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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 13, 1994 - August 19, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Remarks:

CIBA-GEIGY Limited

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

228-985-4

EC Name:

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

Cas Number:

6386-38-5

Molecular formula:

C18H28O3

IUPAC Name:

methyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate

Details on test material:

- Analytical purity: >98%

Test animals

Species:

rat

Strain:

other: Tif: RAI f (SPF), hybrids of RII/1 x RII/2

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Production, WST-455, CIBA-GEIGY Limited, 4332 Stein, Switzerland
- Age at study initiation: Males: 15 to 16 weeks; Females: 10 to 11 weeks
- Weight at study initiation: (P) Males: 400 - 508 g; Females: 207 - 269 g
- Housing: individually (except during mating) in Macrolon cages with wire mesh tops and standardized granulated soft wood bedding material
- Diet: Pelleted, certified standard feed (Nafag No. 890, Tox; Nafag, Naehr- und Futtermittel AG, Gossau, Switzerland) was provided ad libitxim.
- Water: Tap water was provided ad libitum in plastic bottles
- Acclimation period: seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): about 16 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours of light per day

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% (W/W) aqueous solution in 0.1% (W/W) Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test substance-vehicle mixtures were prepared with a high-speed homogenizer (polytron, PT6000, Kinematica AG, 6014 Littau, Switzerland) and kept refrigerated when not in use. Homogeneity of the mixtures during administration was maintained with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 1, 10 and 25 mg/ml mixture
- Amount of vehicle (if gavage): 10 ml mixture/kg actual body weight

Details on mating procedure:

- M/F ratio per cage: 1/1, during mating periods.
- Length of cohabitation: Each mating pair was housed in a single Macrolon Type 3 cage, separated by an aluiminum divider containing a closed sliding door. Pairs were so cohabited from Thursday or Friday until midnight Sunday, when restricted daily mating access was allowed by programmed automatic opening of the door. Beginning at 6.30 a.m. the following morning, females were examined for presence of vaginal plugs or sperm in a vaginal smear (=positive mating). Positively mated females were removed from the mating cage and housed individually; in remaining females, the door is closed to separate the mating pair and the restricted daily mating access procedure repeated for a maximum of 14 days.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear, referred to as day 0 postcoitum
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test substance-vehicle mixtures were taken on the day the dosing of the animals started and one day after the mating period, once before and once after dosing and analyzed by HPLC. The samples from before dosing were taken from the top, middle and bottom of the container; the samples from after dosing were taken from the middle of the container. Samples were taken in duplicate, together with 10 ml of vehicle and approximately 2.0 g of test substance they were transported frozen to the analytical laboratory for analysis.

Duration of treatment / exposure:

Males: dosing started two weeks prior to mating, during the mating period (which lasted until mating was successful or for 14 days, whichever came first), and approximately two weeks post mating.
Females: dosing started two weeks prior to mating and continued throughout the mating period (which lasted until mating was successful or for 14 days, whichever came first), during gestation and for four days postpartum).

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 100, 250 mg/kg bodyweight
Basis:
nominal conc.

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a rangefinding rat reproduction toxicity screening test (body weight gain and food consumption were severely reduced at 500 mg/kg and reduced by approximately 20% at 250 mg/kg).
- Rationale for animal assignment: computer-generated randomization

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily (normally a.m.; repeated p.m. if signs observed)

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION: Yes
- Time Schedule: once weekly premating; as appropriate days 0, 7, 14 and 21 postcoitum, and days 0, 4 postpartum.
- Food consumption for each animal determined and mean daily diet consumption calculated: Yes

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Litter Size and Sex, Live Birth, Viability, Mortality, Clinical signs, Body weights.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Males were necropsied approximately two weeks post mating
- Maternal animals: Females were necropsied on day 4 post partum.

GROSS NECROPSY
- Gross necropsy consisted of macroscopic pathological examination of the main organs of the thoracic and abdominal cavities, in particular the reproductive organs. And for adult females: number of corpora lutea in each ovary, number of implantation sites per uterus.

HISTOPATHOLOGY / ORGAN WEIGHTS
Tissues prepared for microscopic examination and weighed, were:
Liver
Ovaries
Testes
Epididymides
Full histopathological examination were performed on these organs in all control and high dose animals. All organs/- tissues demonstrating histopathology in the high dose group were then histopathologically examined in all animals from the lower dose groups. Changes in liver weights at 100 and 250 mg/kg were noted. To clarify these changes, liver was histopathological examined in all groups (males and females).

Postmortem examinations (offspring):

All Pups were killed on day 4 postpartum by decapitation. The gross necropsy examination consisted of a macroscopic pathological examination of the main organs of the thoracic and abdominal cavities, with special attention directed to the organs of the reproductive system.

Statistics:

Statistical analysis of continuous data (e.g. body weight, food consumption) was performed using the Analysis of Variance Procedure (ANOVA) followed by Dunnett's t-Test in case of a significant result in the ANOVA.

Categorical data (e.g. malformation counts) were analysed using Chi-Square test followed by Fisher's Exact test in case of a significant result in the Chi-Square test.

Non-parametric data (e.g. mean percent affected fetuses/litter) were analysed using the Kruskal-Wallis nonparametric analysis of variance test followed by Mann-Whitney U-test.

In all summary tables with statistics, the p value for the blocking test (ANOVA, Chi-square or Kruskal-Wallis) is given in the control column. P values for subsequent comparisons against controls (Dunnett's, Fisher's Exact or Mann-Whitney U) are given in the appropriate group column, if the blocking test is significant.

No statistics were performed when the number of observations was insufficient (normally n<2).

Reproductive indices:

- Female mating: No. of females positively mated as % of no. of females used for mating
- Female fertility (=fecundity): No. of females pregnant as % of no. of females mated
- Male mating: No. of males with positive mating as % of no. of males used for mating
- Male fertility: No. of males inducing pregnancy as % of no. of males with positive mating
- Number of corpora lutea in each ovary
- Number of implantation sites per uterus

Offspring viability indices:

Litter Size and Sex: number of viable and stillborn pups on day 0 post partum; external sex of viable pups.
Live Birth: Mean pups born alive per litter as % of mean pups born per litter
Viability: Mean pups alive on day 4 per litter as % of mean pups born alive per litter

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
In the control group, one male (no. 7) was found dead on day 14 of treatment. At necropsy, liquid in the lungs and trachea were observed (death was thus attributed to intubation error). In the 100 mg/kg group, one female (no. 135) was found dead on day 19 of gestation. One day prior to death, this animal had piloerection, hunched posture, pushing head through bedding and hypoactivity. At necropsy, mottled kidneys were observed. Neither the type nor the
incidence of premature death was considered to indicate an association with the administration of the test substance.
In group 4 (250 mg/kg), nine males and 12 females displayed discomfort after test article application (pushing head through bedding). This behavior was observed only for one to four days and was considered to be treatment-related. Incidental clinical signs observed occasionally among surviving animals included palpable mass, hair loss, wound and/or crust/scurf, piloerection and chromodacryorrhea with no apparent relationship to treatment.

BODY WEIGHT (PARENTAL ANIMALS)
At 250 mg/kg, for males, there was a retardation of body weight gain during the first week of the treatment period. The resulting lower body weights were significantly different from controls from day 4 to 11. At 250 mg/kg, for females, there was a reduction of body weight gain during the premating period, gestation and lactation. The resulting lower body weights were significantly different from controls from the first week of treatment. At 100 mg/kg, for females, there was a reduction of body weight gain during treatment, only occasional differences in body weights and weight gain attained statistical significance. At 10 mg/kg (both sexes) and 100 mg/kg (males), body weights and body weight gain were similar to that of the control group throughout the study.

FOOD CONSUMPTION (PARENTAL ANIMALS)
At 250 mg/kg, for males, food consumption was reduced during weeks 1 and 2 of treatment. For females at 100 and 250 mg/kg, food consumption was also reduced, differences from the control value attaining statistical significance during premating and gestation. At 10 mg/kg (both sexes) and 100 mg/kg (males) , food consumption was similar to that of the control group throughout the study.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no effect of the test article at any dose level on fertility or mating performance of the males or females. For mated females, mating generally occurred within one estrous cycle.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Mean absolute and relative liver weights were increased at 100 and 250 mg/kg (males and females).

GROSS PATHOLOGY (PARENTAL ANIMALS)
Treatment-related macroscopical findings in parent animals were limited to an enlarged liver in one high dose group male. However, mean absolute and relative liver weights were increased at 100 and 250 mg/kg (males and females).

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopatholgy of the liver showed an increased incidence of hypertrophy of the hepatocytes in both sexes at 100 and 250 mg/kg. In the males, this change was accompanied by cytoplasmic inclusion bodies and was more pronounced than in the females. An increased incidence of cholangiofibrosis of the intreihepatic bile ducts was observed for both sexes at 250 mg/kg and for males at 100 mg/kg. Higher incidences of inflammatory changes in the liver were observed for both sexes at 100 and 250 mg/kg.

OTHER FINDINGS (PARENTAL ANIMALS)
Duration of gestation was between 22.1 and 22.7 days in all groups. One high dose group animal had a gestation period of 24 days, resulting in a longer mean gestation period in this group compared to the controls. The prolonged time of 0.6 days was considered not to be related to treatment. There was no indication of an effect of treatment on parturition.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: reduced body weights and food consumptions, Hypertrophy of hepatocytes in the liver, cholangiofibrosis of the intrahepatic bile ducts and an increase in inflammatory changes

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
At 250 mg/kg, mean litter size was slightly reduced at birth (11.9%; range of historical control values 12.7-15.4). Number of stillborn pups was increased and the incidence of pup loss between days 1 and 4 post partum was significantly higher than that of the control group. In all other test groups, litter size at birth and viability index (pups surviving days 0 to 4 post partum) were similar to that of the control group.

CLINICAL SIGNS (OFFSPRING)
During lactation days 0 to 4, there were no clinical signs for the pups which were considered to be related to the treatment of the dams with the test article.

BODY WEIGHT (OFFSPRING)
Mean birth weight of pups was similar to that of the controls in all treated groups. However, there was a retardation in weight gain in the group treated at 250 mg/kg, such that mean pup body weights on day 4 post partum were significantly lower than controls ( sexes separately and combined).

GROSS PATHOLOGY (OFFSPRING)
There were no treatment-related macroscopical findings at terminal necropsy for pups. Autolysis was generally observed in pups that were found dead.

OTHER FINDINGS (OFFSPRING)
The sex ratios of the pups on day 0 and 4 post partum were similar in all groups.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: litter size at birth was slightly reduced, body weight gain of pups was retarded and pup viability on day 4 post partum was reduced.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the NOAEL for parental rats was established at 10 mg/kg bw, and was established at 100 mg/kg/day for developmental toxicity.

Executive summary:

In a fertility/development screening test according to OECD guideline 421 and in compliance with GLP, young adult male and virgin female rats (15 animals per sex and group ) were orally treated daily (by gavage ) with 0, 10, 100 or 250 mg/kg bw of the test substance for two weeks prior to mating and continued throughout mating, gestation until 4 days post-partum. No treatment-related mortalities occurred, but all high dose males and females showed slightly reduced body weight gain. Food intake was reduced in high dose males and intermediate as well as high dose females. In parent rats, increased relative and absolute liver weight, hypertrophy of hepatocytes accompanied with cytoplasmic inclusion bodies, inflammatory/lymphocytic cell infiltrations as well as increased incidence of intrahepatic bile duct cholangiofibrosis were observed at 100 and 250 mg/kg bw. The alterations found in the liver were more pronounced in males. Mating behavior, fertility, gestation and parturition indices were not affected by the treatment. However, litter size, pub viability on day 4 post partum and mean pup weights were slightly reduced at 250 mg/kg bw. No treatment-related macroscopical changes were seen in the offspring. Pups that died spontaneously were found autolyzed. The NOAEL for parental rats was established at 10 mg/kg bw, and was established at 100 mg/kg/day for developmental toxicity.