coconut oil, reaction products with glycerol esters of 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP- compliant study incorporating all elements needed for OECD testing guideline 415.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The study was a combined one for subchronic toxicity and reproductive toxicity assessment.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

425-400-6

EC Name:

-

Cas Number:

179986-09-5

Molecular formula:

UVCB substance

IUPAC Name:

methyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate; propane-1,2,3-triol; tetradecanoic acid

Details on test material:

- Physical state: colourless paste
- Analytical purity: not indicated by the sponsor; treated as 100% pure
- Storage condition of test material: at room temperature protected from light

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: (P) males 6 wks, females 13-14 wks
- Weight at study initiation: (P) males: 175-177 g; females: 216-221 g
- Housing: males were housed in groups of 5 males/cage in Macrolon cages. The additional males were housed in groups of 2 males/cage in Macrolon cages.
Females:
Pre-mating: Females were housed in groups of 4 animals/sex/cage in Macrolon plastic cages.
Mating: Females were caged together with males from the same treatment group from the 90-day toxicity study on a two-to-one-basis (two females with one male) in Macrolon plastic cages.
Post-mating: Females were individually housed in Macrolon cages.
Lactation: Offspring was kept with the dam until termination.
- Diet (ad libitum): pelleted rodent diet
- Water (ad libitum): tap water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

In-Life phase: 05 March 2008 (Delivery of animals) - 12 June 2008 (Necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: daily, 2 hours prior to dosing

VEHICLE
- Concentration in vehicle: 5, 10, 20 mg/ml
- Amount of vehicle (if gavage): 2 ml/kg bw
- Lot/batch no. (if required): 17644FC6

Details on mating procedure:

- M/F ratio per cage: 1/2
- Length of cohabitation: 15 days
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually

Following a minimum of 14 days ofexposure for the females, females were paired with males from the same treatment group from the 90-day
toxicity study (Part A), avoiding sibling mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Quantitative analysis was based on the analytical method validated for the test substance in NOTOX project 487737.

Duration of treatment / exposure:

males: 12 weeks, females: 8 weeks

Frequency of treatment:

daily

Details on study schedule:

Once daily for 7 days per week, approximately the same time each day with a maximum o f 4 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy

Doses / concentrations

Remarks:

Doses / Concentrations:
14, 56, 222 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

24 females/dose group, 12 males/dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were based on the results of a 28-day toxicity study (RCC Project 636456).
Doses were initially chosen lower, but then it was forgotten to adjust for specific density and it was decided to continue the study with the increased doses.

- Females were treated 14 days prior to mating until the end of weaning.
Females 33, 39, 53, 69, 83 and 91 were not dosed for one occasion as these females were littering at the time of dosing.


- Males were treated

Positive control:

Not needed

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: weekly

OTHER:
- Semen analysis (sperm motility, countand morphology)
- Staging of spermatogenesis (detailed histopathology of the testes)
- Oestrus cycle monitoring

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed to determine the stage of estrus beginning 14 days prior to initiation of the mating period and until evidence of copulation was observed.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight, sperm motility, sperm morphology, sperm concentration

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
- yes, for external and internal abnormalities
- possible cause of death was determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All animals, at the end of the 90-day toxicity study
- Maternal animals: All animals. Females that have delivered were killed at day 21 postpartum or shortly thereafter. Non-pregnant females were killed on day 25-27 post-coitum.

GROSS NECROPSY
After sacrifice, all females were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, special attention was paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
Males
Organ weights of: Adrenal gland, brain, epididymides, heart, kidneys, liver, pituitary gland (weighed when fixed for at least 24 hours), prostate gland (weighed, when fixed for at least 24 hours), seminal vesicles including coagulating gland and fluids, spleen, testes, thymus, thyroid (weighed, when fixed for at least 24 hours)
Histopathology of: AIl tissues collected at the scheduled sacrifice from all main animals of the control and the highest dose group. The additional slides of the testes of all males of the control group and the 222 mg/kg bw dose group to examine staging of spermatogenesis. AII gross lesions.
The investigation was extended to the liver and thyroid for males of the 14 and 56 mg/kg bw dose group because a treatment related effect was suspected.

Females
Organ weights and histopathology of: Liver, ovaries, pituitary gland (weighed, when fixed for at least 24 hours), thyroid (weighed, when fixed for at least 24 hours), uterus

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
Offspring found dead or killed before day 14 of lactation were sexed and externally examined (if practically possible) with emphasis on developmental morphology. The stomach was examined for the presence of milk.
Offspring found dead or killed on or after day 14 of lactation were sexed and subjected to external examination of the cranium, and macroscopic examination of the thoracic and abdominal tissues and organs, with emphasis on developmental morphology. Any abnormal pup was preserved in 10% buffered formalin, bouin or 96% ethanol, as appropriate, for possible further examination. AII gross lesions were fixed in 10% buffered formalin.

Statistics:

The following statistical methods were used to analyse the data:
- lf the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one ranktest) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The T-test was applied for testis and epididymidis concentrations.
- The percentage of motile spermatozoa, progressive motile spermatozoa and sperm with normal morphology was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup difference. Ifthe results ofthe ANOVA were significant (p<0.05), the Wilcoxon test was applied to the data to compare the treated groups to the control group.
AII tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Reproductive indices:

Percentage mating: (Number of females mated/Number of females paired) x 100
Fertility index: (Number of pregnant females/Number offemales paired) x 100
Conception rate: (Number of pregnant females/Number of females mated) x 100
Gestation index: (Number of females bearing live pups/Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Viability index: (Number of live pups on day 4 post partum/Number of pups born alive) x 100
Weaning index: (Number of live pups on day 21 postpartum/Number of live pups on day 4 post partum) x 100

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

Description (incidence and severity):

Body weights, body weight gain and food consumption were reduced for females after mating at 56 and 222 mg/kg bw (see details and attachment)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Body weights, body weight gain and food consumption were reduced for females after mating at 56 and 222 mg/kg bw (see details and attachment)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 56 and 222 mg/kg, treatment related changes were present in the liver (periacinar hepatocytic hypertrophy) and thyroid (follicular epithelial hypertrophy).

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Individual variation in estrous cycles occurred in all groups. The regularity and duration of estrous were not affected by treatment. The mean cycle length was 4.0 for all groups.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Gestation index was slightly reduced about 5 % in the 56 and 222 mg/kg bw dose groups (see details).

Details on results (P0)

The following results, except effects of sperm measurements, are described for the female animals. Effects for male animals are described in the Notox 487736 90-days repeated study.

CLINICAL SIGNS AND MORTALITY
There were no clinical signs of toxicity noted during the observation period.
Salivation was noted in five females treated at 56 mg/kg and twenty females treated at 222 mg/kg. Salivation was considered to be a physiological response to the taste of the formulation rather than a sign of systemic toxicity, considering the nature, minor severity of the effect and its time of occurrence (i.e. after dosing). Therefore, this was considered not toxicologically relevant.
Incidental findings that were noted included scabs, wound and swelling at the left flank, alopecia at several body parts, piloerection, dull eye, pale appearance, opacity ofthe eyes, broken tail apex, and red right ear. These findings are commonly noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

BODY WEIGHT AND FOOD CONSUMPTION (tables given in attachment)
Treatment resulted in a dose-dependent absolute and relative reduced food consumption of dams in all treated groups during the first week of the study. For later time-points, only dams of the high dose group were affected, specifically until post-coitum day 14 and then again during lactation.
The reduced food consumption reflected on statistically lower body weights and body weight gain of dams, beginning prior to implantation, on post-coitum day 4 for the mid and high dose groups. For details it is referred to the tables in the attachment.
At 56 and 222 mg/kg, food consumption (absolute and relative) was decreased.
At 56 mg/kg/day, absolute and relative food consumption was significantly, though transiently, reduced during pre-mating Days 1-8, and lactation Days 1-4. Absolute food consumption was also decreased during post-coitum Days 0-4 without a corresponding reduction in relative consumption.
Absolute and relative food consumption was reduced for animals treated with 222 mg/kg body weight/day during the entire period of quantification including pre-mating, post coitum, and lactation (not always statistical significant).
The statistical significant changes noted for females treated at 14 mg/kg were not considered treatment related as the changes were very slight and were considered within normal limits.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE
Individual variation in estrous cycles occurred in all groups. The regularity and duration of estrus were not affected by treatment. The mean cycle length was 4.0 for all groups.

REPRODUCTIVE FUNCTION: SPERM MEASURES
Sperm examination: At all dose levels, decreased sperm motility and progressive motility was noted (at 222 mg/kg bw: 20 resp. 40%) compared to the average data from three one studies of a different design. As such, evaluation of these two endpoints is not possible.

The number of testicular and epididymidal sperm cells and the percentage of normal sperm cells was similar for the 222 mg/kg bw dose group and control group.
Left testis weight was statistically significantly increased in the animals of the 222 mg/kg bw dose group (about 12%). These values were considered to be within normal limits and as this effect was not observed for the total testes weight it was not considered toxicologically relevant.
In the testes sections which were stained PAS/haematoxylin, in addition to sections stained H&E, no variation was present in the staging of spermatogenesis between the 222 mg/kg bw treated and the control group.

REPRODUCTIVE PERFORMANCE
Percentage mating: 100 % in the control and the test groups.
Fertility index: no effects (70.8 % in the control group; 79.2%, 83.3% and 79.2% in the 14, 56 and 222 mg/kg bw dose groups) (It is noted that the fertility index of the control group is lower than the historical control range for standard one-generation studies of 79.2 - 100%)
Conception rate: no effects (70.8 % in the control group; 79.2%, 83.3% and 79.2% in the 14, 56 and 222 mg/kg bw dose groups)
Gestation index: 100 % in the control group; 100%, 95% and 94.7% in the 14, 56 and 222 mg/kg bw dose groups
At 222 mg/kg, the duration of gestation was significantly longer than controls (21.9 days for Group 4 compared to 21.4 days for the control group).

The number of living pups at first litter check were decreased at 222 mg/kg (8.1 pups/litter for the 222 mg/kg bw dose group compared to 12.0 pups/litter for the control group).

ORGAN WEIGHTS
A statistical significant change note for ovaries weight of animals in the 56 mg/kg bw dose group was not considered treatment related as no dose response relationship was noted.

GROSS PATHOLOGY
At 222 mg/kg, hepatic changes were noted. These consisted of accentuated lobular pattern (five females), enlarged livers (three females) and red-brown discoloration (four females).
Incidental findings included cloudy eyes, alopecia at several body parts, nodule and sore at the left flank, nodule in the uterus horn, foci on the thymus or liver, bent tail apex, and enlarged uterus. In the absence of a treatment-related distribution they were considered changes of no toxicological significance.
Fluid in the uterus, found in females of the control and the 222 mg/kg bw dose group, is related to a stage in the oestrous cycle and is a normal finding. Necropsy of one female of the 56 mg/kg was performed on Day 25 post-coitum: the right uterus horn of this female contained an enlarged foetus (5.8 cm). This isolated finding was not considered treatment related.

HISTOPATHOLOGY
At 56 and 222 mg/kg, treatment related changes were present in the liver (periacinar hepatocytic hypertrophy) and thyroid (follicular epithelial hypertrophy).
Periacinar hepatocytic hypertrophy was seen in one female treated at 56 mg/kg and in nine females treated at 222 mg/kg. Thyroid follicular epithelial hypertrophy was observed for five females of the 56 mg/kg bw and ten females of the 222 mg/kg bw dose group.
The hypertrophic changes in liver and thyroid were both treatment and dosage related changes.
The hepatic enlargement and accentuated lobular pattern recorded at necropsy are gross correlates of periacinar hepatocytic hypertrophy. There was no microscopic evidence of pigmentation to correlate with gross discolouration.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

(parental (systemic) toxicity)

Effect level:

14 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Induction of liver-thyroid axis (weight and histophathology, food consumption and body weight gain affected for liver and thyroid at 56 and 222 mg/kg bw).

2

Dose descriptor:

NOEL

Remarks:

(reproduction)

Effect level:

14 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Slight reduction in the number of implantation sites at 56 mg/kg bw and 222 mg/kg/bw. Reduction in the number of pups at 222 mg/kg bw.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

>= 222 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects occurred via lactation as far as assessed in a one-generation study.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

	control	14 mg/kg bw	56 mg/kg bw	222 mg/kg bw
Corpus Lutea	14.5 ± 3.4	13.6 ± 3.4	13.3 ± 4.6	12.5 ± 3.5
duration of gestation (days)	21.4	21.2	21.5	21.9**
Number of living pups at first litter check	12 ± 2.8	10.9 ± 2.4	10.2 ± 2.9	8.1 ± 3.5**
Number of implantation sites	12.6 ± 2.7	11.6 ± 2.7	10 ± 3.8*	9.7 ± 2.8*
Pre implantation loss (%)	11.6 ± 14.3	13.1 ± 11.8	21.7 ± 24.5	20.7 ± 17.6
Post implantation loss (%)	6.5 ± 7	9 ± 5.7	12.4 ± 25.9	19.1 ± 27.4
**Steel-test, significant at 1% level
*Dunnett-test based on pooled variance significant at 5%
WiIcoxon test (pre-and post implanatation loss)

	control	14 mg/kg bw	56 mg/kg bw	222 mg/kg bw
Percentage mating	100	100	100	100
Fertility index	70.8	79.2	83.3	79.2
Conception rate	70.8	79.2	83.3	79.2
Gestation index	100	100	95	94.7
Weening index	100	100	97.2	100
Viability index	99.5	100	99	99.3

Applicant's summary and conclusion

Executive summary:

No effects on male fertility and on conception rate was observed. The substance caused a slight reduction in the number of implantation sites which matches the reduction in the number of pups. The effect occurred at doses for which the thyroid hormone balance was disturbed via a rat-specific mechanism as indicated by liver and thyroid histopathology findings as well as a reduced food consumption and body weight gain. No effects on pup development were observed.