Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

SAS-296:

In an OECD 422 combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test, there were no evidences of a chemical-related effect on reproduction/developmental parameters. The NOAEL for reproduction/developmental toxicity was considered to be 200 mg/kg bw/day in rats. No chemical-related effects on the estrous cycle, copulation index, fertility index, gestation length, number of corpora lutea, or number of implantation sites were found in dams. Pathological changes were not noted in the male and female reproductive organs. No chemical-related effects on the number, sex ratio, body weight, or viability were found in pups. No external or internal malformations were found in pups at any doses.

1,1 -DPE:

In an OECD 421 reproduction/ developmental toxicity screening test, there were no evidences of a chemical-related effect on reproduction/developmental parameters.

The NOAEL for reproduction/developmental toxicity was considered to be 500 mg/kg bw/day in rats. 1-Phenylethylbenzene does not appear to be toxic when dosed to rats at 500 or 100 mg/kg and does not appear to have a toxicological effect on male or female reproductive performance.

PTE:

In an OECD 422 combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test, there were no evidences of a chemical-related effect on reproduction parameters.

The NOAEL for reproduction toxicity was considered to be 100 mg/kg bw/day in rats.

Link to relevant study records

Referenceopen allclose all

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.
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system 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

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.
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
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
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
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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)
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.
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
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)
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'
Reproductive effects observed:
not specified
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.

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:
2010-11-03 - 2011-08-11
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. Nevertheless, according to the ECHA's practical guide 6: "How to report read-across and categories" the maximum for read-cross is 2.
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Relative humidity was outside protocol range. Cottonseed oil was used as the vehicle instead of corn oil. Pups were sacrificed using Fatal Plus instead of carbon dioxide.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species and strain: Albino rats: Sprague Dawley, Harlan Sprague Dawley

Quantity and Sex: Range finder: five males and five females per group for a total of 25 males and 25 females (nulliparous and non-pregnant).
Definite study: ten males and 10 females per group for a total of 40 males and 40 females (nulliparous and non-pregnant).

Weight: Range finder: Day 0: males 249-279 ; females 179-206 g.
Definite study: Day 1: males 270-309 g; females 194-223 g

Housing: individual
Environmental Controls: Temperature: 19 - 23°C; Relative humidity: 23 - 97 %; 12 hour light/dark cycle; 10-12 air changes/hour
Food: PMI Feeds, Inc, Formulab #5008
Water: Municipal water supply
Route of administration:
oral: gavage
Vehicle:
cotton seed oil
Details on exposure:
The animals were dosed by oral gavage with an appropriately sized stains - Steel ball-tipped dosing needle and syringe since gavage dosing assures the proper dose presentation. Three groups (Groups; II, Ill and IV) of ten males and ten females each were dosed daily at 1000 mg/kg. 500 mg/kg and 100 mg/kg, respectiveiv. A concurrent vehicle control aroup (Group I, ten males and ten females) was dosed with the vehicle, cottonseed oii. Groups were dosed daily at approximately the same time each day and dose amounts were adjusted weekly based on the latest body weight. All animals were dosed at a constant volume of 5 mL/kg.

Males and females were dosed for 14 days prior to mating. Males were dosed for another 14 days through mating, for a total of 28 days. Females were dosed through mating and until one day prior to termination (Lactation Day 4 for para females and Study Day 53 for nuliiparous females).
Details on mating procedure:
After fourteen days of treatment, each female was cohabited with one male from the same group for a period of two weeks or until signs of pregnancy were observed. Each morning, the female was examined for the presence of sperm or a vaginal plug. Gestation Day 0 was the day the plug or sperm was found. A maximum of fourteen days was allowed for mating. After confirmation of mating, the males were returned to their home cages, and females were put in plastic cages and nesting materials were added.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose concentrations were verified weekly.

Equipment:
1. Varian Model CP 3800 Gas Chromatograph
2. Varian Type 1177 injector
3. Varian Model CP-8200 autosampler equipped with a 10µl syringe
4. Galaxie CDS integration software
5. Column: Phenomenex ZB-1 (100 % methylphenyl siloxaine), 30 m lenght, 0.32 mm iner diameter, 1.00 µm film thickness

Preparation of sample solution:
Approximately 50 µL of 20 and 100 mg/mL dose samples and 2µ L of 200 mg/mL dose sample were added to a 10 ml volumetric flask, brought to volume with IP A and sonicated for 15 minutes. The solution was inverted to mix and transferred to an analysis vial. Test substance sample solutions in the range of 19.4 - 305 mg/mL were used. 20 mg/mL sample was diluted 0.05/10 to achieve concentration of 0.1 mg/mL. 100 mg/mL sample was diluted 0.05/10 to achieve concentration of 0.5. 200 mg/mL sampie was diluted 0.002/10 to achieve concentration of 0.04. Reported concentrations are adjusted to pre-diluted levels.

Results:
a) 20 mg/ml
Day 0: 28.4 mg/ml
Day 10: 19.66 mg/ml
Day 16: 20.46 mg/ml
Day 23: 26 mg/ml
Day 30: 22.1 mg/ml
Day 38: 19.4 mg/ml
Day 43: 19.9 mg/ml

b) 100 mg/ml
Day 0: 110.8 mg/ml
Day 10: 103.8 mg/ml
Day 16: 95.0 mg/ml
Day 23: 96 mg/ml
Day 30: 102.7 mg/ml
Day 38: 86.3 mg/ml
Day 43: 87.8 mg/ml

c) 200 mg/ml
Day 0: 286 mg/ml
Day 10: 167.9 mg/ml
Day 16: 159.0 mg/ml
Day 23: 204.5 mg/ml
Day 30: 299.5 mg/ml
Day 38: 305 mg/ml
Day 43: 191.5 mg/ml
Duration of treatment / exposure:
Males and females were dosed for 14 days prior to mating. Males were dosed for another 14 days through mating, for a total of 28 days. Females were dosed through mating and until one day prior to termination (Lactation Day 4 for para females and Study Day 53 for nuliiparous females).
Frequency of treatment:
All animals in all groups werde dosed daily.
Details on study schedule:
After a range-finder was conducted to determine dose levels, eighty rats were randomly divided into four groups with ten males and ten females per group. All animals in all groups were dosed daily. Group I animals received only the vehicle (cottonseed oil) and Groups lI -IV received the test substance orally at 1000 mg/kg, 500 mg/kg and 100 mg/kg, respectively. On Day 14 males were placed with females for mating. On Day 28, the males were sacrificed and necropsied. Tne pregnant females were dosed daily through the gestation period and through Day 4 of the lactation period. At that time, they were sacrificed and necropsied in the same manner as the males. At birth, the litters were examined and findings recorded, and the pops were sacrificed at the some time as their dams. Any females that were not pregnant or did not give birth continued to be dosed through study termination on Day 53, at which time they were sacrificed and necropsiea.
Remarks:
Doses / Concentrations:
100 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Ten males and ten females per dose group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A range finder was conducted using five males and five females per dose level (vehicle control, 2000 mg/kg, 1000 mg/kg, 250 mg/kg and 100 mg/kg. The animals were selected by a weight-stratified randomization procedure. They were dosed five days/week for two weeks. The animals were observed daily and food consumption was measured daily through study termination (Day 14). Body weights were recorded before dosing on Day 0, and on Days 7 and 14.

During the course of this study a clear effect upon weight development was noted in the high dose group (2000 mg/kg). Deaths occured at day 2 in the high dose group. Necropsy findings at 2000 mg/kg included brown crust on abdomen, red crust around muzzle, brown fluid in large intestine, pale kidneys. At 1000 mg/kg effect on weight development, slight to moderate piloerection was observed.

The definitive dose levels were selected by the Sponsor from the data collected from the range finder. The doses selected for the definitive study were 1000 mg/kg. 500 mg/kg and 100 mg/kg, each dosed at a constant volume of 5 mL/kg. The final dose solution concentrations were 200 mg/mL, 100 mg/mL and 20 mg/mL. respectively.
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations:
Body weights were recorded weekly (Days 7, 14, 21 and 28 for the males). Body weights for para females were recorded on Days 7 and 14, and on Gestation Days 0, 4, 7, 11, 14, 17 and 20, and on Lactation Days 1 and 4. Body weights for nulliparous females were recorded on Days 7 and 14, on Presumed Gestation Days 0, 4. 7, 11. 17 and 20 and on an interim day after presumed Gestation Day 20 and prior to Study Day 53 and on Study Day 53. Body weights for the pups were recorded on Lactation Days 1 and 4. Body weights were recorded at the time of discovery after death for animals that died on study.


FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
Food consumption was recorded weekly through the termination of the study with the exception of the cohabitation period.

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


OTHER:
Litter observations:
Parturition and Litter Observations

The day parturition was complete was considered Lactation Day 0. When birth was completed, the litters were sexed, examined for gross malformations, and the number of stillbirths and live pups was recorded. Tne pups were individually marked for identification. Any changes or abnormalities in nesting or nursing behavior were recorded. Body weights for the pups were recorded on Lactation Days 1 and 4, and they were re-sexed at that time. Pups were observed daily for general appearance, behavior and survival.
Postmortem examinations (parental animals):
Terminal Sacrifice

Adult animals were sacrificed by an overdose of carbon dioxide. Males were sacrificed after the mating period on Day 28. Each para female and her litter were sacrificed on their Lactation Day 4, and nulliparous females were sacrificed on Day 53 of the study. Pups were sacrificed by Fatal Plus (Mfg: Vortech. Lot: 2547, Exp: Apr 2011 . Body weights were recorded at time of sacrifice.

Necropsv and Organ Weights

A gross necropsy examination was conducted on each adult animal at the time of discovery after death or at time of sacrifice. Stillborn pups or pups dying on study were necropsied. Pups sacrificed on Lactation Day 4 were carefully examined externally for gross abnormalities. The necropsy of each adult animal included gross observations of external surfaces and all orifices, and gross observations of thoracic and abdominal cavities and their viscera. Special attention was paid to the organs of the reproductive system. The number of implantation sites and corpora lutea were recorded. The testes and epididymides of all sacrificed males and ovaries with oviducts of the sacrificed females were carefully removed, trimmed, and weighed. Ovaries with oviducts, testes, epididymides and accessory sex organs, and all organs showing macroscopic lesions were preserved. Tissues were weighed as soon as possibie to avoid dehydration of the tissues.

Preserved samples of the ovaries, testes and epididymides saved from ail animals that died during the course of the study and all animals sacrificed the control and high-dose groups were sent to Colorado Histo-Prep, P.O. Box 272577, Fort Collins, CO 80527. CLP-compliant pathologists, for analysis.

Histopathology

Histo-Prep performed a full histopathologic examination on the preserved samples of the ovaries, testes and epididymides saved from ail animals that died during the coursc of the study and all animals sacrificed in the control and high-dose groups. Special emphasis was put on tne stages of spermatogenesis In the male gonads and histopathoiogy of interstitial testicular cell structure.

Postmortem examinations (offspring):
Stillborn pups or pups dying on study were necropsied. Pups sacrificed on Lactation Day 4 were carefully examined externally for gross abnormalities.
Statistics:
The findings were evaluated in terms of the observed effects, necropsy, and microscopic findings. The evaluation includes the relationship between the dose of the test substance and the presence or absence, incidence and severity of abnormalities, including gross lesions, identified target organs, infertility, clinical abnormalities, affected reproductive and litter performance, body weight changes, effects on mortality and any other toxic effects. Accepted statistical methods were employed where appropriate, including one-way analysis of variance (ANOVA) and Tukey-Kramer's post test to identify differences between groups.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
6 animals from group II (1000 mg/kg) died on study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group II male body weights for day 7 were significantly lighter then those of control. Group II male body weights for day 28 were significantly lighter than those of group IV. When only surviving males were compared there were no significant differences.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group II male body weights for day 7 were significantly lighter then those of control. Group II male body weights for day 28 were significantly lighter than those of group IV. When only surviving males were compared there were no significant differences.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): 6 animals from group II (1000 mg/kg) died on study(3 males and 3 females).
The only clinical sign noted in males was soft stool in one animal. There were no other observed abnormalities during the general health observations.

In females observations noted prior to the gestation period were stained hair around muzzle and activity decrease. The only observation noted during the gestation period was red stain on the bedding of one animal. There were no other observed abnormalities during the general health observations.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
Male body weights
Group II - 1000 mg/kg male body weights for Day 7 were significantly lighter than those of Group I - Control (p=0.0250). Group II male body weights for Day 28 (Final) (using the terminal weights for the 3 animals that died on test) were significantly lighter than those of Group IV - 100 mg/kg (p=0.0124) and the Group II male body weight gain was significantly less than that of Group IV (p=0.0095). However, when only surviving males were compared there were no significant differences between the groups for the Day 28 body weights or the body weight gains. There were no other significant differences between the groups for the male body weights or the body weight changes.

Female body weights
There were no significant differences between groups for female body weights or body weight changes.




TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): no data

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): no data

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): no data

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
There were 7, 5, 5 and 10 females in Groups I - IV, respectively, that conceived and all had successful births.
There was no significant difference between groups for the number of stillborn births. There was no significant difference between groups for the number of dams that save birth or the number of pups delivered. There were no significant differences in corpora lutea counts among groups.



ORGAN WEIGHTS (PARENTAL ANIMALS):
Group IV - 100 mg/kg testes weights were significantly heavier than the testes weights of Group I - Control and Group II - 1.000 mg/kg. There were no significant differences between groups for the ovary weights, the number of corpora lutea counted at necropsy or the number of implantation sites counted at necropsy.


GROSS PATHOLOGY (PARENTAL ANIMALS):
Males: The only abnormal findings were from the three animals in GroupII - 1000 mg/kg that died on test and included red fluid in the stomach and small intestine and the stomach containing yellow solid. In Group 1 - Control, two males were noted as having a small testis.

Females: The only abnormal findings were from the three animals in Group II - 1000 mg/kg that died on test and included red fluid in small intestine, stomach and small intestine filled with gas and full of black fluid.


HISTOPATHOLOGY (PARENTAL ANIMALS):
The histopathology report concluded. "No test article related lesions were observed in this study. An occasional male rat had a unilateral change in the testicle consisting of a focal area with degeneration of seminiferous tubules and hypospermia in die corresponding epididymis. These are common spontaneous lesions often observed in rats. Most test article related changes are bilateral and not unilateral as observed in this study. Minimal post mortem change was present in the three males and two out of the three females that died on study.


Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mortality and reduced body weight
Clinical signs:
effects observed, treatment-related
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not specified
VIABILITY (OFFSPRING): no effects

CLINICAL SIGNS (OFFSPRING): one pup looked weak in Group II - 1000 mg/kg and one pup looked pale from Group II.

BODY WEIGHT (OFFSPRING): The individual pup weights on both Lactation Day 1 and Lactation Day 4 from Group II - 1000 mg/kg were significantly less than the pup weights from all other groups.

SEXUAL MATURATION (OFFSPRING)

ORGAN WEIGHTS (OFFSPRING): no data

GROSS PATHOLOGY (OFFSPRING): All necropsies performed on pups that were found dead had no observed abnormalities with the exception of one pup from Group IV - 100 mg/kg that had a stomach filled with black fluid.

HISTOPATHOLOGY (OFFSPRING): no data

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight
Reproductive effects observed:
not specified

Parturition and Litter Observations

 Group  Percent Conception Mean M   Mean F Mean Pups  Mean Live Pups  Mean Stillborn 
I - Control  70   6  8 15   14
 II - 1000 mg/kg  71.4  6  7 14   14
 III - 500 mg/kg  50  6  6  12  11
 IV - 100 mg/kg  100  7  7  13  13  0

Group litter total weight mean and pup individual weight mean

Group

Group litter weights mean

 

Individual pup weights mean

 

 

Lact. Day 1

Lact. Day 4

Change

Lact. Day 1

Lact. Day 4

Change

I – Control

 

84.2

122.7

38.5

6.5

10.4

3.9

II – 1000 mg/kg

75.3

104.0

28.6

5.7

8.3

2.6

III – 500 mg/kg

67.7

99.5

31.8

6.8

9.9

3.1

IV – 100 mg/kg

81.0

112.1

31.1

6.5

10.3

3.8

Mean male organ weights (g)

 

Testes

Epididymides

Group I – Control

3.7057

1.2828

Group II – 1000 mg/kg

3.6344

1.3483

Group III – 500 mg/kg

3.9781

1.3412

Group IV – 100 mg/kg

4.1879

1.3980

Mean female ovary weight (g) and number of corpora lutea and implantation sites

 

Ovaries

Corpora

Implant

Group I – Control

0.4091

19

16

Group II – 1000 mg/kg

0.3036

25

15

Group III – 500 mg/kg

0.4329

23

13

Group IV – 100 mg/kg

0.2937

20

15

Conclusions:
This study was conducted to evaluate the potential toxic effects of the test substance 1-Phenylethyibenzene when administered to rats for a minimum of 28 days. It was also conducted to determine the potential of the test substance to affect male and female reproductive performance such as gonodal function, mating behaviour, conception, parturition and early postnatal development. Based on mortality, the Lowest Observed Adverse Effect Level(LOAEL) was determined to be 1000 mg/kg of 1-Phenylethylbenzene. Based on the lack of observed abnormalities and mortality at the lower levels, the No Observed Adverse Effect Level (NOAEL) was determined to be 500 mg/ks of 1-Phenylethylbenzene. There appears to be a relationship between 1 -Phenylethylbenzene dosed at 1000 mg/kg and the mortality of the animais and the reduced individual pup weights. The test substance, 1 -Phenylethylbenzene does not appear to be toxic when dosed to rats at 500 or 100 mg/kg and does not appear to have a toxicologicai effect on male or female reproductive performance.
Executive summary:

This study was conducted to evaluate the potential toxic effects of the test substance, 1-Phenylethylbenzene, when administered to rats for a minimum of 28 days. It was also conducted to determine the potential of the test substance to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development. After a range-finder was conducted to determine dose levels, eighty rats were randomly divided into four groups with ten males and ten females per group. All animals in all groups were dosed daily. Group I animals received only the vehicle (cottonseed oil) and Groups II -IV received the test substance orally at 1000 mg/kg, 500 mg/kg and 100 mg/kg, respectively. On Day 14, males were placed with females for mating. On Day 28, the males were sacrificed ana necropsied. The pregnant females were dosed daily through the gestation period and through Day 4 of the lactation period. At that time, they were sacrificed and necropsied in the same manner as the males. At birth, the litters were examined and findings recorded, and the pups were sacrificed at the some time as their dams. Any females that were not pregnant or did not give birth continued to be dosed through study termination on Day 53, at which time they were sacrificed and necropsied. Observations for mortality were made daily and observations for signs of pharmacologic and/or toxicologic effects were made once daily. Body weights were recorded weekly throughout the study. Food consumption was monitored weekly throughout the study except during the mating period. Conception rates for Groups II-1000 mg/kg, III-500 mg/kg, and IV-lOO mg/kg were 50.0% and 100.0%. respectively and the conception rate for Group I-Control was 70.0%. Delivery was 100% successful for all groups. There was one pup out of a litter of 17 from Group III that was brn deformed. Stillborn births averaged 8%, 0%. 15% and 2% for Groups I-IV, respectively. There were no significant differences between groups for the number of stillborn births, the number of pups born, or conception rate. Six animals from Group II died on study, three males ( found dead on Days 2-4) and three females - found dead on Davs 2 and 3 . Group II average individual pup weights on Lactation Day 1 and on Lactation Day 4 were significantly lighter when compared to average individual pup weight from Groups I, III and IV. Based on mortality, the Lowest Observed Adverse Effect Level (LOAEL) was determined to be 1000 mg/kg of 1 -Phenylethylbenzene. Based on the lack of observed abnormalities and mortality at the lower levels, the No Observed Adverse Effect Level (NOAEL) was determined to be 500mg/kg of 1 -Phenylethylbenzene. There appears to be a relationship between 1-Phenylethylbenzene dosed at 1000 mg/kg and tne mortality of the animals and the reduced individual pup weights. The test substance, 1 -Phenylethylbenzene does not appear to be toxic when dosed to rats at 500 or 100 mg/kg and does not appear to have a toxicological effect on male or female reproductive performance.

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:
2002
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. Nevertheless, according to the ECHA's practical guide 6: "How to report read-across and categories" the maximum for read-cross is 2.
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crj:CD(SD)IGS
Sex:
male/female
Details on test animals or test system and environmental conditions:
no data
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
Exposure period : Males:47 days.
Females:42-45 days from 14 days before mating to day 4 of lactation.
Frequency of treatm. : Once a day
Premating exposure period
Male : 14 days
Female : 14 days
Duration of test : Males: 48 days. Females:from 14 days before mating to day 5 of lactation
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
Exposure period : Males:47 days.
Females:42-45 days from 14 days before mating to day 4 of lactation.
Frequency of treatment:
once a day
Details on study schedule:
Terminal killing: Males, day 50; females, day 4 of
lactation.
Remarks:
Doses / Concentrations:
0, 12.5, 50, or 200 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12 animals/sex/dose
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
Clinical observation performed and frequency: General
condition was observed once a day, body weights were
determined twice a week during treatment period for males
and twice a week before mating and during maiting period and
at days 0, 4, 7, 10, 14, 17 and 21 of gestation period and
at days 0 and 4 of lactation period for females, food
consumption was determined twice a week during treatment
period for males and twice a week before mating and at days
1,4,7,10,14,17 and 21 of gestation period and at days 1 and
4 of lactation for females, but food consumption was
not determined during mating period for males and females.
For all males, urinalysis was carried out at last week of
administration period. For all males and all females
after childbirth, hematology and biochemistry were carried
out at time of necropsy after 50 days for males and at 4
days after delivery for females. The males were fasted
overnight before blood sampling for blood examination.
Organs examined at necropsy.
Oestrous cyclicity (parental animals):
Count of estrus,
estrus cycle, No.of copulated, No.of pregnants, duration of
mating, gestational days, No.of corpora lutea, No.of
implantations, implantation index[(No.of
implanttations/No.of corpora lutea)x100]
Sperm parameters (parental animals):
no data
Litter observations:
No.of newborns,
gestation index[(No.of dam with live newborns/No.of pregnant
females)x100], No.of stillborns, No.of live newborns, birth
index[(No.of live newborns/No.of implantations)x100], sex
ratio of live newborns, body weight of live newborns,
viability ibdex[(No.of live newborns on day 4 after
birth/No.of live newborns)x100], and No.of external
anomalis.
Postmortem examinations (parental animals):
Organ weights measured: Brain, heart, lung, thymus, liver,
spleen, kidney, adrenal, testis and epididymus in males, and
brain, heart, lung, thymus, liver, spleen, kidney, adrenal,
thymus, ovary in females.
Organ weight was determined in 12 males and 12 females in
all dose groups.
Microscopic examination: Brain, pituitary, spinal code,
stomach, thyroid, parathyroid, submaxillary lymph node,
heart, lung, trachea, thymus, liver, spleen, kidney,
adrenal, stomach, duodenum, jejunum, ileum, pancreas, cecum,
colon, rectum, mesentery lymph node, urinary bladder,
seminal vesicle, prostate gland, testis, epididymis, femur,
mammary gland, ovary, uterus, vagina, ischiadic nerve, bone marrow,femoral biceps muscle for 12 males and 12 females in 0 and 200 mg/kg
bw:/day groups, and for liver and adrenal for 12 males and 12 females in
12.5 and 50 mg/kg bw:/day groups.
Postmortem examinations (offspring):
No.of external anomalis
Statistics:
Dunnett's or Scheffe's test for
continuous data, Chi square test for No.of copulated, No.of
impregnated, gestation index and sex ratio, Wilcoxon' test
for implantation index, No.of stillborns, birth index and
viability index.
Reproductive indices:
Count of estrus,
estrus cycle, No.of copulated, No.of pregnants, duration of
mating, gestational days, No.of corpora lutea, No.of
implantations, implantation index[(No.of
implanttations/No.of corpora lutea)x100], No.of newborns,
gestation index[(No.of dam with live newborns/No.of pregnant
females)x100]
Offspring viability indices:
No.of stillborns, No.of live newborns, birth
index[(No.of live newborns/No.of implantations)x100], sex
ratio of live newborns, body weight of live newborns,
viability ibdex[(No.of live newborns on day 4 after
birth/No.of live newborns)x100]
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Mortality: There was no mortality related to the test
substance treatment.
Clinical signs: No effects related to the test article were
apparent on clinical observation.
groups.
Body weight: Depression of body weight gain was observed in
both sexes at 200 mg/kg bw/day.
Food consumption: A tendency to decrease in food consumption
was observed in males at 200 mg/kg bw/day.
Urinalysis: Increases in urine volume and crystals, and
decreases in osmotric pressure and specific gravity in males
at 200 mg/kg bw/day.
Hematology: Extension of prothrombin time was observed in
males at 50 mg/kg bw/day or more.
Blood biochemistry: An increase in total cholesterol in
males at 50 mg/kg bw/day or more, and an increase
in gamma GTP and phospholipids, and a decrease in chlorine
in males at 200 mg/kg bw/day, and an increase in
glucose in females at 200 mg/kg bw/day.
Necropsy: Enlargement of liver was observed in 2 of 12
females at 200 mg/kg bw/day.
Organ weights: Liver weights increased in males at 50
mg/kg bw/day or more and in females at 200
mg/kg bw/day. Adernal weights decreased in males at 12.5
mg/kg bw/day or more.
Histopathology:
LIver:Centrilobular hypertrophy of hepatocytes in males at
50 mg/kg bw/day or more and in females at 200 mg/kg bw/day,
and decreases in incidence of perportal fatty
change of hepatocytes in males at 200 mg/kg bw/day.
Adrenal:Atrophy of zona fasciculata in males at 12.5
mg/kg bw/day or more, and an increase in the incidence
of hypertrophy of zona glomerulosa in males at 200mg/kg bw/day.
Reproductive and developmental parameters: No effects of
this substance were observed on reproductive performance in
males and females or on viability and body weight of
offspring. No malformations were found in offsprings in any
groups.

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Pathological findings in the liver
Dose descriptor:
LOAEL
Effect level:
12.5 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Pathological findings in the adrenal in males at 12.5 mg/kg bw/day and higher
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL for reproduction toxicity
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
Reproductive and developmental parameters: No effects of
this substance were observed on reproductive performance in
males and females or on viability and body weight of
offspring. No malformations were found in offsprings in any
groups.

Dose(mg/kg bw/day) 0 12.5 50 200
No.of newborns Mean 14.42 15.00 13.75 11.00
SD 2.15 2.22 2.01 4.41
Gestation index 100 100 100 100
No.of stillborns(Total) 1 1 0 1
No.of live newborns Mean 14.33 14.92 13.75 10.92
SD 2.19 2.27 2.01 4.29
Birth index 92.97 94.21 93.75 87.92
Sex ratio(Male/female) 1.21 0.85 1.14 1.11
Viability index 98.84 100 99.39 99.24
No.of external anomalis 0 0 0 0
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOAEL for developmental toxicity
Reproductive effects observed:
not specified
Conclusions:
In an OECD combined repeated dose toxicity study with the reproduction/ developmental toxicity
screening test, there were no evidences of the chemical-related effects on
reproduction/developmental parameters. The NOAEL for reproduction/developmental toxicity was
considered to be 200 mg/kg bw/day in rats.
Executive summary:

In an OECD combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test, there were no evidences of the chemical-related effects on reproduction/developmental parameters. The NOAEL for reproduction/developmental toxicity was considered to be 200 mg/kg bw/day in rats. No chemical-related effects on the estrous cycle, copulation index, fertility index, gestation length, number of corpora lutea, or number of implantation sites were found in dams. Pathological changes were not noted in the male and female reproductive organs. No chemical-related effects on the number, sex ratio, body weight, or viability were found in pups. No external or internal malformations were found in pups at any doses. Based on these findings, the NOAEL for reproductive/developmental toxicity was considered to be 200 mg/kg bw/day in rats. At 200 mg/kg/day, some parameters, number of implantations, implantation index, and numbers of newborns and live newborns (24%), were decreased, but not statistically significant. These values are within the range of historical control data.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is GLP compliant and has Klimisch score 1. Lowest NOAEL.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In an OECD 422 combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test with SAS-296, there were no evidences of the chemical-related effects on reproduction/developmental parameters. The NOAEL for reproduction/developmental toxicity was considered to be 200 mg/kg bw/day in rats. No chemical-related effects on the estrous cycle, copulation index, fertility index, gestation length, number of corpora lutea, or number of implantation sites were found in dams. Pathological changes were not noted in the male and female reproductive organs. No chemical-related effects on the number, sex ratio, body weight, or viability were found in pups. No external or internal malformations were found in pups at any doses. Based on these findings, the NOAEL for reproductive/developmental toxicity was considered to be 200 mg/kg bw/day in rats. At 200 mg/kg/day, some parameters, number of implantations, implantation index, and numbers of newborns and live newborns (24%), were decreased, but not statistically significant. These values are within the range of historical control data.

In the OECD Guideline 421 Reproduction/Developmental Toxicity Screening Test with 1,1`-DPE (1,1-Diphenylethane ((phenylethyl)benzene), CAS 38888-98 -1) after oral gavage in the vehicle cotton seed oil clear maternal toxicity (mortality and significantly reduced body weights) was evident at the highest tested dose of 1000 mg/kg bw/d. Therefore it may be argued that the reduced pup weights at 1000 mg/kg bw/d are related to the toxic effect of 1,1`-DPE on the maternal organism such that the observed effects are probably of an indirect (secondary) nature. Conception rates for Groups II-1000 mg/kg, III-500 mg/kg, and IV-100 mg/kg were 50.0% and 100.0% respectively and the conception rate for Group I-Control was 70.0%. Delivery was 100% successful for all groups. There was one pup out of a litter of 17 from Group III that was born deformed. Stillborn births averaged 8%, 0%. 15% and 2% for Groups I-IV, respectively. There were no significant differences between groups for the number of stillborn births, the number of pups born, or conception rate. Based on the lack of observed abnormalities and mortality at the lower levels, the No Observed Adverse Effect Level (NOAEL) was determined to be 500mg/kg of 1 -Phenylethylbenzene.

In an OECD 422 combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test with PTE, 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 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. There were no evidences of a chemical-related effect on fertility parameters up to 100 mg/kg bw/day. The NOAEL for reproductive toxicity was considered to be 100 mg/kg bw/day in rats.

In conclusion, based on the already existing study results for effects on sexual function and fertility for the substances SAS-296, 1,1 -DPE and PTE testing of Benzyltoluene is not justified to gain a validated assessment for this endpoint for two reasons:

- The specifically elaborated read-across seem to be applicable not only from a theoretical point of view but also practically from the already existing data.

- In order not to breach Article 25 of the REACH Regulation ("In order to avoid animal testing, testing on vertebrate animals for the purposes of this Regulation shall be undertaken only as a last resort. It is also necessary to take measures limiting duplication of other tests.") testing of the substance Benzyltoluene for reproductive toxicity is scientifically unjustified and unethical since read-across data indicate that the substance Benzyltoluene is devoid of any reproductive toxicity.

Higher-tier fertility study (two-generation study) is not required at this tonnage band, since there were no adverse effects observed in the repeated dose toxicity studies in reproductive organs or tissues or any adverse effects in the screening studies for reproductive toxicity (OECD 421/422). Therefore, there is no data gap in fertility. There is no reason to believe that results of the screening study would not be relevant for fertility in humans and, therefore, for risk assessment.

"For detailed read across rationale and justification within the diphenylmethane category see section 13, Read across document"


Short description of key information:
1,1-DPE:
Reproduction / Developmental Toxicity Screening: rat (Wistar) male/female, gavage (OECD Guideline 421): NOAEL (P and F1): 500 mg/kg bw/day (male/female)

SAS-296:
Combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test: rat (Wistar) male/female, gavage (OECD Guideline 422): The NOAEL for reproduction/developmental toxicity was considered to be 200 mg/kg bw/day in rats.

PTE:
Combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test: rat (Wistar) male/female, gavage (OECD Guideline 422):
Reproduction NOAEL 100 mg/kg bw/d: based on absence of effects

Justification for selection of Effect on fertility via oral route:
OECD & EC guideline study, no deviations, GLP

Effects on developmental toxicity

Description of key information

In a prenatal developmental toxicity study, the test item Benzyltoluene was administered orally to female rats at dose levels of 30,100 or 300 mg/kg b.w./day from the 6th to 20th day of pregnancy.

Under the test conditions described, the no-observed-adverse-effect level (NOAEL) was 100 mg Benzyltoluene/kg b.w./day for the dams.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was 100 mg Benzyltoluene/kg b.w./day.

No test item-related malformation was noted at any of the test item treated groups. There was no test item-related increase in the incidence of fetal external / internal or skeletal variations at any tested dose level. Under the conditions of the study, Benzyltoluene did not show any teratogenic potential.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-08-26 - 2017-02-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 60 days
- Weight at study initiation: 197.2 - 255.2 g
- Fasting period before study: no
- Housing: singly in MAKROLON cages
- Diet (e.g. ad libitum): Commercial diet ssniff® R/Z V1324
- Water (e.g. ad libitum):ad libitum
- Acclimation period:6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): fifteen to twenty air changes per hour
- Photoperiod (hrs dark / hrs light):12 hours dark/12 hours light cycle

Route of administration:
oral: gavage
Vehicle:
cotton seed oil
Details on exposure:
Route of administration Oral, via gavage

Frequency of administration Once daily

Treatment period Day 6 to 20 of gestation

Vehicle Cotton seed oil

Administration volume 2 mL/kg b.w./day

Selection of route of administration According to OECD guideline 414

The test item formulations were freshly prepared every day.
The test item was diluted in the vehicle to the appropriate concentrations and was ad-ministered orally at a constant volume once daily from the 6th to the 20th day of gestation.
The amount of the test item was daily adjusted to the current body weight of the animal. The control animals received the vehicle at the same administration volume daily in the same way.
The male rats for mating remained untreated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method (HPLC-UV) was validated by the laboratory. The following parameters were determined:
- Linearity
- Accuracy
- Precision
- Sensitivity
- Specifity
- Stability
Details on mating procedure:
Sexually mature ('proved') male rats of the same breed served as partners. The female breeding partners were randomly chosen.
Mating was monogamous: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was taken to check for the presence of sperm. If findings were negative, mating was repeated with the same partner. The day on which sperm was found was considered as the day of conception (day 0 of pregnancy). This procedure was repeated until enough pregnant dams were available for all groups. Rats which did not become pregnant were excluded from the analysis of the results and replaced by other animals. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.
Duration of treatment / exposure:
Day 6 to 20 of gestation
Frequency of treatment:
once daily
Duration of test:
Day 6 to 20 of gestation
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
30 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected in agreement with the Sponsor based on the results of a dose-range-finding study in rats. In the dose-range-finding study, the test item Benzyltoluene was administered orally to female rats at dose levels of 100, 300 or 800 mg/kg b.w./day from the 6th to 20th day of pregnancy.
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 100 mg/kg b.w./day for the dams.
One of 6 dams of the high dose group (800 mg/kg b.w./day) was found dead before the end of the study.
An increase in the incidence, duration and in the number of signs of clinical toxicity (sal-ivation, reduced motility, prone position, piloerection) was noted from the low dose group (100 mg/kg b.w./day) to the high dose group (800 mg/kg b.w./day).
A reduced body weight and body weight gain were noted in the intermediate and high dose level (300 or 800 mg/kg b.w./day) and a reduced food consumption at the high dose level.
No test item-related changes were noted at necropsy.
The no-observed-adverse-effect level (NOAEL) for the fetal organism was also 100 mg/kg b.w./day.
No dead foetuses and no test item-related variations or malformations were noted at any of the tested dose levels during the external examination.
In the intermediate and high dose group (300 or 800 mg/kg b.w./day) a decreased pla-cental and fetal weight was noted.
Together with the fact that no external malformations or variations were noted for the fetuses, dose levels of 30, 100 or 300 mg Benzyltoluene/kg b.w./day were selected in agreement with the Sponsor for the present main study.
Maternal examinations:
Clinical signs:
Individual animals were observed daily for behavioural changes, reaction to treatment, or illness.
Viability:
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals.
Body weight:
The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighing - always at the same time of the day.
The body weight gain was calculated in intervals (i.e. day 0-3, 3 6, 6-9, 9-12, 12-15, 15-18 and 18-21), for the whole study (gestation day 0 - 21) and for the period after the start of dosing (gestation day 6 to gestation day 21). Furthermore the carcass weight and the net weight gain from day 6 is given.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes / No / No data
- Number of corpora lutea: Yes /
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
Statistics:
Parametrical data: The statistical evaluation of the parametrical values was done by Provantis.
Non-parametrical data: The statistical evaluation of non-parametrical values was done using the FISHER or Chi test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Slight or moderate - in one dam pronounced - salivation was noted in 16 of 22 high dose dams (300 mg Benzyltoluene/kg b.w./day) on 1 to 7 test days. Salivation started immediately to 5 minutes after administration and disappeared within 20 to 60 minutes after administration. In addition, an increased drinking water consumption was noted (by visual appraisal) in 5 of 22 high dose dams on 1 or 2 test days. Salivation and increased drinking water consumption are considered to be test item-related findings.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg Benzyltoluene/kg b.w./day, a stagnation in body weight was noted for the dams of the high dose group during the first days of treatment (first dosing on gestation day 6) due to reduced food consumption. Thereafter, the body weight improved but remained below the values of the control group by up to 4% or 5%. Statistically significant differences from control (at p ≤ 0.05) were noted on gestation days 10, 17, 18 and 19. A more distinct reduction of body weight (significant at p ≤ 0.01) was noted towards the end of test on gestation days 20 and 21. The maximal difference (11.1% below the value of the control group) was reached on gestation day 21 (day of laparotomy). This finding was partly due to reduced food intake and partly due to a lower gravid uterus weight
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At the high dose level (300 mg Benzyltoluene/kg b.w./day), the start of treatment evoked slight but statistically significant (p ≤ 0.01) reductions in food consumption on every day between gestation days 7 and 11 (between 17.6% and 26.4% below the values of the control group). Thereafter, the food consumption of the dams from the high dose group recovered. However, marginal reductions (significant at p ≤0.05) in food consumption were noted again on gestation day 16-17 (12.5% below the values of the control group). Severe statistically significant (p ≤ 0.01) reductions in food consumption were noted on gestation day 20-21.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
An increased water consumption was noted for 5 of 22 dams of the high dose group (300 mg Benzyltoluene/kg b.w./day) by visual appraisal on one or two test days and considered as test item-related
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic inspection during necropsy revealed gastric changes (haemorrhagic focus/foci, thin walls) in 4 of 22 high dose dams (300 mg Benzyltoluene/kg b.w./day).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
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
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg Benzyltoluene/kg b.w./day, statistically significant decreases (at p ≤ 0.01) were noted for the fetal weights (by approx. 26% below the control) and for the placental weights (by approx. 13% or 14% below the control).
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): At 300 mg Benzyltoluene/kg b.w./day, statistically significant decreases (at p ≤ 0.01) were noted for the fetal weights (by approx. 26% below the control) and for the placental weights (by approx. 13% or 14% below the control).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
other: skeletal and soft tissue variations (see summary)
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
In this prenatal developmental toxicity study, the test item Benzyltoluene was adminis-tered orally to female rats at dose levels of 30, 100 or 300 mg/kg b.w./day from the 6th to 20th day of pregnancy.
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 100 mg Benzyltoluene/kg b.w./day for the dams.
Treatment with 300 mg Benzyltoluene/kg b.w./day caused salivation and increased intake of drinking water on a few or individual test days as well as reductions in the body weight, the body weight gain and the food consumption.
A few dams with gastric changes (haemorrhagic focus/foci, thin walls) were noted at the high dose level during the macroscopic inspection at necropsy.
The no-observed-adverse-effect level (NOAEL) for the fetal organism was 100 mg Benzyltoluene/kg b.w./day.
At the materno-toxic dose of 300 mg Benzyltoluene/kg b.w./day, pronounced decreases were noted for fetal body weights (by approx. 26%) and placental weights (by approx. 14%).
At 300 mg Benzyltoluene/kg b.w./day, an increased incidence was noted for retarded skeletal ossification (sternum, caudal vertebral bodies, metacarpalia and metatarsalia). These changes are regarded to be a secondary effect related to the maternal toxicity (Carney and Kimmel, 2007).
In addition, an increased incidence was noted for slight soft tissue variations of the kidney (uni- or bilateral dilatation of renal pelvis) and the 4th cerebral ventricle (dilatation). According to the ECETOC guidance document these effects are regarded to represent a 'low level of concern'. Considering the maternal and fetal toxicity at the high dose level the final conclusion according to the ECETOC guidance document for these findings is 'no concern'.
No test item-related malformation was noted at any of the test item treated groups. There was no test item-related increase in the incidence of fetal external / internal or skeletal variations at any tested dose level.
Under the conditions of the study, Benzyltoluene did not show any teratogenic potential.

Executive summary:

In this prenatal developmental toxicity study, the test item Benzyltoluene was administered orally to female rats at dose levels of 30,100or300 mg/kg b.w./day from the 6th to 20th day of pregnancy.

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 100 mg Benzyltoluene/kg b.w./day for the dams.

Treatment with 300 mg Benzyltoluene/kg b.w./day caused salivation and increased intake of drinking water on a few or individual test days as well as reductions in the body weight, the body weight gain and the food consumption.

A few dams with gastric changes (haemorrhagic focus/foci, thin walls) were noted at the high dose level during the macroscopic inspection at necropsy.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was 100 mg Benzyltoluene/kg b.w./day.

At the materno-toxic dose of 300 mg Benzyltoluene/kg b.w./day, pronounced decreases were noted for fetal body weights (by approx. 26%) and placental weights (by approx. 14%).

At 300 mg Benzyltoluene/kg b.w./day, an increased incidence was noted for retarded skeletal ossification (sternum, caudal vertebral bodies, metacarpalia and metatarsalia). These changes are regarded to be a secondary effect related to the maternal toxicity (Carney and Kimmel, 2007).

In addition, an increased incidence was noted for slight soft tissue variations of the kidney (uni- or bilateral dilatation of renal pelvis) and the 4th cerebral ventricle (dilatation). According to the ECETOC guidance document ("Identification and assessment of the effects of chemicals on reproduction and development (Reproductive Toxicology)", Monograph No. 5, December 1983) these effects are regarded to represent a 'low level of concern'. Considering the maternal and fetal toxicity at the high dose level the final conclusion according to the ECETOC guidance document for these findings is 'no concern'. No test item-related malformation was noted at any of the test item treated groups. There was no test item-related increase in the incidence of fetal external / internal or skeletal variations at any tested dose level.

Under the conditions of the study, Benzyltoluene did not show any teratogenic potential.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is GLP compliant and has Klimisch score 1.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

There is conclusive data available for the non-classification of the test substance with regard to sexual function and fertility. The substance is not classified for this endpoint in accordance to Directive 67/548/EEC or the CLP Regulation (EC) No 1272/2008.

Following study results from members of the diphenylmethane category are available for reproductive toxicity:

In an OECD 422 combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test with SAS-296, there were no evidences of the chemical-related effects on reproduction/developmental parameters. The NOAEL for reproduction/developmental toxicity was considered to be 200 mg/kg bw/day in rats. No chemical-related effects on the estrous cycle, copulation index, fertility index, gestation length, number of corpora lutea, or number of implantation sites were found in dams. Pathological changes were not noted in the male and female reproductive organs. No chemical-related effects on the number, sex ratio, body weight, or viability were found in pups. No external or internal malformations were found in pups at any doses. Based on these findings, the NOAEL for reproductive/developmental toxicity was considered to be 200 mg/kg bw/day in rats. At 200 mg/kg/day, some parameters, number of implantations, implantation index, and numbers of newborns and live newborns (24%), were decreased, but not statistically significant. These values are within the range of historical control data.

In the OECD Guideline 421 Reproduction/Developmental Toxicity Screening Test with 1,1`-DPE (1,1-Diphenylethane ((phenylethyl)benzene), CAS 38888-98 -1) after oral gavage in the vehicle cotton seed oil clear maternal toxicity (mortality and significantly reduced body weights) was evident at the highest tested dose of 1000 mg/kg bw/d. Therefore it may be argued that the reduced pup weights at 1000 mg/kg bw/d are related to the toxic effect of 1,1`-DPE on the maternal organism such that the observed effects are probably of an indirect (secondary) nature. Conception rates for Groups II-1000 mg/kg, III-500 mg/kg, and IV-100 mg/kg were 50.0% and 100.0% respectively and the conception rate for Group I-Control was 70.0%. Delivery was 100% successful for all groups. There was one pup out of a litter of 17 from Group III that was born deformed. Stillborn births averaged 8%, 0%. 15% and 2% for Groups I-IV, respectively. There were no significant differences between groups for the number of stillborn births, the number of pups born, or conception rate. Based on the lack of observed abnormalities and mortality at the lower levels, the No Observed Adverse Effect Level (NOAEL) was determined to be 500mg/kg of 1 -Phenylethylbenzene.

In an OECD 422 combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test with PTE, 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 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

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. There were no evidences of a chemical-related effect on fertility parameters up to 100 mg/kg bw/day. The NOAEL for reproductive toxicity was considered to be 100 mg/kg bw/day in rats.

There is also conclusive data available for the non-classification of the substance with regard to developmental toxicity.The available test data for Benzyltoluene does not substantiate a classification of the substance as developmental toxicant.

Higher-tier fertility study (two-generation study) is not required at this tonnage band, since there were no adverse effects observed in the repeated dose toxicity studies in reproductive organs or tissues or any adverse effects in the screening studies for reproductive toxicity (OECD 421/422). Therefore, there is no data gap in fertility. There is no reason to believe that results of the screening study would not be relevant for fertility in humans and, therefore, for risk assessment.

For developmental toxicity following results were obtained from an OECD 414 pre-natal developmental toxicity study:

The test item Benzyltoluene was administered orally to female rats at dose levels of 30,100 or 300 mg/kg b.w./day from the 6th to 20th day of pregnancy.

Under the test conditions described, the no-observed-adverse-effect level (NOAEL) was 100 mg Benzyltoluene/kg b.w./day for the dams.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was 100 mg Benzyltoluene/kg b.w./day.

No test item-related malformation was noted at any of the test item treated groups. There was no test item-related increase in the incidence of fetal external / internal or skeletal variations at any tested dose level. Under the conditions of the study, Benzyltoluene did not show any teratogenic potential.

Therefore, no classification for developmental or reproductive toxicity for the substance Benzyltoluene is required.

Additional information