropsy. Subsequent histopathological examination revealed that the nodular contents were former implantation sites located in the adjacent part of the female reproductive tract; the uterus. This latter finding together with the thickened uterus was considered to be related to the earlier pregnancy. Other isolated findings noted among control and/or treated animals were considered to be of no toxicological significance, since they were seen in control animals only, occurred without a doserelated distribution and/or remained within the range of biological variation for rats of this age and strain treated under comparable conditions. These findings included a yellowish soft nodule on the epidymides, reduced size of testes and preputial glands (each both sides), accentuated lobular pattern of the liver, tan or red-brown discoloration of the thymus, irregular surface of the glandular mucosa of the stomach, alopecia of the different parts of the body, red-brown or reddish foci on the clitoral gland, and enlarged mandibular lymph nodes.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related microscopic findings were present in (paired) Main females in the spleen.
In the spleen, hemopoietic foci were decreased in Main Group 4 females treated at 100 mg/kg bw/day (2/4 minimal, 2/4 slight) compared to control Main Group 1 females (2/5 moderate, 3/5 marked), Main Group 2 females at 10 mg/kg bw/day (2/5 slight, 2/5 moderate, 1/5 marked) and Main Group 3 females at 30 mg/kg bw/day (2/5 slight, 3/5 moderate). The rather subtle decrease in hemopoietic foci in spleens for females from Main Group 1 to Main Groups 2 and 3 was not considered to be an adverse effect but rather a normal variation seen as a consequence of pregnancy. However, in the (nulliparous) Recovery Group females there was no difference in hemopoietic foci between the control Recovery Group 1 (3/5 minimal) and Recovery Group 4 (2/5 minimal, 1/5 slight) animals. Pregnancy normally leads to an increase in hemopoietic foci in the spleens of female rats as seen in Main Groups 1 to 3. The hemopoietic foci seen in Main Group 4 treated females more closely resembles the amount present in nulliparous females. This may indicate the normal increase seen at pregnancy is blocked by the test item. Since the Recovery Group animals were not mated, the possible recovery from this could not be adequately checked.
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY
No substance related mortality occurred.

CLINICAL SIGNS
Four main females and One Recovery female (1st cohort) treated at 100 mg/kg bw/day showed hunched posture and/or piloerection on several days of treatment. There were no clinical signs of toxicological relevance for females up to 30 mg/kg bw/day.
Several animals showed increased salivation after dosing, which was not considered to be a sign of systemic toxicity.
Several incidents were noted, but they were found not to be related to exposure to the test substance:
(1) one Recovery female (2nd cohort) at 100 mg/kg bw/day had a single adenoma in the female mammary gland area.
(2) one Recovery female (2nd cohort) at 100 mg/kg bw/day suffered from an eye abnormality, most probably caused by damage during orbital blood sampling at the end of the treatment period.

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
Changes in motor activity were not dose related, consisted of increase in females, and were not accompanied by clinical signs and were thus considered not to be toxicologically significant.

BODY WEIGHTS
There were no changes in body weights up to 100 mg/kg bw/day that were considered toxicological relevant. Reduced terminal body weights for Main females at 100 mg/kg bw/day were slight and were not considered to be toxicologically relevant.

FOOD CONSUMPTION
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted.

HAEMATOLOGY
Treatment-related changes of haematology parameters were noted for (paired) Main females only at end of treatment. These changes included:
- Slightly higher red blood cells at 10 mg/kg bw/day (not statistically significant), 30 and 100 mg/kg bw/day.
- Lower reticulocytes at 10 and 30 mg/kg bw/day (not statistically significant), and 100 mg/kg bw/day.
- Lower red blood cell distribution width (RDW) at 30 and 100 mg/kg bw/day.
- Slightly higher haemoglobin at 10, 30 and 100 mg/kg bw/day.
- Lower platelets for females at 100 mg/kg bw/day.
No comparable changes were seen for (nulliparous) Recovery females. All other statistically significant changes recorded during this study were considered to be of no toxicological relevance as they were only very slight, remained within the range considered normal for rats of this age and strain and/or were noted at the end of the recovery period only. These changes included higher eosinophils and lower activated partial thromboplastin time (APTT) for 2nd cohort Recovery females at 100 mg/kg bw/day (all at end of treatment), and lower mean corpuscular haemoglobin (MCH) for 1st cohort Recovery females at 100 mg/kg bw/day (all at end of recovery). The changes in blood parameters noted for Main females at the lower dose levels of 10 and 30 mg/kg bw/day were not considered adverse due to the absence of any corroborative changes at the organ level.

CLINICAL BIOCHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
End of treatment
- Lower glucose for Main females at 100 mg/kg bw/day.
- Lower potassium for Main females at 30 and 100 mg/kg bw/day.
- Lower calcium for Main females at 100 mg/kg bw/day.
In the absence of any correlating organ weight changes and/or microscopic findings, these clinical biochemistry alterations are not considered adverse.

URINALYSIS
Urinalysis did not reveal any toxicologically relevant change in females up to 100 mg/kg bw/day.

MACROSCOPIC EXAMINATION
Macroscopic examination at necropsy did not reveal any toxicologically relevant alterations that were related to treatment up to 100 mg/kg bw/day.
A thickened uterus and nodular yellowish contents in the cervix were noted for all four females with total litter loss at necropsy. Subsequent histopathological examination revealed that the nodular contents were former implantation sites located in the adjacent part of the female reproductive tract; the uterus. This latter finding together with the thickened uterus was considered to be related to the earlier pregnancy. Other isolated findings noted among control and/or treated animals were considered to be of no toxicological significance, since they were seen in control animals only, occurred without a doserelated distribution and/or remained within the range of biological variation for rats of this age and strain treated under comparable conditions. These findings included a yellowish soft nodule on the epidymides, reduced size of testes and preputial glands (each both sides), accentuated lobular pattern of the liver, tan or red-brown discoloration of the thymus, irregular surface of the glandular mucosa of the stomach, alopecia of the different parts of the body, red-brown or reddish foci on the clitoral gland, and enlarged mandibular lymph nodes.

ORGAN WEIGHTS
The following (statistically significant) changes in organ weights distinguished treated animals from control animals at end of treatment:
- Increased thyroid weights (absolute and relative to body weight) for females at 10, 30 and 100 mg/kg bw/day. At the end of the two weeks recovery period, all organ weights for treated animals were in the normal range again. No microscopic findings correlating to the changes in organ weights were recorded, therefore the increased thyroid weights (absolute and relative to body weight) for Main females at 10, 30 and 100 mg/kg bw/day at end of treatment were not regarded as toxicologically relevant. In addition, the thyroid weight values of all groups were within normal limits with the concurrent control values at the lower end.

MICROSCOPIC EXAMINATION
Treatment-related microscopic findings were present in (paired) Main females in the spleen.
In the spleen, hemopoietic foci were decreased in Main Group 4 females treated at 100 mg/kg bw/day (2/4 minimal, 2/4 slight) compared to control Main Group 1 females (2/5 moderate, 3/5 marked), Main Group 2 females at 10 mg/kg bw/day (2/5 slight, 2/5 moderate, 1/5 marked) and Main Group 3 females at 30 mg/kg bw/day (2/5 slight, 3/5 moderate). The rather subtle decrease in hemopoietic foci in spleens for females from Main Group 1 to Main Groups 2 and 3 was not considered to be an adverse effect but rather a normal variation seen as a consequence of pregnancy. However, in the (nulliparous) Recovery Group females there was no difference in hemopoietic foci between the control Recovery Group 1 (3/5 minimal) and Recovery Group 4 (2/5 minimal, 1/5 slight) animals. Pregnancy normally leads to an increase in hemopoietic foci in the spleens of female rats as seen in Main Groups 1 to 3. The hemopoietic foci seen in Main Group 4 treated females more closely resembles the amount present in nulliparous females. This may indicate the normal increase seen at pregnancy is blocked by the test item. Since the Recovery Group animals were not mated, the possible recovery from this could not be adequately checked.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
body weights were slightly lower for pups at 30 mg/kg bw/day (males only) and 100 mg/kg bw/day (both sexes) as compared to control pups on lactation Day 1.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
Treatment related effects on early postnatal pup development were noted. The number of dead and living pups at first litter check, postnatal loss and viability index were affected at 100 mg/kg bw/day:
- The average number of dead pups per litter at first litter check was 3.0 compared to 0.0 in the control group.
- The average number of living pups per litter at first litter check was 6.7 compared to 10.7 in the control group.
- Postnatal loss comprised 18 pups of 5 litters compared to 1 pup of 1 litter in the control group.
- The viability index was 61.7% compared to 99.1% in the control group.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Treatment related effects on early postnatal pup development were noted. The number of dead and living pups at first litter check, postnatal loss and viability index were affected at 100 mg/kg bw/day:
- The average number of dead pups per litter at first litter check was 3.0 compared to 0.0 in the control group.
- The average number of living pups per litter at first litter check was 6.7 compared to 10.7 in the control group.
- Postnatal loss comprised 18 pups of 5 litters compared to 1 pup of 1 litter in the control group.
- The viability index was 61.7% compared to 99.1% in the control group.
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
Treatment related effects on early postnatal pup development were noted. The number of dead and living pups at first litter check, postnatal loss and viability index were affected at 100 mg/kg bw/day:
- The average number of dead pups per litter at first litter check was 3.0 compared to 0.0 in the control group.
- The average number of living pups per litter at first litter check was 6.7 compared to 10.7 in the control group.
- Postnatal loss comprised 18 pups of 5 litters compared to 1 pup of 1 litter in the control group.
- The viability index was 61.7% compared to 99.1% in the control group.
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Early postnatal pup development:
Treatment related effects on early postnatal pup development were noted. The number of dead and living pups at first litter check, postnatal loss and viability index were affected at 100 mg/kg bw/day:
- The average number of dead pups per litter at first litter check was 3.0 compared to 0.0 in the control group.
- The average number of living pups per litter at first litter check was 6.7 compared to 10.7 in the control group.
- Postnatal loss comprised 18 pups of 5 litters compared to 1 pup of 1 litter in the control group.
- The viability index was 61.7% compared to 99.1% in the control group.
In addition, body weights were slightly lower for pups at 30 mg/kg bw/day (males only) and 100 mg/kg bw/day (both sexes) as compared to control pups on lactation Day 1.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
Remarks on result:
other: Body weights were slightly lower for pups at 30 mg/kg bw/day (males only) and 100 mg/kg bw/day (both sexes) as compared to control pups on lactation Day 1. At 100 mg/kg bw/day, the numbers of dead and living pups were increased and decreased, respectively

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
10 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

REPRODUCTIVE DATA

No toxicologically relevant effects on reproductive parameters were noted. All paired females were mated, resulting in a mating index of 100% for treated and control groups. There were 10, 10, 10, and 8 pregnant females in the control, 10, 30, and 100 mg/kg bw/day groups, respectively. Examination of the reproductive organs of the non-pregnant females and their paired males did not reveal any abnormalities. Assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis. Therefore, the lower fertility and conception indices for the high dose group was not regarded as treatment related. Precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

GESTATION

The gestation index and duration of gestation were unaffected by treatment up to 100 mg/kg bw/day. There were only four females at 100 mg/kg bw/day with life offspring (8 pregnant females in total), the gestation index for the high dose group was only 50%. Examination of the reproductive organs of the nonpregnant females and their paired males did not reveal any abnormalities. Assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis. Therefore, the incidence of two non-pregnant females in the high dose group was not regarded as treatment related.

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 and no deficiencies in maternal care were observed.

EARLY POSTNATAL PUP DEVELOPMENT

Treatment related effects on early postnatal pup development were noted. The number of dead and living pups at first litter check, postnatal loss and viability index were affected at 100 mg/kg bw/day:

- The average number of dead pups per litter at first litter check was 3.0 compared to 0.0 in the control group. - The average number of living pups per litter at first litter check was 6.7 compared to 10.7 in the control group.

- Postnatal loss comprised 18 pups of 5 litters compared to 1 pup of 1 litter in the control group.

- The viability index was 61.7% compared to 99.1% in the control group. In addition, decreased body weights of pups (both sexes) were noted at 30 and 100 mg/kg bw/day.

MORTALITY PUPS

At 100 mg/kg bw/day, in total 21 pups of 3 litters were found dead at first litter check and 18 pups of 5 litters were found dead, killed in extremis or missing during the first two days of lactation. In addition, no pups were found for one female. This latter female had been pregnant as indicated by the increased body weights and confirmed at necropsy by the presence of 12 implantation sites in the uterus. No late resorptions were found. Therefore, it is most likely that she had cannibalized her litter shortly after delivery. In one litter only 2 out of 13 pups were found alive at first litter check. Due to the bad condition of these two pups (they were cold and had no milk in the stomach), they were euthanized on lactation Day 1. Other pups (indicated as “missing”) were lost by cannibalism during the lactation period. In total 4 litters were lost completely. At the lower dose levels of 10 and 30 mg/kg bw/day, 1 pup/1 litter and 2 pups/2 litters, respectively, were found dead or missing. In the control group, only one pup was missing. This occurrence was within normal limits.

CLINICAL SIGNS PUPS

Clinical signs of pups found dead or missing consisted of little or no milk in the stomach, and cold appearance. For one surviving control pup a missing tiptoe on the right foot was noted from lactation Day 4 onwards. This finding was not related to treatment. Another pup at 30 mg/kg bw/day had a blue spot on the head from lactation Day 4 to Day 6. This finding is more often seen for pups of this age. At the single occurrence in this study and in the absence of a treatment-related distribution, it was considered to be of no toxicological relevance.

BODY WEIGHT PUPS

Body weights were slightly lower for pups at 30 mg/kg bw/day (males only) and 100 mg/kg bw/day (both sexes) as compared to control pups on lactation Day 1. On Day 4 of lactation, body weights of treated and control pups (both sexes) were within comparable ranges.

MACROSCOPY PUPS

Absence of milk in the stomach was recorded for all dead pups, except for three dead pups in one litter from group dosed to 100 mg/kg bw/day. These latter dead pups could not be evaluated due to either severe autolysis or cannibalism. No macroscopic abnormalities were noted for the two pups that were killed in extremis. Incidental macroscopic findings among surviving pups included the absence of milk in the stomach. 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.

Applicant's summary and conclusion

Conclusions:
In an oral OECD 422 screening study with rats, the parental NOAEL was determined to be 30 mg/kg bw/day, while the developmental NOAEL was determined to be 10 mg/kg bw/day. Parental toxicity was evident at 100 mg/kg bw/day as seen by clinical signs, changes in haematology parameters and concurring decreased hemopoietic foci in the spleen. Developmental toxicity was observed at 30 and 100 mg/kg bw/day, as seen by an increased number of dead pups at 100 mg/kg bw/ day, clinical signs and decreased body weight for pups at 30 and 100 mg/kg bw/ day.
Executive summary:

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted according to OECD guidelines and GLP principles. Phenyl-tolyl-ethane was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 10, 30 and 100 mg/kg bw/day. No significant and toxicologically relevant changes in body weight gain were noted for any of the groups. At 100 mg/ kg bw/ day, heamatological parameters including increased red blood cells with corresponding slightly increased haemoglobin and decreased reticulocytes with corresponding decreased red blood cell distribution width (RDW), and decreased platelets were noted in females, which correlated with decreased hematopoietic foci in the spleen. Based on these data, the parental NOAEL for PTE was established to be 30 mg/kg bw/ day. Mortality of the pups occurred at 100 mg/kg bw day and reduced body weight of pups was found at 30 and 100 mg/ kg bw/ day, therefore the NOAEL for Development was established at 10 mg/ kg bw/ day.