Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to OECD guideline with GLP, acceptable with restrictions (male reproductive parameters, oestrous cycle, and developmental milestones of pups and fertility index/gestation index were not examined)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

male reproductive parameters, oestrous cycle, and developmental milestones of pups and fertility index/gestation index were not examined

GLP compliance:

yes

Remarks:

Huntingdon Research Centre Ltd., England

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate

EC Number:

218-216-0

EC Name:

Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate

Cas Number:

2082-79-3

Molecular formula:

C35H62O3

IUPAC Name:

octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate

Details on test material:

- Physical state: a white powder

Test animals

Species:

rat

Strain:

other: Crl:COBS CD (SD) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd., Manston Road, Margate, Kent
- Age at study initiation: (P) x 6 wks; (F1) x 4 wks (±4 days)
- Diet (e.g. ad libitum): Spratt's Laboratory Diet No. 2 (low fat)
- Water (e.g. ad libitum): drinking water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 50±20
- Air changes (per hr): 13
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh batches of each concentration of diet were prepared weekly throughout the study. Diet remaining in the food hoppers at the end of each week was discarded.
- Mixing appropriate amounts with (Type of food): An appropriate quantity of material as supplied was weighed and ground directly into a weighed amount of sieved Spratt's Laboratory Diet No. 2 and stirred by hand to give a premix of suitable strength. The dietary concentrations required were obtained from this premix by direct dilution with further quantities of diet, homogeneity being achieved by mixing for 7 minutes in a rotary double-cone blender.
- Storage temperature of food: The diets were then stored until use in heat-sealed opaque polythene bags.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 20 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

- Premating Exposure Period: 10 weeks
- Treatment of the F0 males and females by dietary inclusion began when they were 6 weeks of age, and continued until all F1 litters had been weaned. Direct treatment of the F1 males and females was considered to commence when they were four weeks (± 4 days) of age, and continued until all F2 litters had been weaned.

Frequency of treatment:

continuously

Details on study schedule:

- F1 parental animals not mated until 4 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
500, 1500 and 5000 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
32.09, 96.48 and 315.12 mg/kg bw/d in males and 39.00, 111.26 and 373.04 mg/kg bw/d in females
Basis:
actual ingested

No. of animals per sex per dose:

F0 generation: 28
F1 generation: 24

Control animals:

yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations: signs and mortalities

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal of the F0 generation was determined initially (i.e. week -1) and subsequently at weekly intervals. All pups from the main mating phase of each generation (Fl and F2) were weighed at birth, and on Days 4, 8, 12 and 21 post partum. Pups derived at re-mating were weighed at birth and on Days 4 and 8. Animals retained for the Fl generation were weighed weekly from selection. During the mating period, all females were additionally weighed on alternate days throughout. The occurrence of a positive indication of mating (i.e. sperm or plug) was considered as Day 0 of pregnancy and thereafter females were also weighed on presumed Days 0, 7, 14, 17 and 20 of gestation. Weights of pregnant animals without positive indication of mating were obtained by retrospective calculation from birth (assuming a 22- day gestation period. Dams that littered were weighed as soon as possible after parturition and on Days 7, 14 and 21 post partum.

OTHER:
- Pregnancy rate was determined as the percentage of paired females that became pregnant.
- Vaginal smears were taken during the mating period to enable the number of animals that mated on specific days to be determined, this information was used:
(i) to detect whether pregnancy was interrupted after mating
(ii) to detect marked anomalies of the oestrous cycle
(iii) to determine the median pre-coital time for the group
- The duration of gestation was taken as the time between the day of successful mating and parturition.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no, as soon as possible after parturition

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 (main mate pups were also weighed on Days 4, 8, 12 and 21 post partum; pups from the re-mated females were weighed on Days 4 and 8), 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 [after the majority of litters had been weaned]
- Maternal animals: All surviving animals [3 weeks after weaning of their litter]

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues indicated below were prepared for microscopic examination: adrenals, aorta, bone (femur), bone marrow (sternum), brain, cranial vault (for lachrymal glands, teeth, nasal turbinates, inner ear), caecum, colon, duodenum, eyes, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (cervical/ mesenteric), mammary gland, oesophagus, ovaries, pancreas, pituitary, prostate with seminal vesicle (adults only), salivary gland, sciatic nerve, Skeletal muscle, skin, spinal column (vertebral column), spleen, stomach, testes with epididymides, thymus (if present), thyroids, tongue, trachea (with larynx, pharynx), urinary bladder, uterus with vagina.
- Selected organs were weighed prior to preservation, where appropriate, organs were weighed as a pair. When one of a pair of organs was abnormal,
both organs were weighed individually.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 23 days of age.
- These animals were subjected to postmortem examinations (macroscopic and microscopic examination).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
- The tissues indicated below were prepared for microscopic examination: adrenals, aorta, bone (femur), bone marrow (sternum), brain, cranial vault (for lachrymal glands, teeth, nasal turbinates, inner ear), caecum, colon, duodenum, eyes, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (cervical/ mesenteric), mammary gland, oesophagus, ovaries, pancreas, pituitary, prostate with seminal vesicle (adults only), salivary gland, sciatic nerve, Skeletal muscle, skin, spinal column (vertebral column), spleen, stomach, testes with epididymides, thymus (if present), thyroids, tongue, trachea (with larynx, pharynx), urinary bladder, uterus with vagina.
- Selected organs were weighed prior to preservation, where appropriate, organs were weighed as a pair. When one of a pair of organs was abnormal,
both organs were weighed individually.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
There were no obvious signs of reaction to treatment. Five adult mortalities occurred, one at 1500 ppm and four at 5000 ppm. There was no clear indication of a direct effect of treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
At 5000 ppm mean weekly bodyweight gain of males and females in both generations tended to be slightly lower than among control animals during the first part of the treatment period. Subsequently, towards the end of each generation there was a tendency among males for parity with control animals to be regained. Occasional statistically significant differences from control animals were recorded (p<0.001 during 0 to 3 weeks in males and females; p<0.01 during 0 to 10 weeks in females). Weight gain of females at 5000 ppm during gestation tended to be slightly lower than among control animals, in both generations. At 500 and 1500 ppm there were no consistent or obvious dosage-related differences among mean weekly bodyweights. At both concentrations, mean weight gain of F0 females during gestation was slightly lower than among control animals in the F1 generation, weight gain during gestation was similar to that of control animals. At 5000 ppm there appeared to be a slight general reduction in mean food consumption (-6.4% in males and -7.3 % in females of F0; -2.8% in males and -6% in females of F1). There was no clear effect on mean food consumption at 500 or 1500 ppm; marginally lower values did occur but not consistently and there were no statistically significant differences. There was no difference in the mean food conversion ratios in the treatment and control groups of both F0 and F1 generations.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
F0 generation: 32.09, 96.48 and 315.12 mg/kg bw/d in males and 39.00, 111.26 and 373.04 mg/kg bw/d in females of the 0, 500, 1500 and 5000 ppm dose levels, respectively.
F1 generation: 41.92, 127.60 and 435.21 mg/kg bw/d in males and 46.37, 141.14 and 469.39 mg/kg bw/d in females of the 0, 500, 1500 and 5000 ppm dose levels, respectively.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
Mating performance, as judged by the median pre-coital time and vaginal smear records, and pregnancy rate, as indicated by the proportion of females that became pregnant, were similar for all groups in both generations. The duration of gestation was similar for all groups in both generations.

ORGAN WEIGHTS (PARENTAL ANIMALS)
5000 ppm: Statistically significant (p<0.01) increases in liver weight (+10% in males and +12% in females of F0; +9% in males and +10% in females of F1) and reductions in spleen weight (-12% in males and no effect in females of F0; -17% in males and females of F1). Although significantly lower brain weight was recorded among F1 adults, the reduction in adjusted mean weights was slight (approximately 3.5%) and F0 adults were unaffected.
1500 ppm: Organ weight analyses of F0 and F1 adults showed statistically significant (p<0.05) reductions in spleen weight (-7% in males and -1% in females of F0; -6% in males and -11% in females of F1). Liver weight was not adversely affected.
500 ppm: there was no effect on adults other than for statistically significant (p<0.05) reductions in spleen weight among F0 males (-10%) and F1 females (-10%).

GROSS PATHOLOGY (PARENTAL ANIMALS):
Among F0 generation males at 5000 ppm there was a slight increase in the incidence of animals showing macroscopically enlarged livers (5/28 compared with 0/28 among control animals). No other macroscopic changes attributable to treatment were observed at terminal autopsy of F0 or F1 generation adults.

HISTOPATHOLOGY (PARENTAL ANIMALS):
Minimal centrilobular hepatocyte enlargement was observed in 7/24 male and 11/19 female rats in the 5000 ppm treatment group of F1 generation compared to the controls. This change was considered to be treatment related. Similar changes were not observed in any rat examined from the 1500 ppm treatment group. Histology examination of spleens and brain sections from F1 adult animals showed no treatment-related abnormality.

OTHER FINDINGS (PARENTAL ANIMALS): There was no clear or consistent effect on mean water consumption.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

Fertility

Effect level:

>= 5 000 ppm (nominal)

Sex:

female

Basis for effect level:

other: highest dose tested (5000 ppm is equivalent to ca. 373.04 mg/kg bw/day in F0 generation)

2

Dose descriptor:

NOEL

Remarks:

Systemic toxicity

Effect level:

1 500 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: histological changes and organ weight (1500 ppm is equivalent to ca. 96.48 mg/kg bw/d in males and 111.26 mg/kg bw/d in females of F0 generation)

3

Dose descriptor:

NOEL

Effect level:

< 500 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: organ weight

Remarks on result:

other: Generation: F1 and F2 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

VIABILITY (OFFSPRING):
At 5000 ppm mean total litter size at birth in the F0 generation was significantly lower than among control animals (P<0.05), and in the F1 generation although not significantly affected (P>0.05) was both below the concurrent control value and slightly below the recent laboratory control range. Post partum pup mortality at 5000 ppm in the F0 generation appeared slightly but significantly higher than among control animals even with the exclusion of dams showing total litter loss (P<0.05). In the F1 generation there was no obvious effect on pup mortality. At 500 and 1500 ppm, mean total litter sizes at birth in the F0 generation were lower than the control value to a similar extent as at 5000 ppm. In the F1 generation mean litter size at birth at 1500 ppm was similar to the control value and at 500 ppm slightly lower. In the absence of a consistent pattern of differences across the two generations and taking into account the extent of variation in litter size among control groups in recent studies, there was no clear indication of a treatment-related effect on initial litter size at these two dietary inclusions in either generation.

BODY WEIGHT (OFFSPRING):
At all three dietary concentrations there was no obvious effect on mean pup weight at birth in either generation. Subsequently during lactation at 5000 ppm in the F0 generation there was a tendency for lower pup weight gain with significantly (p<0.05 to p<0.001) lower values occurring at Days 12 and 21 post partum (-9 and -14%, respectively). In the F1 generation at 5000 ppm there was no significant effect on pup weight although marginally lower values were recorded despite the lower litter size and hence reduced intra-litter competition. The mean litter weight during lactation at 5000 ppm was significantly reduced in both generations (P<0.05 to P<0.001). During lactation at 500 and 1500 ppm, there was no clear effect on mean pup weight in either generation. Significantly lower values for litter weight at both concentrations in the F0 generation (P<0.05 to P<0.01) mainly reflected initial differences in total litter size at birth rather than an effect on mean pup weight.

ORGAN WEIGHTS (OFFSPRING)
Brain: At 5000 ppm slightly lower mean brain weights in comparison with corresponding control values were observed among F1 weanling males [-5.1%] and females [-2.8%] with differences attaining statistical significance (P<0.05 to P<0.01). Among F2 weanlings however, mean values appeared unaffected by treatment. At 1500 ppm slightly but significantly lower (P<0.05) brain weights were recorded for F1 weanling males [-2.4%] and females [-3.2%]. There was no apparent effect on values for F2 weanlings. At 500 ppm F1 weanling males showed slightly [-3.0%] but significantly lower brain weights; there were no other significant differences (P>0.05).
Liver: At 5000 ppm male and female adults and weanlings of both generations showed significantly higher liver weights [+15 to +27%] in comparison with control values (P<0.01). At 500 and 1500 ppm significantly higher liver weights [+9 to +15%] occurred among weanlings of both generations (P<0.05 to P<0.01).
Spleen: At 5000 and 1500 ppm, weanlings of both generations showed significantly lower spleen weights [-10 to -23%] in comparison with control animals (P<0.05 to P<0.01). At 500 ppm F2 weanling females showed significantly lower spleen weights [-3 to 10%] (P<0.05); occasional other mean values were slightly lower than among control animals but were not statistically significant (P>0.05).

GROSS PATHOLOGY (OFFSPRING):
Macroscopic examination of offspring showed no obvious increase in the incidence of deformities.

HISTOPATHOLOGY (OFFSPRING):
No microscopic abnormality was detected in any brain, liver or spleen examined of F2 generation weanlings.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In this investigation at 5000 ppm, the highest dose level, reaction to treatment was indicated by differences among adult observations during the study, litter data and, at termination, organ weight differences among adults and weanlings. Histological examinations showed centrilobular hepatocyte enlargement in the livers of Fl adults. At 500 and 1500 ppm reaction to treatment among adults and offspring was confined to statistically significant organ weight differences. These differences however occurred in the apparent absence of histological change. Mating performance, pregnancy rate and the duration of gestation at all three dietary concentrations were unaffected by treatment. Based on the results, the NOAEL for fertility in the parental generation was considered to be 5000 ppm (highest dose tested). The histological changes in the highest dose or organ weight difference can be due to the adaptive response to the treatment, however no such information is provided. Therefore the NOEL was identified for systemic toxicity in the parental generation, which was considered to be 1500 ppm. Similarly, the NOEL for F1 and F2 generations was considered to be <500 ppm.