Benzoic acid, 4-[[[4-[(1-oxo-2-propenyl)oxy]butoxy]carbonyl]oxy]-, 2-methyl-1,4-phenylene ester

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Fertility was investigated at the screening level (OECD 421 of 28 July 2015, GLP). Rats were dosed by gavage with 100, 300 and 1000 mg/kg bw. Offspring was observed until postnatal day 14.

No adverse effects on fertility were observed at any dose level. The NOAEL is 1000 mg/kg bw.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016/2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

not applicable for OECD 421

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 11-12 weeks (males) and 10 weeks (females) at receipt
- Weight at study initiation: (P) Males: 348.5 g - 392.2 g; Females: 200.4 g - 234.5 g
- Fasting period before study: no
- Housing: single caging, unless during mating
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: June 2016 To: August 2016

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material is weighed into calibrated beaker and dopped up with corn oil to achieve the required concentration.
The mixture is intensely mixed with a homogenizer. Before and during administration, the preparations are kept homogenous with a magnetic stirrer.


VEHICLE
- Justification for use and choice of vehicle (if other than water): The substance is not soluble in water.
- Amount of vehicle (if gavage): 4 ml/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days or shorter (if proof of mating)
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after one unsuccessful attempts: no
- After successful mating each pregnant female was caged in individual cages

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in corn oil at room temperature over a period of 96 hours had been verified prior to the start of the study.

At the beginning (during premating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. These samples were used as a concentration control at the same time. At the above mentioned time points additionally one sample from the mid concentration was taken for concentration control analysis

Duration of treatment / exposure:

males: 4 weeks
females: 57 days

Frequency of treatment:

daily (exception: no dosing of animals in labor)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No adverse effects observed at the limit dose in the existing 28-day study (OECD 407).

Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1.

At necropsy on PND 4 and PND 13, the pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings. On PND 13 thyroid glands and parathyroid glands were fixed for possible further processing. All surviving male pups were examined for the presence or absence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen were counted.

Blood samples for hormone measurements were taken from all surplus pups at PND 4 as well as from one male and one female pup per litter at PND 13 during necropsy of pups.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity.
During the administration period all animals was checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after
the administration. Abnormalities and changes were documented for each animal.
The parturition and lactation behavior of the dams was generally evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular
findings (e.g. inability to deliver or umbilical cord not cut) would be documented on an individual dam basis.
On weekdays (except Saturdays, Sundays and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.


DETAILED CLINICAL OBSERVATIONS: See above


BODY WEIGHT: Yes
- Time schedule for examinations: Once per week. During the mating period, the females were weighed on the day of positive
evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 0) and on PND 4, 7, 10 and 13


:

Oestrous cyclicity (parental animals):

In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to mating and
throughout cohabitation until there is evidence of sperm in the vaginal smear.
Additionally, on the day of scheduled sacrifice, the estrous status will was determined in all parental female rats.

For all females of the pool estrous cycle normality was valuated before the beginning of the administration period (The estrous cycle data of these individuals are reported and can be found in the raw data).

Sperm parameters (parental animals):

Parameters examined in all Pmale parental generations:
testis weight, epididymis weight, special attention was given on the stages of spermatogenesis in the testes during histopathology.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups]

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities.
If applicable and possible, cause of death for pups that died prematurely was determined.

Other: On PND 4, as a result of standardization, the surplus pups, were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid
hormone concentrations
On PND 13, one selected male and one female pup per litter were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid
hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after 28 days
- Maternal animals: All surviving animals at the end of postnatal day 13

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively.
Organ weight determination:
1. Anesthetized animals
2. Epididymides
3. Ovaries
4. Prostate
5. Seminal vesicles with coagulating glands
6. Testes
7. Thyroid glands (fixed)
8. Uterus (with cervix)

Organs for microscopic examination:
1. All gross lesions
2. Cervix
3. Coagulating glands
4. Epididymides (modified Davidson's solution)
5. Ovaries (modified Davidson's solution)
6. Oviducts
7. Prostate
8. Seminal vesicles
9. Testes (modified Davidson's solution)
10. Thyroid glands
11. Vagina
12. Uterus

Postmortem examinations (offspring):

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

HISTOPATHOLOGY / ORGAN WEIGTHS
No histopathology and organ weight examination was performed for offspring.

Statistics:

Included, statistical test depending on parameter

Reproductive indices:

Mating indices, fertility indices, gestation index

Offspring viability indices:

viability index, survival index

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
Remark: High-dose male No. 37 and female No. 137 are not used for the calculation of the fertility parameters, as the male animal No. 37 was found dead on mating day 1.
Subsequently high-dose female No. 137 had no sperm in vaginal smear (no GD 0) and consequently no implants or pups.
The mean duration until sperm was detected (GD 0) varied between 2.2 and 2.4 days without any relation to dosing.
All female rats delivered pups or had implants in utero with the following exceptions:
• Control female No. 105 (mated with male No. 5) did not become pregnant.
• High-dose female No. 138 (mated with male No. 38) did not become pregnant.
The fertility index varied between 100% in test groups 1 and 2, 90% in test group 0 and 88.9% in test group 3. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.

The mean duration of gestation was similar in all test groups: 22.2 days in test groups 1 and 3, 22.3 days in test group 0 and 22.4 days in test group 2.
The gestation index varied between 90% (test group 2) and 100% (test groups 0, 1 and 3).

One female animal (No. 125; 300 mg/kg bw/d) had all pups stillborn (complete litter loss on PND 0) and was found dead on PND 2. The death of this animal is considered as a spontaneous finding which cannot be attributed to the test substance administration.

Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (13.1 / 11.0 / 13.0 and 13.5 implants/dam in test groups 0-3, respectively).

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed at the limit dose

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Gross pathological findings:

no effects observed

There were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (12.9 / 10.6 / 11.9 and 12.8 pups/dam in test groups 0-3, respectively).
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99.1% / 98.9% / 89.1% and 100% in test groups 0-3.

The mean number of delivered F1 pups per dam and the rates of liveborn F1 pups were evenly distributed among the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
The number of stillborn pups as well as perinatal loss in test group 2 was increased (without attaining statistical significance). This finding is incidental and mainly caused by one litter (No. 125) who has all pups stillborn and died thereafter.

The viability index indicating pup mortality during early lactation (PND 0-4) varied between 97.1% (test group 1), 97.2% (test group 2), 98.2% (test group 3) and 99.3% (control) without
relation to dosing. The survival index indicating pup mortality PND 4-13 was 100% in all test groups.

One male runt was seen in the control, two male and two female runts were seen in test group 1 and one female runt was seen in test group 3.

Neither on anogenital distance nor anogenital index test substance-related effects were noted in all treated F1 offspring (100, 300 and 1000 mg/kg bw/d]).
The slightly but statistically significantly increased anogenital index of female pups in test group 2 (300 mg/kg bw/d) is considered as spontaneous in nature due to the lack of doseresponse relationship.

The number of low-, mid- and high-dose male pups having areolae was not influenced by the test substance when examined on PND 13.
The percentage of male pups in test groups 1-3 having areolae was not influenced by the test substance when examined on PND 13.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects at the limit dose

Remarks on result:

other: F1 raised until PND 21

Critical effects observed:

no

Reproductive effects observed:

no

Analyses confirmed the overall accuracy of the prepared concentrations and the homogeneity of the test substance in the vehicle. The stability of these preparations was also demonstrated over a period of 96 hours under ambient conditions.

During the mating and post-mating period in some high dosed male animals salivation after treatment was seen. From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Developmental toxicity was investigated at the screening level (OECD 421 of 28 July 2015, GLP). Rats were dosed by gavage with 100, 300 and 1000 mg/kg bw. Offspring was observed until postnatal day 13.

No adverse effects on offspring were observed at any dose level. The NOAEL is 1000 mg/kg bw.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available screening study is reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for fertility or developmental toxicity under Regulation (EC) No. 1272/2008,as amended for the seventh time in Regulation (EC) No 2015/1221.

During the 13 days covered in the screening study, no effects via lactation were observed.