Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, 2-ethylhexyl ester

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 12 July 2000 to 03 April 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Version / remarks:

1983

Deviations:

no

Principles of method if other than guideline:

Study performed according to OECD guideline and according to GLP. The analytical investigation at 5mg/mL were carried out as part of a preliminary study which was in compliance with GLP. In the current report no compliance was claimed for this data. However, this did not affect the validity of the report.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

406-040-9

EC Name:

-

Cas Number:

125643-61-0

Molecular formula:

C24-26 H40-44 O3

IUPAC Name:

C7-9-(branched)-alkyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate

Details on test material:

Description of test substance: brown liquid
Stability of formulations: 15 days

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature protected from light
- Stability of formulations: 15 days

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation (P): approximately 5 weeks
- Weight at study initiation (P): 22,4 to 35,1 g for males and 18,2 to 27,1 g for females
- Housing: Mice were housed singly in MT2 cages (29cm x 11.5cm x 11cm) from North Kent Plastics Limited, Erith, Kent, England throughout the study (except during cohabitation for pairing and when females were with their litter during lactation).
- Diet: commercially available laboratory animal diet (VRF-1), ad libitum. During week 11 of the study, the diet in four food hoppers (group 4 males) was found to be contaminated with mould. Contaminated food hoppers were replaced with food hoppers containing a new batch of diet. Upon completion of the study data from these animals were scrutinized and no effect on the outcome of the study was seen.
- Water: water from the public supply, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25°C
- Humidity: 40-70%
- Air changes: The animal room had its own supply of filtered air, under positive pressure, which was passed to the atmosphere without recirculation.
- Photoperiod: 12 hrs/12 hrs.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% carboxymethylcellulose in 0.1% Tween 80

Details on exposure:

The dosages of the test item were prepared weekly as a mixture in the vehicle. For each concentration the required quantity of test item was weighed, and added to the vehicle and then mixed thoroughly until it was visibly homogeneous. The formulations were re-mixed before and during dosing using a magnetic stirrer.
The animals were dosed daily for 8 weeks prior to pairing until termination at a volume-dosage of 10 mL/kg bw/day. Control animals received the vehicle at the same volume-dosage during the treatment period. The volume administered daily to each animal was based on the last scheduled bodyweight for males throughout the study and for females during the pre-pairing period. For females after mating the volume administered daily was based on the bodyweight recorded before dose administration.

Details on mating procedure:

After eight weeks of treatment, males and females from the same treatment groups were paired on a one-to-one basis. Each morning following pairing the females were examined for the presence of a copulation plug. The day on which positive evidence of mating was found was designated Day 0 of gestation.
One male was killed before pairing and the prospective female of this male was paired with a male that mated successfully within that group. One female at 150 mg/kg/day showed no positive evidence of mating after 14 days of pairing. Therefore, the original male partner was replaced by a male that successfully mated within that group.
Once mating had been detected the males and females were separated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of the test substance during ambient temperature storage for 2 days and refrigerated storage for 15 days were confirmed at nominal concentrations of 5 and 50 mg/mL in a preliminary study (Huntingdon Life Sciences Ltd. 000054). A copy of this analytical report was attached to the current report.
The homogeneity and stability during ambient temperature storage for 2 days and refrigerated storage for 15 days were confirmed at a nominal concentration of 60 mg/mL as part of the present study. The storage period represented the maximum time from preparation to completion of use. In addition, the stability of discrete (1 ml) samples was confirmed during freezer storage for 8 days.The mean concentrations of test substance analysed during the study (Weeks 1, 9 and 13) were between 1.3% below and 3.6% above nominal concentrations, confirming the accuracy of formulation.

Duration of treatment / exposure:

The animals were dosed daily by the oral route (gavage) for 8 weeks prior to pairing and until termination at a volume-dosage of 10 mL/kg bw/day.

Frequency of treatment:

once daily

Details on study schedule:

After eight weeks of treatment, 13 weeks old males and females from the same treatment groups were paired on a one-to-one basis. Each morning following pairing the females were examined for the presence of a copulation plug. The day on which positive evidence of mating was found was designated Day 0 of gestation. From Day 17 after mating females were inspected three times each day for the onset, progress and completion of parturition. All offspring were examined at approximately 24 hours after birth.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10 animals/sex and group

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CLINICAL SIGNS AND MORTALITY
All animals were examined at least twice daily throughout the study and any visible signs of reaction to treatment were recorded. Detailed clinical observations were recorded daily during the first week of treatment and weekly thereafter.
Animals sacrificed or found dead were examined macroscopically and specimens of tissues considered abnormal were retained.

BODYWEIGHT
Males were weighed twice weekly for the first two weeks of the study and weekly thereafter. Females were weighed twice weekly for the first two weeks of the study and then weekly until mating was detected. After mating females were weighed on Days 0, 6, 12 and 17 after mating and on Days 1, 4, 7, 14 and 21 of the lactation period.

FOOD CONSUMPTION
Food consumption was recorded weekly for F0 males and females until the animals were paired for mating. Food consumption for females after mating was recorded for Days 0, 6, 12 and 17 of gestation and Days 1, 4, 7 and 14 of the lactation period.

BLOOD SAMPLING FOR PLASMA T3 AND T4
Immediately before termination each animal was bled under isoflurane anaesthesia from the orbital sinus using lithium heparin as an anticoagulant. Approximately 0.5 ml of whole blood was taken from each animal. The samples were then centrifuged and the plasma divided into 2 aliquots, one of which was assayed for total thyroxine (T4) and the other assayed for total tri-iodothyronine (T3) by radioimmunoassay.

ASSESSMENT OF REPRODUCTIVE PERFORMANCE
The time elapsing between initial pairing and detection of mating was recorded.
From Day 17 after mating females were inspected three times each day for the onset, progress and completion of parturition. The time elapsing between the detection of mating and commencement of parturition was reported. Dams were observed daily for evidence of abnormal maternal behaviour.

Sperm parameters (parental animals):

Immediately after sacrifice the left vas deferens, epididymis and testis of each male was removed and the epididymidis and testis were weighed.
A sample of sperm was expressed from the vas deferens into pre-warmed (37°C) medium Ml99, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). At least 200 sperm per animal were analysed for:
a) The percentages of motile and progressively motile sperm for all groups,
b) Sperm morphology after staining with Nigrosine and Eosin.

The left cauda epididymis of each male was weighed and homogenised in 1 ml of a mixture of 0.9% saline, 0.01% merthiolate and 0.05% Triton X-100 (SMT-mixture) An aliquot of this mixture was assessed for sperm count. The concentration (Million/g) and total number of sperm were reported for Groups 1 and 4 only.
Homogenisation-resistant spermatids
The left testis of each male was weighed and frozen. Prior to analysis, the testis was allowed to thaw, 1 ml of SMT-mixture was added and the testis was homogenised. An aliquot of this mixture assessed for homogenisation-resistant spermatid count. The concentration (Million/g) and total number of spermatids were reported for Groups 1 and 4 only.

Litter observations:

POST-NATAL OBSERVATIONS (Fl OFFSPRING)
Observations at Day 1 of age
All offspring were examined at approximately 24 hours after birth (Day 1 of age) and the a) Number of offspring (live and dead), b) Bodyweights of live offspring - weighed individually on Days 1, 4 (before culling), 7, 14, and 21 of age, c) Sex ratio and d) Observations on individual offspring were recorded for each litter.
Observations after Day 1:
Litters were observed daily for evidence of abnormal appearance or behaviour and mortality of pups. Any offspring found dead were examined externally and internally. Litters containing 11 or more offspring were culled by random selection to 10 (where possible 5 males and 5 females per litter) on Day 4 of age. Culled offspring were killed and discarded without necropsy.
Group mean litter size and litter weights were calculated from the individual litter values. Group mean offspring bodyweight was calculated separately for male and female offspring from the individual litter values.

Postmortem examinations (parental animals):

NECROPSY, F0 GENERATION
The males were sacrificed following successful littering of the females and the females following weaning of their progeny. Immediately before termination each animal was bled under isoflurane anaesthesia from the orbital sinus using lithium heparin as an anticoagulant. Approximately 0.5 mL of whole blood was taken from each animal. The samples were then centrifuged and the plasma divided into 2 aliquots, one of which was assayed for total thyroxine (T4) and the other assayed for total tri-iodothyronine (T3) by radioimmunoassay. After blood sampling the animals were sacrificed by inhaled carbon dioxide and each animal was subjected to a detailed macroscopic examination.

ORGAN WEIGHTS
The following organs, taken from each F0 parental animal were weighed (paired organs were weighed separately): Adrenal glands, Prostate (ventral lobe, males), Brain, Seminal vesicles and Coagulating gland (males), Epididymides (males), Spleen, Kidneys, Testes (males), Liver, Thyroid, Ovaries (with oviduct, females), Uterus with cervix (females), Pituitary.

TISSUES PRESERVED FOR EXAMINATION
Samples of Adrenal glands, Brain, Epididymides (right), Kidneys, Liver, Mammary glands (caudal), Ovaries (with oviduct), Pituitary, Prostate (ventral lobe), Seminal vesicles and Coagulating gland, Spleen, Testis (right), Thyroid, Uterus with cervix, and Vagina, as well as tissues with abnormalities were preserved in 10% neutral buffered formaldehyde (NBF), except for the testes and epididymides, which were preserved in Bouin's fluid for at least 24 hours before transfer to 70% industrial methylated spirit.

HISTOPATHOLOGY
Following tissue samples from parental animals of group 0 and 4 were dehydrated, embedded in paraffin wax, sectioned, stained with Haematoxylin and Eosin and investigated microscopically: Epididymides(right, caput, corpus and cauda), Liver, Ovaries with oviduct(left and right), Prostate (ventral lobe), Seminal vesicles and Coagulating gland, Testis (right), Uterus with cervix, Vagina and tissues with abnormalities.
Testes were stained using a standard Periodic Acid-Schiff (PAS) method. Examination of the ovary of F0 females was limited to a qualitative assessment of the presence of primordial follicles, growing follicles and corpora lutea.
Tissues with abnormalities from all mice should have been subjected to histopathological examination. Since findings were seen across all groups and were clearly not treatment related, microscopic examinations were not performed on these tissues.

Postmortem examinations (offspring):

NECROPSY, F1 OFFSPRING (Day 21 post natal)

ORGAN WEIGHT
Brain, Liver, Spleen and Thymus were weighed from the first male and the first female selected from each litter. In addition, following organs were retained from these animals:
Brain, Epididymides, Liver, Ovaries (with oviduct), Prostate (ventral lobe), Seminal vesicles and coagulating gland, Spleen, Testes, Thymus, Uterus with cervix, Vagina, Tissues with abnormalities.

HISOPATHOLOGY
As no treatment related findings were observed at macroscopic examination in the offspring, tissues from these animals were not examined microscopically.

Reproductive indices:

For each group and sex the Percentage mating, Conception rate, Fertility index, Gestation length and Gestation index were calculated.

Offspring viability indices:

Survival indices for each group were calculated from individual litter values: Post-implantation survival index, Live birth index, Viability index, Lactation index.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment related clinical signs were seen at any dose group. No treatment related mortality was observed. However, one male of the high dose group was sacrificed moribund; one female of the high dose group and one females of the control group was found dead. A further female of the control group was sacrificed moribund. None of these mortalities was treatment related.
One total litter loss was seen in each of the treatment groups on or before Day 1 of age. These litter losses were considered to be not treatment related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No treatment related mortality was observed. However, one male of the high dose group was sacrificed moribund; one female of the high dose group and one females of the control group was found dead. A further female of the control group was sacrificed moribund. None of these mortalities was treatment related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effects on body weight were seen in any dosage group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption of males and females remained unaffected by treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

An increased incidence of hepatocyte hypertrophy of the centrilobular regions of the liver was seen in 23/32 males and 10/32 females at 600 mg/kg bw/day. The high incidence of centrilobular hepatocytic hypertrophy was considered to be an adaptive response to treatment.
No further treatment related findings were seen upon histopathology. There were no apparent effects on the reproductive organs of males and females.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

PLASMA T3 AND T4 (details see table 1)
T3 level were slightly increased in animals of both sexes, while T4 level were slightly decreased only in male animals at 600 mg/kg bw/d when compared to control. Only the decreased T4 levels of males at 600 mg/kg bw/day were statistically significant. Since the T3 level was elevated in both sexes and effects on T3 and T4 Level were only seen in the highest dose group these changes in T3/T4 levels could be treatment related.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No effects on any parameter examined (motility, progressive motility, epididymal sperm count, homogenisation-resistant spermatids from the testis and sperm morphology) were seen at 600 mg/kg bw/d. Due to the absence of effects at the highest dose group sperm analysis was not conducted at the lower treatment groups.

Reproductive performance:

no effects observed

Description (incidence and severity):

No effects on mating performance, fertility, gestation length, parturition and gestation indices were seen at any dosage level.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: At 150 mg/kg bw/day liver weights were increased in both sexes. Since no histopathological correlates were seen at this dose this effect was considered to be not adverse.

2

Key result

Dose descriptor:

NOAEL

Remarks:

(reproduction performance and fertility)

Effect level:

600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Since no effects on mating performance, fertility, gestation length, parturition and gestation indices were seen at the highest dose level tested, the NOAEL was set at 600 mg/kg bw/day.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were seen in the offspring of parental animals from any dose group.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No effects on survival to Day 21 were seen.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No differences in bodyweights of offspring at birth and no effects on bodyweight change during lactation were seen.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No effects on brain, spleen liver or thymus weights were seen.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment related effects were seen upon macroscopic examination of pups dying before weaning or pups sacrificed at weaning.

Histopathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

The number of uterine implantation sites (recorded at termination), litter size at birth and survival of offspring to Day 4 showed minor intergroup variation without dose response relationship.
The sex ratio from Day 1 to Day 4 and to Day 21 after birth was comparable throughout the study.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

(developmental)

Generation:

F1

Effect level:

600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Littering, survival and development of the F1 progeny were similarly unaffected at dose levels up to and including 600 mg/kg bw/day.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1: Blood T3 and T4 level in males and females

		Dose (mg/kg bw/d)
		0	50	150	600
MALES
Total T3	nmol/L	1.41 +/ -0.16	1.43 +/- 0.17	1.42 +/- 0.17	1.51 +/- 0.14
Total T4	nmol/L	52 +/- 11	53 +/- 12	49 +/- 13	44 +/- 11*
FEMALES
Total T3	nmol/L	1.38 +/- 0.19	1.32 +/- 0.20	1.37 +/- 0.19	1.4 +/- 0.22
Total T4	nmol/L	62 +/- 20	59 +/- 13	57 +/- 18	54 +/- 15
*, p<0,05

Applicant's summary and conclusion

Conclusions:

The No-Observed-Effect-Level for reproductive function and survival, growth and development of the offspring to weaning in the context of this study was 600 mg/kg/day.

Executive summary:

The test substance was administered to male and female CD-1 mouse in a one-generation study according to OECD guideline 415 (1983) via gavage in doses of 50, 150 and 600 mg/kg bw/d for a total of 16 weeks.

No treatment-related mortalities, no clinical signs and no effects on bodyweight and food consumption were seen in parental animals of both sexes even at the highest dose group.

At 600 mg/kg bw/d the relative liver weights of males and females were increased and at histopathology centrilobular hepatocytic hypertrophy was seen in these animals. Liver weights were also increased at 150 mg/kg bw/day in both sexes. Since no histopathological correlates were seen at this dose this effect was considered to be not adverse.

Thyroid weights were increased in males at 600 mg/kg bw/day. T3 level were slightly increased in animals of both sexes, and T4 level were slightly decreased only in male animals at 600 mg/kg bw/d when compared to control. Although only the decreased T4 levels of males reached statistically significance, these changes in T3/T4 levels could be treatment related.

Due to the increased liver weights with histopathological correlates and the increased thyroid weights which possibly resulted in the changes in T3/T4 levels, the NOAEL for parental toxicity was considered to be 150 mg/kg bw/day.

 No effects on mating performance, fertility, the ability of the females to successfully rear a litter to weaning and the condition, survival and development (to weaning) of the offspring were seen even at the highest dose group. No treatment related macroscopic findings were seen. Thus, the NOAEL for reproduction and developmental toxicity was 600 mg/kg bw/day, respectively.