Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-11-03 - 2011-08-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The GLP study was conducted according to an internationally accepted guideline.
Qualifier:
according to
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 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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 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 fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

For PTE, developmental toxicity was manifested when given at a dosage which does not indicate clearly recognizable general maternal toxicity. Therefore, classification of PTE for developmental toxicity into category 1B based on non-maternal mediated pup toxicity is justified.

In the reproduction/developmental screening test with 1,1`-DPE after oral gavage in the vehicle cotton seed oil (which should allow for appropriate dissolution of the test substance) clear maternal toxicity (mortality and significantly reduced body weights) was evident at the highest tested dose of 1000 mg/kg bw/d. Therefore it can be stated 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 probablyof an indirect (secondary) nature.

The OECD Guideline 422 screening test with “SAS-296” revealed no evidence of substance related effects on reproduction/developmental parameters even though body weight gain was decreased also for females by 5-8 %. No substance related effects on the number, sex ratio, body weight, or viability were found in pups. Furthermore no external or internal malformations were found in pups at any dose.

In the dose-range-finding study for the prenatal developmental toxicity study with the test item 1,1-Diphenylethanethe (maternal) no-observed-adverse-effect level (NOAEL) was 100 mg 1,1-Diphenylethane/kg b.w./day for the dams.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was 400 mg 1,1-Diphenylethane/kg b.w./day.

A slightly but statistically significant reduced fetal body weight was noted at the materno-toxic dose level of 800 mg 1,1-Diphenylethane/kg b.w./day.

In the pre-natal developmental toxicity study with 1,1 -DPE no (non-maternal mediated) developmental effects were seen.

Under the test conditions, the no-observed-adverse-effect level (NOAEL) was 100 mg1,1-Diphenylethane/kg b.w./day for the dams. The no-observed-adverse-effect level (NOAEL) for the fetal organism was 100 mg1,1-Diphenylethane/kg b.w./day.

The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item.

No dead fetuses and no malformations were noted.

At the maternal toxic dose levels of 400 and 800 mg1,1-Diphenylethane/kg b.w./day a reduced fetal body weight and a variation in the form of an increased incidence of wavy ribs were noted.

At 800 mg1,1-Diphenylethane/kg b.w./day a retardation in the form of an increased incidence of incompletely ossified thoracic vertebral bodies was noted additionally.

The retarded ossification and the wavy ribs are regarded to be secondary effects related to the maternal toxicity ( CARNEY E.W. and KIMMEL C.A.: “Interpretation of skeletal variations for human risk assessment: delayed ossification and wavy ribs”, Birth Defects Research (Part B) 80, 473-496 (2007).

In summary, no (non-maternal mediated) developmental effects were seen in any of these well conducted studies with 1,1 -DPE.

Therefore 1,1 -DPE has not to be classified for 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.

 


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)
Dose range finding study OECD 414: maternal NOAEL: 100 mg/kg bw; fetal NOAEL 400 mg/kg bw

Pre-natal developmental toxicity study: rat (CD) male/female (OECD Guideline 414): NOAEL (p): 100 mg/kg bw, NOAEL (F1): 100 mg/kg bw

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
Developmental NOAEL 10 mg/kg bw/d: based on pup death and total litter loss at 100 mg/kg and decreased pup weight at 30 mg/kg

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
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)
Dose range finding study OECD 414: maternal NOAEL: 100 mg/kg bw; fetal NOAEL 400 mg/kg bw
Pre-natal developmental toxicity study: rat (CD) male/female (OECD Guideline 414): NOAEL (p): 100 mg/kg bw, NOAEL (F1): 100 mg/kg bw
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
Developmental NOAEL 10 mg/kg bw/d: based on pup death and total litter loss at 100 mg/kg and decreased pup weight at 30 mg/kg
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-08-21 - 2016-04-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The GLP-study was performed according to an internationally accepted guideline.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: CD/ Crl:CD (SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany

- Age at study initiation: 60 days (on day 0 of pregnancy)
- Weight at study initiation: 185.2 - 236.6 g
- Fasting period before study: no
- Housing: Except during the mating period, the dams were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm.
- Diet (e.g. ad libitum): Commercial diet ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food. This food was offered daily ad libitum.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 15% (maximum range)
- Air changes (per hr): between 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:
PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared every day. The test item was diluted in the vehicle to the appropriate concentration 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.


DIET PREPARATION
- Rate of preparation of diet (frequency): The food was offered daily ad libitum.
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 99.1% - 100.8%
- Amount of vehicle (if gavage):Administration volume: 2 mL/kg b.w./day
- Lot/batch no. (if required): Batch no. 0123486, supplier: Kräuter Schulte, 76593 Gernsbach, Germany
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle formulations, samples of approximately 10 mL were taken at the following times and stored at -20°C or colder until analysis at LPT by HPLC.
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:
from the 6th and lasting until the 20th day of pregnancy (the 'critical' phase of organogenesis and the fetal development)
Frequency of treatment:
once daily
Duration of test:
Day 6 to 20 of gestation
Remarks:
Doses / Concentrations:
0, 100, 400, 800 mg/kg bw
Basis:
analytical conc.
No. of animals per sex per dose:
30 females per group
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 for a prenatal developmental toxicity study in pregnant rats (LPT study no. 32122).
In the dose-range finding study, 1,1-Diphenylethane was administered to pregnant female rats at dose levels of 100, 400 or 800 mg/kg b.w./day orally, by gavage, once daily from gestation day 6 to 19.
The no-observed-adverse-effect level (NOAEL) was 100 mg 1,1-Diphenylethane/kg b.w./day for the dams.
At 400 mg 1,1-Diphenylethane/kg b.w./day, an increased drinking water consumption, a slightly reduced body weight gain and a transient reduction of food intake were noted.
After oral treatment with 800 mg 1,1-Diphenylethane/kg b.w./day individual to all dams revealed increased salivation, increased drinking water consumption and/or pale faeces as well as reductions in the mean body weight, body weight gain and in food consumption in comparison to the control group all of which were statistically significant.
No premature deaths were noted. Necropsy revealed no changes for the dams of any test group.
The no-observed-adverse-effect level (NOAEL) for the fetal organism was 400 mg 1,1-Diphenylethane/kg b.w./day.
A slightly but statistically significantly reduced fetal body weight was noted at the maternal-toxic dose level of 800 mg 1,1-Diphenylethane/kg b.w./day.
No dead fetuses were noted and the external macroscopic inspection of the fetuses at laparotomy revealed no malformations and no test item-related variations at any of the tested dose levels.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Individual animals were observed daily for behavioural changes, reaction to treatment, or illness.
Immediately after administration, any signs of illness or reaction to treatment were rec-orded. In case of changes, the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m.
On Saturdays and Sundays, the animals were checked regularly starting from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: 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 change from day 6 is given.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each rat was recorded daily. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment day.
The relative food consumption (g/kg b.w./day) was calculated using the following formula:
Daily food consumption [g/kg b.w./day]= Total food intake in g/Body weight in kg


WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study. Dehydration of the dams was avoided.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of sacrifice or on the day on which the animals were found dead. In case of macroscopical findings, the affected maternal tissues were preserved in 7% buffered formalin for possible future histopathological examination.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Weight of placenta: yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- 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 using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Non-parametrical data
The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01)
or
Chi2 test, n ≤ 0.01 (p ≤ 0.05 and p ≤ 0.01)

The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy) or an internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).
Note:
The statistical evaluation of the pre- and post-implantation index (per group) using the number of corpora lutea, implantation sites and/ or fetuses per group (see table 7-1 Reproduction Data - Summary - Values per Group) was done using StatXact 4.0.1 software, as such a calculation is not possible in Provantis.
Historical control data:
Results of 59 last embryotoxicity studies in Sprague-Dawley rats (Charles River Deutschland GmbH) performed at the lab in the years 2000 to December 2015 are included in the study report.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical signs:
At 400 mg 1,1-Diphenylethane/kg b.w./day salivation was noted for all dams (22 of 22), Piloerection for 4 of 22 dams and an increased drinking water consumption (by visual appraisal) for 2 of 22 dams.
Salivation was also noted for all dams (22 of 22) of the high dose level (800 mg 1,1-Diphenylethane/kg b.w./day). An increased drinking water consumption (by visual appraisal) was noted for 10 of 22 dams, whereas piloerection was only noted for 1 of 22 dam.

Body weight and body weight gain:
At 400 mg 1,1-Diphenylethane/kg b.w./day statistically significant (p ≤ 0.05 or 0.01) reductions in body weight were noted from gestation day 7 onwards until the end of the study with a maximum value on gestation day 21 (10.1% below the value of the control group).
Similar reductions in body weight were noted for the dams of the high dose group (800 mg 1,1-Diphenylethane/kg b.w./day). The maximal reduction was noted on gestation day 21 with a body weight that was 11.8% (p ≤ 0.01) below the value of the control group.
Body weight gain for the whole study period for the dams of the intermediate and the high dose group (400 or 800 mg 1,1-Diphenylethane/kg b.w./day) was 20.7% or 28.0% (p ≤ 0.01) below the value of the control group.

Food consumption:
At 100 mg 1,1-Diphenylethane/kg b.w./day a statistically significant (p ≤ 0.01) reduction in food consumption was noted for 1 day after the start of treatment (19.5% below the value of the control group).
At 400 mg 1,1-Diphenylethane/kg b.w./day the start of treatment evoked a statistically significant reduction in food consumption for 3 days. The food consumption was 41.5% (gestation day 6, p ≤ 0.01) to 15.0% (gestation day 9, p ≤ 0.01) below the value of the control group.
A slightly more pronounced reduction as for the dams of the intermediate dose group was noted for the dams of the high dose group (800 mg 1,1-Diphenylethane/kg b.w./day): 53.7% to 21.4% below the value of the control group between gestation days 6 and 9 (p ≤ 0.01).
Beside the reduction in food consumption that was noted after the start of treatment further statistically significant (p ≤ 0.05 or 0.01) reductions in food consumption were noted for the dams of the intermediate and/or the high dose group (400 or 800 mg 1,1-Diphenylethane/kg b.w./day) between gestation days 11 and 14 and between gestation days 17 to 21 with maximum values on gestation day 21 (26.1% or 26.2% below the value of the control).

Drinking water consumption (by visual appraisal):
An increased drinking water consumption was noted for 2 of 22 dams of the intermediate and for 10 of 22 dams of the high dose group by visual appraisal.

Necropsy findings:
A reduction in spleen size was noted for 3 of 22 dams of the high dose group (800 mg 1,1-Diphenylethane/kg b.w./day) during the macroscopic inspection at necropsy.

Uterus and carcass weights:
Statistically significant reductions (p ≤0.05 or 0.01) in the gravid uterus weight were noted for the dams of the intermediate and the high dose group (400 or 800 mg 1,1-Diphenylethane/kg b.w./day) (8.5% or 14.7% below the value of the control group).
The carcass weight of the dams of the intermediate and the high dose group (400 or 800 mg 1,1-Diphenylethane/kg b.w./day) was statistically significantly (p ≤0.01) reduced in comparison to the control group by 10.6% (intermediate dose level) or 10.8% (high dose level).

Reproduction data:
No influence on the reproductive parameter (number of implantation sites, resorptions and fetuses) was noted.



Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Body weight of the fetuses and the placentae:
A statistically significant reduction (p ≤0.01) in the fetal body weight was noted for the dams of the intermediate and the high dose group (400 or 800 mg 1,1-Diphenylethane/kg b.w./day) (12.3% or 15.3% below the value of the control group for the male and female fetuses together).

Fetal alterations:
Malformations:
No malformation was noted during the macroscopic examination at laparotomy (external inspection and inspection of the organs and tissues for gross lesions). The skeletal examination according to DAWSON and the soft tissue examination according to WILSON also revealed no test item-related malformations.

Variations:
The macroscopic examination at laparotomy and the soft tissue examination according to WILSON revealed no test item-related variations.
A test item-related variation was noted during the skeletal examination according to DAWSON in the form of a slight, but statistically significantly (p ≤0.05 or 0.01) increased incidence of wavy ribs that was noted for the fetuses of the intermediate and the high dose group (400 or 800 mg 1,1-Diphenylethane/kg b.w./day) (4.2% or 6.5% fetuses with wavy ribs in the intermediate and the high dose group in comparison to 0.7% in the control group).

Retardations:
At 800 mg 1,1-Diphenylethane/kg b.w./day a statistically significant (p ≤0.05) increase was noted in the incidence of incompletely ossified thoracic vertebral bodies (thoracic vertebral body/bodies bipartite) (9.4% of the high dosed fetuses showed a bipartite ossification pattern of one or more thoracic vertebral body/bodies in comparison to 2.9% in the control group).
Abnormalities:
not specified
Developmental effects observed:
not specified

Behaviour, external appearance,faeces

Test item-related observations in group 3

(400 mg 1,1-Diphenylethane/kgb.w./day)

Observation

Affected

dams

Incidence

(number of test days the observation was noted for the individual dams)

First to last seen

(gestation days)

Salivation

22 of 22

Between 4 and 7 consecutive or non-consecutive test days.

13 - 20

Piloerection

4 of 22

On one or 2 test days.

20 - 21

Increased water consumption

2 of 22

On 2 or 3 consecutive test days.

19 - 21

 

 

 

Test item-related observations in group 4

(800 mg 1,1-Diphenylethane/kgb.w./day)

Observation

Affected

dams

Incidence

(number of test days the observation was noted for the individual dams)

First to last seen

(gestation days)

Salivation

22 of 22

Between 5 and 9 consecutive or non-consecutive test days.

12 -20

Piloerection

1 of 22

Only on one test day.

21

Increased water consumption

10 of 22

On 2, 3 or 4 consecutive test days.

18 - 21

 

 

Body weight and body weight gain

 

1,1-Diphenylethane - Mean body weight gain

Group

Time interval

Gestation day 6 - 21

(after the start of treatment)

Time interval

Gestation day 0 - 21

(whole study period)

 

Gain

in g

Gain

in %

Difference to control %

Gain

in g

Gain

in %

Difference to control %

Control

120.5

48.7

n.e.

155.6

73.5

n.e

Group 2

100 mg/kg

119.8

49.3

- 0.6

153.0

72.9

-1.7

Group 3

400 mg/kg

90.3 **

37.6

- 25.1

123.4 **

59.8

- 20.7

Group 4

800 mg/kg

75.8 **

30.5

- 37.1

112.1 **

53.0

- 28.0

*/**:

Statistically significant at p ≤ 0.05/ 0.01 (ANOVA/DUNNETT test)

 

 

n.e.:

Not evaluable

Gravid uterus and carcass weight

Group /

Dose level

% change in comparison to control

Gravid uterus weight

Carcass weight

Group 3 (400 mg/kg)

- 8.5*

- 10.6**

Group 4 (800 mg/kg)

- 14.7**

- 10.8**

*/**:

p≤ 0.05/0.01, ANOVA/DUNNETT test.

 

Reproduction data of the dams

Parameter

Group 1

Control

(n=22)

Group 2

100

mg/kg

(n=22)

Group 3

400 mg/kg

(n=22)

Group 4

800 mg/kg

(n=22)

 

Corporalutea

total

mean per dam

301

13.7

314

14.3

307

14.0

307

14.0

 

Implantation sites

total

mean per dam

285

13.0

301

13.7

296

13.5

283

12.9

 

Resorptions

total

mean per dam

5

0.2

6

0.3

10 #

0.5

7

0.3

 

Early resorptions

total

mean per dam

5

0.2

6

0.3

6

0.3

7

0.3

 

Late resorptions

total

mean per dam

0

0.0

0

0.0

4

0.2**

0

0.0

 

Live fetuses

total

mean per dam

280

12.7

295

13.4

287

13.0

276

12.5

 

Dead fetuses

total

0

0

0

0

 

Pre-implantation loss [%]

per group ##1

mean per dam

5.3

5.0

4.1

3.9

3.6

3.1

7.8

7.0

 

Post-implantation loss [%]

per group ##2

mean per dam

1.8

1.8

2.0

2.0

3.0

3.4

2.5

2.5

 

 

*/**:

 p ≤ 0.05/ 0.01

Statistical analyses were performed for the mean values per dam using an ANOVA/DUNNETT test.

 

##1

The statistical comparison of the pre-implantation loss per group was done by comparing the values of implantation sites/corporaluteaof the test group with the ratio of implantation sites/corporaluteaof the control group using the Chi2test.

 

##2

The statistical comparison of the post-implantation loss per group was done by comparing the values of live fetuses/implantation sites of the test group with the ratio of fetuses/implantation sites of the control group using the Chi2test.

 

#

A twin (consisting of a late resorption and a live fetus) was noted at implantation site R02 from dam no. 78

 

Weight of fetuses

Group /

Dose level

Fetal weight

(% change in comparison to control)

Male

fetuses

Female

fetuses

Male + Female

fetuses

Group 3

(400 mg/kg)

- 12.8 **

- 11.6 **

- 12.3 **

Group 4

(800 mg/kg)

- 15.4 **

- 15.0 **

- 15.3 **

*/**:

p≤ 0.05/0.01, ANOVA/DUNNETT test.

 

 

Skeletal variations


Skeletal variations

Values observed

in this study

(LPT Report 32123)

 

[fetal incidence in% per group]

LPT Background Data#1

Mean value ±SD

(range)

of the individual control or test groups

[fetal incidence in % per group]

(n=59 control or

n= 150 test item groups;

Data taken from 2000 to

December 2015)

Rib(s) wavy

Control:

0.7

5.2 ± 7.2

(0.0 - 27.9)

control groups

Group 2:

1.4

Group 3:

4.2*

5.4 ±9.2

(0.0 - 52.9)

test item groups

Group 4:

6.5**

Total fetal skeletal

variations

Control:

0.7

8.2 ± 8.5

(0.0 - 38.0)

control groups

Group 2:

3.4

Group 3:

6.3 **

8.3 ± 9.6

(0.0 - 55.0)

test item groups

Group 4:

8.7 **

 

Test item-related findings are marked in bold.

#1:

not audited by QAU

*/**:

(p ≤ 0.05 / p ≤ 0.01) Fisher or Chi2- test

 

Skeletalretardations


Skeletal

retardations

Values observed

in this study

(LPT Report 32123)

 

[fetal incidence in % per group]

LPT Background Data#1

Mean value ±SD

(range)

of the individual control or test groups

[fetal incidence in % per group]

(n=59 control or

n= 153 test item groups;

Data taken from 2000 to

December 2015)

Absence of ossification inmetacarpalia2 to 5

Control:

7.1

36.3 ± 36.5

(0.0 - 94.9)

control groups

Group 2:

16.2*

Group 3:

13.2

36.1 ± 36.0

(0.0 - 97.6)

test item groups

Group 4:

15.2*

Absence of ossification inmetatarsalia2 to 5

Control:

4.3

4.5 ± 8.8

(0.0 - 42.3)

control groups

Group 2:

12.2**

Group 3:

6.9

4.0 ± 8.5

(0.0 - 37.6)

test item groups

Group 4:

5.8

Caudal vertebral bodies,

all bodiesunossified

Control:

0.0

5.6 ±9.0

(0.0 - 37.4)

control groups

Group 2:

0.0

Group 3:

2.8*

4.4 ± 6.9

(0.0 - 29.6)

test item groups

Group 4:

0.0

Skull,

incomplete ossification

Control:

8.6

23.8 ± 17.6

(1.4 - 71.3)

control groups

Group 2:

16.2*

Group 3:

17.4*

23.7 ±17.3

(1.4 - 79.5)

test item groups

Group 4:

21.7**

Thoracic vertebral body/

bodies bipartite

Control:

2.9

2.7 ± 2.7

(0.0 - 13.7)

control groups

Group 2:

2.0

Group 3:

3.5

2.6 ± 2.5

(0.0 - 14.6)

test item groups

Group 4:

9.4*

 

Test item-related findings are marked in bold.

#1:

not audited by QAU

*/**:

(p ≤ 0.05 / p ≤ 0.01) Fisher or Chi2- test

 

Conclusions:
In this prenatal developmental toxicity study, the test item 1,1-Diphenylethane was administered orally to female rats at dose levels of 100, 400 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 1,1-Diphenylethane/kg b.w./day for the dams.
None of the dams died prematurely.
At 100 mg 1,1-Diphenylethane/kg b.w./day a slight reduction in food consumption was noted for 1 day after the start of treatment. As this incidence led to a slightly and only temporary reduction in body weight gain between gestation days 6 and 9 and was without any effect on the whole body weight gain, it was considered as non-adverse (for review see Lewis et al, 2002).
Changes in behavior (salivation, increased drinking water consumption), the external appearance (piloerection), reductions in the body weight and a reduced food consumption were noted at the intermediate and the high dose level (400 or 800 mg 1,1-Diphenylethane/kg b.w./day).
A few dams with a reduced spleen size were noted at the high dose level (800 mg 1,1-Diphenylethane/kg b.w./day) during the macroscopic inspection at necropsy.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was 100 mg 1,1-Diphenylethane/kg b.w./day.
The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item.
No dead fetuses and no malformations were noted.
At the maternal toxic dose levels of 400 and 800 mg 1,1-Diphenylethane/kg b.w./day a reduced fetal body weight and a variation in the form of an increased incidence of wavy ribs were noted.
At 800 mg 1,1-Diphenylethane/kg b.w./day a retardation in the form of an increased incidence of incompletely ossified thoracic vertebral bodies was noted additionally.
The retarded ossification and the wavy ribs are regarded to be secondary effects related to the maternal toxicity (CARNEY E.W. and KIMMEL C.A.: “Interpretation of skeletal variations for human risk assessment: delayed ossification and wavy ribs”, Birth Defects Research (Part B) 80, 473-496 (2007).
Executive summary:

In this prenatal developmental toxicity study, the test item 1,1-Diphenylethane was administered orally to female rats at dose levels of 100, 400 or 800 mg/kg b.w./day from the 6th to 20th day of pregnancy.

Findings

Examination of the dams:

 

Mortality

No test item-related premature deaths were noted for the dams treated orally with 100, 400 o r800 mg 1,1-Diphenylethane/kg b.w./dayfrom gestation day 6 to 20.

 

 

Clinical signs

At 400 mg1,1-Diphenylethane/kg b.w/day salivation was noted for all dams (22 of 22),Piloerectionfor 4 of 22 dams and an increased drinking water consumption (by visual appraisal) for 2 of 22 dams.

Salivation was also noted for all dams (22 of 22) of the high dose level (800 mg1,1-Diphenylethane/kg b.w./day). An increased drinking water consumption (by visual appraisal) was noted for 10 of 22 dams, whereas piloerection was only noted for 1 of 22 dam.

 

 

Body weight and

body weight gain

At 400 mg1,1-Diphenylethane/kg b.w./day statistically significant (p ≤ 0.05 or 0.01) reductions in body weight were noted from gestation day 7 onwards until the end of the study with a maximum value on gestation day 21 (10.1% below the value of the control group).

Similar reductions in body weight were noted for the dams of the high dose group (800 mg1,1-Diphenylethane/kg b.w./day). The maximal reduction was noted on gestation day 21 with a body weight that was 11.8% (p ≤ 0.01) below the value of the control group.

Body weight gain for the whole study period for the dams of the intermediate and the high dose group (400 or 800 mg1,1-Diphenylethane/kg b.w./day) was 20.7% or 28.0% (p ≤ 0.01) below the value of the control group.

 

 

The 3-day interval of body weight gain revealed statistically significant reductions in body weight gain after the start of treatment between gestation days 6 and 9 for all dose groups (100,400 or 800 mg1,1-Diphenylethane/kg b.w./day) (4.8 g to minus 2.6 g in comparison to 9.9 g in the control group).

 

Food consumption

 

At 100 mg1,1-Diphenylethane/kg b.w./day a statistically significant (p ≤ 0.01) reduction in food consumption was noted for 1 day after the start of treatment (19.5% below the value of the control group).

At 400 mg1,1-Diphenylethane/kg b.w./day the start of treatment evoked a statistically significant reduction in food consumption for 3 days. The food consumption was 41.5% (gestation day 6, p ≤ 0.01) to 15.0% (gestation day 9, p ≤ 0.01) below the value of the control group.

A slightly more pronounced reduction as for the dams of the intermediate dose group was noted for the dams of the high dose group (800 mg1,1-Diphenylethane/kg b.w./day): 53.7% to 21.4% below the value of the control group between gestation days 6 and 9 (p ≤ 0.01).

Beside the reduction in food consumption that was noted after the start of treatment further statistically significant (p ≤ 0.05 or 0.01) reductions in food consumption were noted for the dams of the intermediate and/or the high dose group (400 or 800 mg1,1-Diphenylethane/kg b.w./day) between gestation days 11 and 14 and between gestation days 17 to 21 with maximum values on gestation day 21 (26.1% or 26.2% below the value of the control).

 

 

Drinking water consumption

(by visual appraisal)

An increased drinking water consumption was noted for 2 of 22 dams of the intermediate and for 10 of 22 dams of the high dose group by visual appraisal.

 

 

Necropsy findings

A reduction in spleen size was noted for 3 of 22 dams of the high dose group (800 mg1,1-Diphenylethane/kg b.w./day) during the macroscopic inspection at necropsy.

 

 

Uterus and carcass weights

Statistically significant reductions (p ≤0.05 or 0.01) in the gravid uterus weight were noted for the dams of the intermediate and the high dose group (400 or 800 mg1,1-Diphenylethane/kg b.w./day)(8.5% or 14.7% below the value of the control group).

 

The carcass weight of the dams of the intermediate and the high dose group (400 or 800 mg1,1-Diphenylethane/kg b.w./day)was statistically significantly (p ≤0.01) reduced in comparison to the control group by 10.6% (intermediate dose level) or 10.8% (high dose level).

Reproduction data

No influence on the reproductive parameter (number of implantation sites, resorptions and fetuses) was noted.

 

Examination of the fetus

Mortality

No dead fetuses were noted in any of the test groups.

 

Body weight of the fetuses

and the placentae

A statistically significant reduction (p ≤0.01) in the fetal body weight was noted for the dams of the intermediate and the high dose group (400 or 800 mg1,1-Diphenylethane/kg b.w./day)(12.3% or 15.3% below the value of the control group for the male and female fetuses together).

Fetal alterations

 

Malformations

No malformation was noted during the macroscopic examination at laparotomy (external inspection and inspection of the organs and tissues for gross lesions).

The skeletal examination according to DAWSON and the soft tissue examination according to WILSON also revealed no test item-related malformations.

 

 

Variations

The macroscopic examination at laparotomy and the soft tissue examination according to WILSON revealed no test item-related variations.

A test item-related variation was noted during the skeletal examination according to DAWSON in the form of a slight, but statistically significantly     (p ≤0.05 or 0.01) increased incidence of wavy ribs that was noted for the fetuses of the intermediate and the high dose group (400 or 800 mg1,1-Diphenylethane/kg b.w./day) (4.2% or 6.5% fetuses with wavy ribs in the intermediate and the high dose group in comparison to 0.7% in the control group).

 

Retardations

At 800 mg 1,1-Diphenylethane/kg b.w./day a statistically significant (p ≤0.05) increase was notedin the incidence of incompletely ossified thoracic vertebral bodies (thoracic vertebral body/bodies bipartite) (9.4% of the high dosed fetuses showed a bipartite ossification pattern of one or more thoracic vertebral body/bodies in comparison to 2.9% in the control group).

 

Conclusions:

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL : 100 mg/kg bw/day.

Developmental NOAEL : 100 mg/kg bw/day.

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
Additional information

For PTE, developmental toxicity was manifested when given at a dosage which does not indicate clearly recognizable general maternal toxicity. Therefore, classification of PTE for developmental toxicity into category 1B based on non-maternal mediated pup toxicity is justified.

In the reproduction/developmental screening test with 1,1`-DPE after oral gavage in the vehicle cotton seed oil (which should allow for appropriate dissolution of the test substance) clear maternal toxicity (mortality and significantly reduced body weights) was evident at the highest tested dose of 1000 mg/kg bw/d. Therefore it can be stated 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 probablyof an indirect (secondary) nature.

The OECD Guideline 422 screening test with “SAS-296” revealed no evidence of substance related effects on reproduction/developmental parameters even though body weight gain was decreased also for females by 5-8 %. No substance related effects on the number, sex ratio, body weight, or viability were found in pups. Furthermore no external or internal malformations were found in pups at any dose.

In the dose-range-finding study for the prenatal developmental toxicity study with the test item 1,1-Diphenylethanethe (maternal) no-observed-adverse-effect level (NOAEL) was 100 mg 1,1-Diphenylethane/kg b.w./day for the dams.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was 400 mg 1,1-Diphenylethane/kg b.w./day.

A slightly but statistically significant reduced fetal body weight was noted at the materno-toxic dose level of 800 mg 1,1-Diphenylethane/kg b.w./day.

In the pre-natal developmental toxicity study with 1,1 -DPE no (non-maternal mediated) developmental effects were seen.

Under the test conditions, the no-observed-adverse-effect level (NOAEL) was 100 mg1,1-Diphenylethane/kg b.w./day for the dams. The no-observed-adverse-effect level (NOAEL) for the fetal organism was 100 mg1,1-Diphenylethane/kg b.w./day.

The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item.

No dead fetuses and no malformations were noted.

At the maternal toxic dose levels of 400 and 800 mg1,1-Diphenylethane/kg b.w./day a reduced fetal body weight and a variation in the form of an increased incidence of wavy ribs were noted.

At 800 mg1,1-Diphenylethane/kg b.w./day a retardation in the form of an increased incidence of incompletely ossified thoracic vertebral bodies was noted additionally.

The retarded ossification and the wavy ribs are regarded to be secondary effects related to the maternal toxicity ( CARNEY E.W. and KIMMEL C.A.: “Interpretation of skeletal variations for human risk assessment: delayed ossification and wavy ribs”, Birth Defects Research (Part B) 80, 473-496 (2007).

In summary, no (non-maternal mediated) developmental effects were seen in any of these well conducted studies.

Therefore 1,1 -DPE has not to be classified for developmental toxicity.

 


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

Justification for classification or non-classification

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

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