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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2011-11-28 - 2012-07-26
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.Nevertheless, according to the ECHA's practical guide 6: "How to report read-across and categories" the maximum for read-cross is 2.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Molecular formula C15H16
Molecular weight 196.29
CAS Number 40766-30-1
Description Clear colourless liquid (determined at NOTOX)
Batch PTE-01
Purity ≥ 95%
Test substance storage At room temperature in the dark under nitrogen
Stability under storage conditions Stable

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 11 wks
- Weight at study initiation: (P) Males: 295-300 g; Females: 197-209 g
- Fasting period before study: no
- Housing: Pre-mating: groups of 5 animals/sex/cage; Mating: one-to-one-basis
- Diet (e.g. ad libitum):Free access to pelleted rodent diet
- Water (e.g. ad libitum):Free access to tap-water
- Acclimation period:5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C):18.6 – 23.1°C
- Humidity (%):27 - 88%)
- Air changes (per hr):15
- Photoperiod (hrs dark / hrs light):12 hours artificial light and 12 hours darkness per day

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
Method: Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Frequency: Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females,
one female was cohabitated with one male of the same treatment group,
avoiding sibling mating. Detection of mating was confirmed by evidence
of sperm in the vaginal lavage or by the appearance of an intravaginal
copulatory plug. This day was designated Day 0 post-coitum. Once
mating occurred, the males and females were separated.
Recovery animals were not mated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (22 February 2012), according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
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.
Females nos. 53 (Group 1), 79 and 83 (Group 3) and 93 (Group 4) were not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day
with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 30 and 100 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
4 main groups: ten male and ten female per dose
2 recovery groups: five males and five females
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:twice daily, at least conducted within 30 minutes after dosing
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations:Males and females were weighed on the first day of exposure and weekly thereafter. Mated main 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 AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OTHER:
Clinical laboratory investigations were performed at the following time points:
End of treatment
- Haematology and clinical biochemistry parameters for selected Main animals/sex/group,
Recovery males, and 2nd cohort Recovery females
- Urinalysis for selected Main males/group, Recovery males, and 2nd cohort Recovery females;
no urinalysis was performed for the selected Main females/group)
End of recovery
- Haematology, clinical biochemistry and urinalysis parameters for Recovery males, 1st cohort
and 2nd cohort Recovery females.
Sperm parameters (parental animals):
From the selected 5 Main males of the control and high dose group (see Allocation), and all males
suspected to be infertile, additional slides of the testes were prepared to examine staging of
spermatogenesis.
Litter observations:
Each litter was examined to determine the following, if practically possible:
Mortality / Viability: 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. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe,
were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).
Clinical signs: At least once daily, detailed clinical observations were made for 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.
Postmortem examinations (parental animals):
All males (Main and Recovery), Recovery females and the selected Main females/group were deprived of food overnight (with a minimum of 20 hours) prior to planned necropsy, but water was provided. Non-selected Main females were not deprived of food.
Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The numbers of former implantation sites and corpora lutea were recorded for all paired Main females. Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral
phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).

Selected Main animals/sex/group, all Recovery animals and male no. 40 (Main Group 4) that died spontaneously:
Ovaries
Adrenal glands (Pancreas)
(Aorta) Peyer's patches [jejunum, ileum] if detectable
Brain - cerebellum, mid-brain, cortex Pituitary gland
Caecum Preputial gland
Cervix Prostate gland
Clitoral gland Rectum
Colon (Salivary glands - mandibular, sublingual)
Coagulation gland Sciatic nerve
Duodenum Seminal vesicles
Epididymides Skeletal muscle
Eyes (with optic nerve (if detectable) and Harderian
gland)
(Skin)
Spinal cord -cervical, midthoracic, lumbar
Female mammary gland area Spleen
Femur including joint Sternum with bone marrow
Heart Stomach
Ileum Testes
Jejunum Thymus
Kidneys Thyroid including parathyroid if detectable
(Lacrimal gland, exorbital) (Tongue)
(Larynx) Trachea
Liver Urinary bladder
Lung, infused with formalin Uterus
Lymph nodes - mandibular, mesenteric Vagina
(Nasopharynx) All gross lesions

All remaining animals, Main females which failed to deliver and Main females with total litter loss4:
Cervix Preputial gland
Clitoral gland Prostate gland
Coagulation gland Seminal vesicles
Epididymides Testes
Mammary gland area Uterus
Ovaries Vagina
All gross lesions
Postmortem examinations (offspring):
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.
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.
Reproductive indices:
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant 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.
Following reproductive parameters were determined:
Mating index (%)
Fertility index (%)
Conception index (%)
Gestation index (%)
Duration of gestation
Percentage live males at
First Litter Check
Percentage live females at First Litter Check
Percentage of postnatal loss
Days 0-4 of lactation
Offspring viability indices:
(Number of live pups on Day 4 post-partum/Number of pups born alive) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
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 males up to 100 mg/kg bw/day and females
up to 30 mg/kg bw/day.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
There were no changes in body weights up to 100 mg/kg bw/day that were considered toxicological
relevant.No toxicologically relevant changes in food consumption before or after allowance for body weight
were noted.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS):
No toxicologically relevant effects on reproductive parameters were noted.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS):
Assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.
No toxicologically relevant effects on reproductive parameters were noted.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
No toxicologically relevant effects on reproductive parameters were noted.

ORGAN WEIGHTS (PARENTAL ANIMALS):
The following (statistically significant) changes in organ weights distinguished treated animals from
control animals at end of treatment:
- Increased liver weights (absolute and relative to body weight) for males at 100 mg/kg bw/day.
- Decreased epididymides weights (absolute) for males at 100 mg/kg bw/day.
- 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.

GROSS PATHOLOGY (PARENTAL ANIMALS):
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.

HISTOPATHOLOGY (PARENTAL ANIMALS):
Treatment-related microscopic findings were present in (paired) Main females in the spleen and in
males in the liver. 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.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.
In male liver, centrilobular hypertrophy was present at increased incidence in Main Group 4 males at
100 mg/kg bw/day (5/6 minimal) compared to control Main Group 1 (1/5 minimal) and Main Group 2
and Main Group 3 males at 10 and 30 mg/kg bw/day, respectively (both 0/5). After the 14-day
treatment free recovery period this finding was completely absent in the Recovery Group animals.

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproduction toxicity was observed up to 100 mg/kg bw/day.

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
effects observed, treatment-related

Details on results (F1)

VIABILITY (OFFSPRING):
Treatment related pup mortality was observed at 100 mg/kg bw/day.At 100 mg/kg bw/day, in total 21 pups of 3 litters (nos. 88, 90 and 95) were found dead at first litter
check and 18 pups of 5 litters (nos. 88, 90, 93, 94 and 95) were found dead, killed in extremis or
missing during the first two days of lactation. In addition, no pups were found for one female (no. 87).
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 (no. 88)
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 (nos. 87,
88, 94 and 95) 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.

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.

CLINICAL SIGNS (OFFSPRING):
Clinical signs of pups found dead or missing consisted of little or no milk in the stomach, and cold
appearance.

BODY WEIGHT (OFFSPRING):
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.

SEXUAL MATURATION (OFFSPRING):
The sex ratio was unaffected by treatment up to 100 mg/kg bw/day.

GROSS PATHOLOGY (OFFSPRING):
Absence of milk in the stomach was recorded for all dead pups, except for three dead pups in litter 94.
These latter dead pups could not be evaluated due to either severe autolysis or cannibalism.
No macroscopic abnormalities were noted for the two pups in litter 88 that were killed in extremis (for
details on clinical signs, see previous section Mortality).

HISTOPATHOLOGY (OFFSPRING):
The mammary gland area from three out of four females
with total litter loss was examined at the microscopic level and no abnormalities were noted that could
indicate an impaired milk production. All examined mammary glands had proteinaceous contents in
their ducts.

OTHER FINDINGS (OFFSPRING)

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL : 30 mg/kg bw/day.
Reproduction NOAEL : 100 mg/kg bw/day.
Developmental NOAEL : 10 mg/kg bw/day.
Executive summary:

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of Phenyl-tolyl-ethane in rats by oral gavage followed by a 14-day recovery period was performed.

Parental toxicity was evident at 100 mg/kg bw/day. At 100 mg/kg bw/day, five females had hunched posture and/or piloerection on several days during treatment. Toxicologically relevant changes in haematology parameters were noted for (paired) Main females at 100 mg/kg only. These changes included slightly increased red blood cells with corresponding slightly increased haemoglobin. In addition, decreased reticulocytes with corresponding decreased red blood cell distribution width (RDW), and decreased platelets were noted. These latter changes are in line with the microscopic observation of decreased hemopoietic foci in the spleen of Main females at 100 mg/kg bw/day. It should be noted that for (nulliparous) Recovery females, there were no haematology changes. Moreover, no difference was noted in hemopoietic foci in the spleen from control Recovery females (3/5 minimal) and Recovery females at 100 mg/kg bw/day (2/5 minimal, 1/5 slight). 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 females at 100 mg/kg bw/day 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 investigated. 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. Several clinical biochemistry parameters were affected by treatment with Phenyl-tolyl-ethane. Decreased cholesterol, glucose, potassium and calcium, and higher creatinine, chloride and sodium were recorded for males or Repro females at 30 and/or 100 mg/kg bw/day at end of treatment, and higher chloride for males at 100 mg/kg bw/day at end of recovery. In the absence of any correlating organ weight changes and/or microscopic findings, these clinical biochemistry alterations were not considered adverse. The increased liver weights (absolute and relative to body weight) recorded for males at 100 mg/kg bw/day at end of treatment correlated to the microscopic finding of increased centrilobular hypertrophy. No underlying mechanistic cause for centrilobular hypertrophy of the liver could be established based on the examinations conducted in this study. Most likely it was an adaptive response to the treatment (enzyme induction). Complete recovery of the liver was observed after the 14-day treatment free period. Without any correlating microscopic findings, the decreased epididymides weights (absolute) for males at 100 mg/kg bw/day and 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. No parental toxicity was observed at 10 and 30 mg/kg bw/day. Reproductive results: No reproduction toxicity was observed up to 100 mg/kg bw/day. Developmental results: Developmental toxicity was observed at 30 and 100 mg/kg bw/day. At 100 mg/kg bw/day, the numbers of dead and living pups were increased and decreased, respectively, at first litter check. In addition, postnatal loss was increased with consequently decreased viability index. These findings were primarily due to four dams with total litter loss on Days 1 and 2 of lactation.

Clinical signs of pups at 100 mg/kg bw/day found dead or missing (after first litter check) consisted of little or no milk in the stomach, and cold appearance. At necropsy, absence of milk in the stomach was recorded for all dead pups that could be evaluated. No underlying cause could be established based on the examinations conducted in this study. The mammary gland area from three out of four females with total litter loss was examined at the microscopic level and no abnormalities were noted that could indicate an impaired milk production. All examined mammary glands had proteinaceous contents in their ducts. Furthermore, there were no signs for dams neglecting their 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 were within comparable ranges. No developmental toxicity was observed at 10 mg/kg bw/day. In conclusion, treatment with Phenyl-tolyl-ethane by oral gavage in male and female Wistar Han rats at dose levels of 10, 30 and 100 mg/kg bw/day bw/day revealed parental toxicity at 100 mg/kg bw/day and developmental toxicity at 30 and 100 mg/kg bw/day. No Reproductive toxicity was observed up to 100 mg/kg bw/day. Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived: Parental NOAEL : 30 mg/kg bw/day. Reproduction NOAEL : 100 mg/kg bw/day. Developmental NOAEL : 10 mg/kg bw/day.