Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproduction toxicity (oral) in rat (OECD 416). NOAEL: 450 mg/kg bw/day

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany.
- Age at study initiation: (P) 37 ± 1 days
- Weight at study initiation: (P) Males: 123.4 (113.1 - 135.2) g; Females: 108.0 (96.4 - 119.0) g
- Housing:
individually in type DK lll stainless steel wire mesh cages supplied by BECKER& CO., Castrop-Rauxel, Germany (floor area of about 800 cm2)
overnight matings: male and female mating partners housed together in type DK lll cage
gestation day 18- lactation day 21: pregnant animals and their litters housed in Makrolon type M lll cages.
nesting material (cellulose wadding) toward the end of gestation
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 hours (12h light from 6.00 a.m. to 6.00 p.m. and 12 h darkness from 6.00 p.m. to 6.00 a.m.)

IN-LIFE DATES: From: 28.Feb.2002 To: 03.Dec.2002
Route of administration:
oral: feed
Vehicle:
other: unchanged, mixed with diet
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): ground Kliba maintenance diet mouse/rat "GLP" meal
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight matings for 4d (F0), 6d (F1)
- Proof of pregnancy: sperm in vaginal smear referred to as day 0
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. Mating indices were 100%
- Further matings after two unsuccessful attempts: no, mating indices were 100%
- After successful mating each pregnant female was caged (how):
From gestation day 18 to lactation day 21: pregnant animals and their litters were housed in Makrolon type Mlll cages and nesting material (cellulose wadding) was provided.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Feed samples were extracted with acetonitrile. Aliquots of the extracts were used for HPLC-analysis.
Duration of treatment / exposure:
F0 generation: continuous administration of the test substance until or up to
about 16 hours before they were sacrificed (Feb.28 - Jul.17.2002)
F1 generation: After weaning, continuous administration of the test substance until or up to
about 16 hours before they were sacrificed. (Jul.3. - Nov.11.2002)
F2 generation: After weaning, continuous administration of the test substance until or up to
about 16 hours before they were sacrificed (Nov.7. - Dec.3.2002)
Frequency of treatment:
continuous administration via diet
Details on study schedule:
- F1 parental animals not mated until at least 74 days after selected from the F1 litters.
for further details, see appendix 1
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
in diet
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
in diet
Dose / conc.:
450 mg/kg bw/day (nominal)
Remarks:
in diet
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
in diet
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: parental animals daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
Exeptions for female animals:
During gestation period parental females were weighed on day 0, 7, 14, 20 post coitum.
Females showing no positive evidence of sperm in vaginal smears were not weighed during the mating interval.
Females with litter were weighed on the day after parturition day 1, 4, 7, 14 and 21 post partum.
Females without litter were not weighed during the lactation phase.
After weaning female F0 parental animals were weighed again once weekly (in parallel to male) until scheduled sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculation:
ITx = (FCx*C)/BWy
ITx = TS intake on day x(mg/kg bw/day)
FCx = daily food consumption day x (g)
C = TS concentration (ppm)
BWy = body weight day y (last weighing before day x)
Oestrous cyclicity (parental animals):
Estrous cycle length and normality were evaluate daily (F0 and F1 parental animals) for a minimum of 3 weeks prior to mating and were continued throughout the mating period until the female exhibited evidence of mating.
Sperm parameters (parental animals):
Parameters examined in [all/P/F1/F2] male parental generations:
testis weight,
epididymis weight,
sperm count in testes,
sperm count in epididymides,
sperm motility,
sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4/sex/litter as nearly as possible. Standardisation was not performed <= 8 pups /litter

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
number and sex of pups
stillbirths,
live births
postnatal mortality
presence of gross anomalies
weight gain
physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrifieced after weaning period of respective offspring
- Maternal animals: All surviving animals were sacrifieced after weaning period of respective offspring

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Appendix 2 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed after standardization or weaning (14 - 28 d), except for the group of reared F2 pups, which were sacrificed shortly after achieving sexual maturity (43 - 56 d).

- These animals were subjected to postmortem examinations as follows:
Pups were examined externally and eviscerated their organs were assessed macroscopically

ORGAN WEIGTHS
brain, spleen and thymus of 1 pup/sex and litter from the F1 and F2 pups were weighed.
Statistics:
For statistics of clinical examinations see appendix 3
For statistics of pathology see appendix 4
Reproductive indices:
Male mating Index (%) = (number of males with confirmed mating* / number of males placed with females) x 100
Male fertility Index (%) = (number of fertile males**/number of males placed with females) x 100
Female mating index (%) = (number of females mated*/number of females placed with males) x 100
Female fertility index (%) = (number of females pregnant***/ number of females mated*) x 100
* animals with vaginal sperm or that gave birth to a litter or with pups/implantations in utero
** defined by a female giving birth to a litter or with pups/implantation in utero
*** defined as the number of females that gave birth to a litter or with pups/implantations in utero
Offspring viability indices:
Gestation Index (%) = (number of females with live pups on the day of birth/number of females pregnant*) x 100
* defined as the number of females that gave birth to a litter or with pups/implantations in utero
Live birth Index (%) = (number of liveborn pups at birth/total number of pups born) x 100
Post lmplantation loss (%) = ((number of implantations - number of pups delivered)/number of implantations) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
F0: Urine-smeared fur around the anogenital region in 2 animals (1000 mg/kg bw/day)
F1: Urine-smeared fur around the anogenital region in 3 animals (1000 mg/kg bw/day)
Mortality:
mortality observed, non-treatment-related
Description (incidence):
F0: One low dose female (150 mg/kg body weight) was found dead: not considered as substance related.
F1: Mortality in two mid dose females (found dead/ sacrificed for to humane reasons): not considered as substance related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw /day:
Males F0: significantly decreased mean bodyweights (16 % below control) and body weight gain (average 22 % below control)
Males F1: significantly decreased mean body weights (14 % below control) and body weight gain (average 14 % below control)
Females F0: decreased mean body weights during premating (4 %), gestation (8 %) and early lactation (6 % below control)
Females F1: decreased mean body weights during premating (6 %), gestation (10 %) and lactation (6 % below control)
Females F0: decreased body weight gain during premating (9 %) and gestation (18 % below control)
Females F1: decreased body weight gain during gestation (17 % below control)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males F1: Significantly decreased mean food consumption (4 % below control)
Females F1: significantly decreased mean food consumption during gestation (9 %) and mid lactation (11 %) below control.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day
F0/F1: eosinophilic homogeneous appearance of the liver cell cytoplasm indicative of enzyme induction in males/females
F0/F1: increased amount of hemosiderin in the spleen of males
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
F0: mean number of implantation sites was statistically significantly lower for the high dose group (12.0; 12.3; 11.3; 10.0 implants/dam for test group 0; 150; 450; 1000 mg/kg bw/day, respectively)
F1: mean number of implantation sites was statistically significantly lower for mid/ high dose group (12.4; 11.5; 10.7; 10.3 implants/dam for test group 0; 150; 450; 1000 mg/kg bw/day, respectively)
F0: mean number of F1 pups delivered/dam was statistically significantly lower for the high dose group (11.0 ; 11.9; 10.9; 9.2 for test group 0; 150; 450; 1000 mg/kg bw/day, respectively)
Key result
Dose descriptor:
NOAEL
Remarks:
systemic parental toxicity
Effect level:
450 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects: body weight; gross pathology; organ weights; histopathology;
Remarks on result:
other: Generation: P/F1
Key result
Dose descriptor:
NOAEL
Remarks:
Fertility and reproduction parameters
Effect level:
450 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects: secondary number of implantation sites; secondary delayed sexual maturation
Remarks on result:
other: Generation: P/F1
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
450 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects: pup weights
Remarks on result:
other: Generation: F1/F2
Key result
Critical effects observed:
not specified
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day
F1: significantly decreased mean body weights (13 % below control at weaning) and body weight gain (15 % below control) males/females during lactation
F2: significantly decreased mean body weights (16 % below control at weaning) and body weight gain (20 % below control) males/females during lactation.
F2 reared: significantly decreased mean body weights (final weight 12 % below control) and body weight gain (average 10 % below control) in males
F2 reared: significantly decreased mean body weights (final weight 6 % below control) in females
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day
F1: slight delay of preputial separation in males (average age 45.4d vs.43.6 d in control) exceeds historical control (42.5 - 45.0 d), secondary to delayed body weight development
F2 reared: slight delay of preputial separation in males (average age 45.1d vs.42.8 d in control) exceeds historical control (42.5 - 45.0 d), secondary to delayed body weight development
F1: apparent slight delay of vaginal patency in females (average age 33.8d vs. 31.3d in control) within historical control (30.8d - 33.8 d), secondary to delayed body weight development
F2 reared: apparent slight delay of vaginal patency in females (average age 35.5d vs. 32.9d in control) within historical control (30.8d - 33.8 d), secondary to delayed body weight development.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not specified
OTHER FINDINGS (OFFSPRING)
F2 reared: Significantly decreased mean food consumption (10 % below control) in males
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: significantly decreased mean body weights (13% below control at weaning) and body weight gain (15% below control) males/females during lactation
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Sex:
male
Basis for effect level:
sexual maturation
Remarks on result:
other: slight delay of preputial separation in males (average age 45.4 d vs.43.6 d in control) exceeds historical control (42.5 - 45.0 d), secondary to delayed body weight development
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Sex:
female
Basis for effect level:
sexual maturation
Remarks on result:
other: apparent slight delay of vaginal patency in females (average age 33.8 d vs. 31.3 d in control) within historical control (30.8 d - 33.8 d), secondary to delayed body weight development
Key result
Reproductive effects observed:
not specified
Endpoint:
screening for reproductive / developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because a two- (or multi-) generation reproductive toxicity study is available
Effect on fertility: via oral route
Dose descriptor:
NOAEL
450 mg/kg bw/day
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 the two-generation reproduction study (BASF SE, 2007, Schneider et al., 2005, according to OECD 416), a NOAEL of 450 mg/kg bw/day was established based on secondary number of implantation sites and secondary delayed sexual maturation.

Effects on developmental toxicity

Description of key information

- Developmental toxicity (oral) in rat (equivalent of OECD 414). NOAEL: 1000 mg/kg bw/day

- Developmental toxicity (oral) in rabbit (equivalent of OECD 414). NOAEL: 1000 mg/kg bw/day

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 January 1983 - 4 February 1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study is performed according to internationally accepted guidelines (FDA & CSM) and similar to the current OECD 414. The study is performed under GLP.
Qualifier:
according to
Guideline:
other: Food and Drug Administration (1966). Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use.
Deviations:
no
Qualifier:
according to
Guideline:
other: Committee on Safety of Medicines. Guidelines on Reproduction Studies for Guidance of Applicants for Product Licences and Clinical Trial Certificates, June 1974.
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
The number of pregnant animals in the high dose groups is too low (13 of 20) and slightly over 10 % of the animals in the high dose group died during the study (3 of 20).
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
other: Swiss hare
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Institute for Biological and Medical Research, CH-4414 Fiillinsdorf, Switzerland
- Age at study initiation: no data
- Weight at study initiation: 2510 - 3670 g
- Housing: stainless steel cages
- Diet (e.g. ad libitum): Nafag 814 cubes ad libitum, analysed for contaminants
- Water (e.g. ad libitum):ad libitum (periodically analysed)
- Acclimation period: a minimum of 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): about 18 °C
- Humidity (%): about 50 %
- Air changes (per hr): fully airconditioned
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: SSV: 0.5 % Carboxymethylcellulose, 0.5 % Benzyl-EtOH, 0.4 % TWEEN 80, 0.9 % NaCl
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test compound was formulated freshly each week in the vehicle. High shear mixing was used during formulation and the suspension was kept homogeneous during dosing by the use of a magneto stirrer.

VEHICLE
- Justification for use and choice of vehicle: solubility
- Amount of vehicle: 2 mL/kg
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not relevant
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: 4 - 6 hours
- the mounted females were mated again with another male and were then randomly allocated to the dosage groups.
- Proof of pregnancy: Day of copulation referred to as day 1 of pregnancy
Duration of treatment / exposure:
Day 7 - Day 20 of gestation.
Frequency of treatment:
Once daily.
Duration of test:
Day 1 - Day 30 (necropsy) of gestation.
No. of animals per sex per dose:
20 mated females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a dose range finding study for maternal toxicity and embryotoxicity of the test substance, doses of 200 and 500 mg/kg were administered to pregnant rabbits. No adverse effects could be observed except a slight impairment of body weight development of dams treated with 500 mg/kg. In respect to these findings we chose doses of 80, 200 and 500 mg/kg for the main study.
- Rationale for animal assignment: After successful mating the females were distributed to the dosage groups by the use of a computer generated randomization table.
Maternal examinations:
OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: all changes in behaviour, general condition, signs of pharmacological action, etc., rabbits which died during the experiment were autopsied.

BODY WEIGHT: Yes
- Time schedule for examinations: on day of gestation 1, 7, 20 and 30.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 30
- Organs examined: Only uterus examined
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
All the young were tested for their 24 h viability in an incubator at 34 °C.
Statistics:
1. Descriptive Statistics
For each group, median and interquartilerange are indicated.
2. Significance tests
For the statistical examination of each quantity, the following testalgorithm is applied:
a) Trend test
b) Simultaneous test without ordered alternative
c) Two sample tests for each dosage group against control
3. Experimental unit
The dam is considered the independent experimental unit within all significant tests.
Indices:
4. Relative numbers
The numbers of resorptions and all numbers of pubs reported are transformed for the test calculations. For each dam the relative number (relative number = absolute number/number of implantations) is determined.
Therefore, significant test results refer to the relative number, which can be interpreted as a rate, the number of implantations being 100 %.
Historical control data:
Yes, present at facility
Details on maternal toxic effects:
Maternal toxic effects: no effects

Details on maternal toxic effects:
No obvious signs of toxicity in treated females could be observed at any dosages except a slight Impairment of the body weight gain and an slightly increased frequency of constipated and anorectic dams in the 500 mg/kg group. All maternal deaths which occured during the study resulted from either broken vertebral column, enteritis or from application accidents. Therefore, the cause of deaths was not considered to be drug related.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
The parameters which could not have been influenced by the treatment, i.e. % of pregnant females, average number of corpora lutea and average number of iinplantations sites were within normal limits in all groups. The number of resorptions in % of implantations is lower in all treatment groups (12.5, 32.5 and 18.8 %) than in concurrent control (34.1 %). The control group of the present study represent an extremly high resorption rate when compared with the mean value of the historical control data (17.8%). This is mainly due to 1 dam (No. 127) exhibiting 10 complete resorptions.
The relatively high resorption rate which resulted from the group treated with 200 mg/kg is due to two dams (No 326 and 336) which both resorbed their implantations completely and therefore contributed 19 out of a total of 40 resorptions to this group (200 mg/kg). However, the distribution of resorptions does not follow a dose effect relation and it is therefore considered that there is no drug related effect on intrauterine death.
Two dams (No 433 and 439) of the highest dosage group (500 mg/kg) aborted their fetuses and died prior to term or were moribund at the autopsy day. The abortions are considered to be due to the poor health status of the mother animals and not to an abortive property of the test compound.
Both animals are excluded from statistical evaluation.

In the highest dosage group (500 mg/kg) the median individual body weight of fetuses was decreased significantly, but was comparable between the other dosage groups and the control. Whether this effect is directly due to a drug action or indirectly to the poor bodyweight gain of dams can not be definitely established from the available data.

Examination of fetuses at necropsy or following skeletal processing revealed no drugrelated deviations. All macroscopically observed abnormalities such as spina bifida, missing or rudimentary tail, torsion in limbs or ectopy of intestines are isolated findings, distributed randomly amongst all groups and are known to arise spontaneously in this rabbit strain. The examination of the head using a serial sectioning method revealed no drugrelated abnormalities at any dose groups.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Basis for effect level:
changes in postnatal survival
skeletal malformations
visceral malformations
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
not specified
Conclusions:
It may be concluded that under the conditions of the present experiment 2-Ethylhexyl 4-methoxycinnamate is neither embryotoxic nor teratogenic in rabbits at oral doses up to 500 mg/kg bw/day. A NOAEL of 500 mg/kg bw/day was therefore established for both maternal and developmental toxicity, indicating that the substance does not need to be classified as reprotoxic based on the criteria outlined in Annex I of 1272/2008/EC.
Executive summary:

2-Ethylhexyl 4-methoxycinnamate, a UV-B filter for topical use, was tested for embryotoxic and teratogenic action in rabbits according to the guidelines of the American FDA and the English CSM. Doses of 80, 200 and 500 mg/kg bodyweight were administered by oral gavage from Day 7 to Day 20 inclusive of gestation. A control group received the vehicle only (ssv, 2 mL/kg). All females were sacrificed on Day 30 of gestation. Fetuses were removed by ovariohysterectomy, tested for viability (24 h) and examined for skeletal and visceral abnormalities.

A slight decrease of maternal bodyweight gain during the treatment period and a slightly increased frequency of constipated and anorexic animals was observed in the 500 mg/kg group exclusively. No other drug related clinical sings of toxicity could be observed in treated female rabbits. The reproductive parameters were within the normal range of the rabbit strain, however 30 day old fetuses of the highest dosage group (500mg/kg) were slightly lighter when compared with the concurrent control offspring as well as with historical controls. None of the fetuses showed any drug related skeletal or visceral abnormalities at any dosage group. All adverse findings are isolated, distributed randomly amongst groups and are known to arise spontaneously in the rabbit strain.

The 24h survival rate of fetuses was not impaired by the treatment of mother animals during gestation.

It may be concluded that under the conditions of the present study 2-Ethylhexyl 4-methoxycinnamate is neither embryotoxic nor teratogenic in rabbits at oral doses up to 500 mg/kg . Whether the lighter fetuses observed in the highest dosage group (500 mg/kg) resulted from a direct intrauterine drug action during gestation or are secondarily correlated to the reduced bodyweight gain of mother animals can not be definitely established from the available data. A NOAEL of 500 mg/kg bw/day was therefore established for both maternal and developmental toxicity, indicating that the substance does not need to be classified as reprotoxic based on the criteria outlined in Annex I of 1272/2008/EC.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start January 10, 1983
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to internationally accepted guidelines and in compliance with GLP.
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
other: FDA (1966) Guidelines for reproduction studies for safety evaluation of drugs for human use
Qualifier:
according to
Guideline:
other: CSM (june 1974) Guidelines on reproduction studies for guidance of applicants for product licences and clinical trial certificates.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Fullinsdorf albino
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Institute of Biological and Medical Research, Fullinsdorf
- Age at study initiation: no data
- Weight at study initiation: ca 200 grams (day 1 of gestation)
- Housing: Wire mesh cages during acclimatization and premating period macrolon cages with dust free wood shavings during gestation and lactation.
- Diet (e.g. ad libitum): NAFAG 850 cubes ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca 22 degrees Celsius
- Humidity (%): ca. 50 %
- Air changes (per hr): fully airconditioned
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: Standard Solvent Vehicle (SSV)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Frequency: Freshly each week
Storage: Stored in a refrigerator
High sphere mixing during preparation

VEHICLE
- Justification for use and choice of vehicle: SSV (standard solvent vehicle )
- Amount of vehicle: 10 mL/kg
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not relevant
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- The mating procedure was repeated until enough mated females were available.
- Proof of pregnancy: vaginal plug referred to as day 1 of pregnancy
Duration of treatment / exposure:
From day 7 through day 16 of gestation
Frequency of treatment:
Once daily
Duration of test:
Day 1 of gestation - Day 23 of lactation
No. of animals per sex per dose:
36 mated females females per dose group (test # 4R83)
20 mated females (test # 33R83)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: preliminary study with a dose of 1000 mg/kg did not produce any maternal effects or embryotoxic or teratological effects.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
All changes in behaviour and general condition, signs of pharmacological effects etc. Animals which died during the experiment were necropsied.

BODY WEIGHT: Yes
- Time schedule for examinations:On days of gestation 1, 7, 17 and 21 for all females and on parturition (within 24 hours after beginning of parturition) and on days of lactation 4, 12 and 23 for the females of the rearing group.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21 (Subgroup 1)
- Sacrifice after lactation day # 23 (Subgroup 2)
- Organs examined: uteri
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
-Number of resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter (15 litters per dose group)
- Skeletal examinations: Yes: half per litter (15 litters per dose group)
Statistics:
- Descriptive statistics:
In the tables for each group, median and interquartile-range are indicated.
Significance test:
For the statistical examination of each quantity, the following test
algorythm is applied:
a) Trend test.
b) Simultaneous test without ordered alternative.
C) Two sample tests for each dosage group against control.
Experimental unit:
The dam is considered the independent experimental unit within all significant tests.
Indices:
- Relative numbers:
The numbers of resorptions and all numbers of pups reported are transformed for the test calculations. For each dam the relative number = absolute number/number of implantations is determined. Therefore, significant test results refer to the relative number, which can be interpreted as a rate,
the number of implantations being 100 %.
Historical control data:
Yes, control data on used strain available at test facility.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No drug-related maternal mortality and no signs of maternal toxicity were observed in this study . In the highest dose group the weight development over the whole gestation period was slightly reduced.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
In the highest dose group the incidence of skeletal variations known at the test facility with this strain of rats was slightly increased but no malformations were observed indicating that the test substance was not teratogenic.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
not specified
Conclusions:
It may be concluded that under the conditions of this study the test substance was not embryotoxic and not teratogenic up to the dose of 1000 mg/kg bw/day (NOAEL). Therefore, ethylhexyl methoxycinnamate does not need to be classified as reprotoxic according to the criteria outlined in Annex I of 1272/2008/EC.
Executive summary:

2-ethylhxyl 4 -methoxycinnamate was tested for embryotoxic and teratogenic action in rats in accordance with the guidelines of the American FDA and the English CSM. Doses of 0, 250, 500 and 1000 mg/kg were administered orally in SSV from day 7 through day 16 of gestation. No drug-related maternal death and no signs of maternal toxicity were noted in all dose groups. There was no indication of any embryotoxic or teratogenic action of 2-ethyl-p-methoxy cinnamate in all 3 dose groups tested. The rearing experiment showed no indication of any functional abnormalities in all dose groups.

It may be concluded that under the conditions of this study the test substance was not embryotoxic and not teratogenic up to the dose of 1000 mg/kg bw/day (NOAEL). Therefore, ethylhexyl methoxycinnamate does not need to be classified as reprotoxic according to the criteria outlined in Annex I of 1272/2008/EC.

Effect on developmental toxicity: via oral route
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
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

The two key studies were chosen because of the dosing ranges that were tested in different species (Burgin, 1983, Kistler, 1983). Both supporting studies were limit tests with a similar design. In one supporting study (Burgin, 1986, equivalent of OECD 414) for oral developmental toxicity (limit test) in rabbits, no effects were found at 500 mg/kg bw/day. In another supporting study in rats (Eckhardt, 1985, equivalent of OECD 414) for the same endpoint, no effects at 1000 mg/kg bw/day were observed. This justifies the NOAEL of 1000 mg/kg bw/day as was established in the key studies.

Toxicity to reproduction: other studies

Description of key information

A Rodent Hershberger assay (BASF SE, 2003) and an Uterotrophic Assay (BASF SE, 2001) were available as other supporting studies for the endpoint of toxicity to reproduction. The first study exposed rats for 10 days to doses up to 1000 mg/kg bw/day and found that ethylhexyl methoxycinnamate had no anti-androgen efficacy. The second study found no uterotrophic (estrogenic) effect of the submission substance up to an oral dose of 1000 mg/kg bw/day for 3 days in rats.

A further supporting study undertaken was a Hormone study in female Wistar rats (BASF SE, 2004; Robust Study Summary see section 7.5.1) with administration at 1000 mg/kg bw/day in the diet over 4 weeks. Multiple hormone assays were undertaken. The rat is considered a very sensitive species for indirect thyroid hormone effects in comparison to man. Serum thyroid stimulating hormone (TSH) and total triiodothyronine (T3) were not affected. Slightly increased serum thyroxine (T4) concentrations were observed but, in the absence of effects on TSH or T3, ethylhexyl methoxycinnamate was not considered to be a true thyroid modulator or thyroid toxicant. In the other hormone investigations, in particular for estradiol and progesterone, no treatment-related effects were observed.

These studies support the findings in the two generation study which indicate that in vivo ethylhexyl methoxycinnamate does not have an adverse effect on reproductive function or endocrine function up to high dosages.

Link to relevant study records

Referenceopen allclose all

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study
Qualifier:
according to
Guideline:
other: Rodent Hershberger assay
GLP compliance:
yes (incl. certificate)
Type of method:
in vivo
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany

- Age at study initiation: 56 ± 1 days
- Weight at study initiation: mean weights per group 199.8 - 205.7 g
- Fasting period before study: no
- Housing: Singly in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm2). Underneath the cages, disposable waste trays were used.
- Diet : ground Kliba maintenance diet rat-mouse meal, ad libitum
- Water : ad libitum
- Acclimation period: 14d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 hours light from 06.00 a.m. - 06.00 p.m., 12 hours dark from 06.00 p.m. - 06.00 a.m.

IN-LIFE DATES: From: 23.04.2003 To: 28.05.2003
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
VEHICLE
- Concentration in vehicle: 6, 20 g/100 mL
- Amount of vehicle: 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
10 days
Frequency of treatment:
daily
Duration of test:
4 weeks
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Statistics:
Food consumption, body weight, body weight change, food efficiency: A comparison of each group with control group was performed using DUNNETT's test (two-sided).
Organ weight parameter: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). lf the resulting p-value was equal or less than 0.05, a pairwise comparison of dose groups with control groups using the WILCOXON test for the hypothesis of equal medians was performed.

- Mortality: No animal died prematurely.

- Clinical examinations/ Food-Water consumption / Body weight data / Food efficiency: No substance-related findings were obtained.

- Hormones:

In the serum of castrated male rats testosterone levels were significantly decreased and luteinizing hormone concentrations slightly increased compared to the values of castrated animals receiving 0.4 mg/kg bw/day of testosterone propionate as reference androgen. No significant difference in serum dihydrotestosterone concentrations was seen between both test groups. No effects on testosterone, dihydrotestosterone and luteinizing hormone concentrations were found in the serum of castrated males treated with 1000 mg/kg bw/day of Uvinul MC 80 N and 0.4 mg/kg bw/day of testosterone propionate for 10 days when compared with the hormone levels of animals given testosterone propionate, only.

- Pathology:

Absolute liver weight was significantly increased (1000 mg/kg bw / day). Relative liver weights were significantly increased (300, 1000 mg/kg bw / day).

Absolute and relative weights of the accessory sex organs were significantly reduced (vehicle untreated) compared to testosterone proprionate animals. Histologically, prostate, seminal vesicle and the bulbo-urethral gland were immature. Absolute weight of the ventral prostate (fresh) was significantly decreased (1000 mg/kg bw / day). Absolute weights of the ventral prostate (fixed) were dose-related significantly decreased in (300, 1000 mg/kg bw / day). The relative weights of these organs did not show significant differences, when compared to control group 1.

The terminal body weight was slightly but not significantly decreased: 300 mg/kg bw / day (-2.3%) and 1000 mg/kg bw / day (-5.5%). The decreased weights of the ventral prostate are considered as consequence of the slightly reduced terminal body weight of these animals and do not represent an adverse compound-related effect. Absolute and relative weights of the other accessory sex organs were not significantly reduced. Histology of prostate, seminal vesicle and the bulbo-urethral gland was comparable to control.

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: non GLP, only brief summary
Qualifier:
according to
Guideline:
other: Uterotrophic Assay
GLP compliance:
no
Type of method:
in vivo
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: 20 + 1 days
- Weight at study initiation: 30.0 - 40.0 g
- Housing: Makrolon Cages, type M-Il (Becker & Co., Castrop-Rauxel), two animals per cage; Bedding material: SSNIFF (type 3/4)
- Diet (e.g. ad libitum): Pelleted Kuba maintenance diet rat/mouse/hamster; ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: min 1 day before study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 hours light (6.00 to 18.00 hours), 12 hours darkness (18.00 to 6.00 hours)

IN-LIFE DATES: From: 22.01.2001 To: 26.01.2001
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
VEHICLE
- Concentration in vehicle: 50, 200 mg/mL
- Amount of vehicle: 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC analysis
Duration of treatment / exposure:
3 days
Frequency of treatment:
daily
Duration of test:
5 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Statistics:
Dunnett-test (two-sided)
Wilcoxon-test (two-sided)

- Clinical examinations: No clinical signs or symptoms were detected

- Body weights: No statistically significant differences were found.

- Body weight gain: Statistically significantly increased between days 2 and 3 (250 mg/kg bw/day). Statistically significantly reduced between days 0 and 1 (1000 mg/kg bw/day). No statistically significant difference in the positive control.

- Uterus weights: No increased uterus weights compared to carrier control (250 and 1000 mg/kg bw/day). Positive control DES-DP (5 µg/kg) used led to a statistically significant increase in the absolute and relative uterus weights with a 4.7- and 4.9-fold increase, respectively, compared to carrier control.

- Histopathology: No changes in the uterus detected (250 and 1000 mg/kg bw/day). Positive control (5 µg/kg DES-DP) morphologically showed the changes in the uterus:

1. Endometrial epithelium extended and hypertrophic

2. Uterus lumen partially clearly dilated

3. Uterus wall thickened without any of the muscular layers or particularly the stroma being prominent

Justification for classification or non-classification

Based on the results, the substance Ethylhexyl Methoxycinnamate does not need to be classified as reprotoxic based on the criteria outlined in Annex I of 1272/2008/EC.