Corn oil (Zea mays, Gramineae), peroxidised

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:

from 15 April 2011 to XXX

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to OECD guideline and GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

corn oil, peroxidised

IUPAC Name:

corn oil, peroxidised

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Velaz Prague, Czech Republic
- Age at study initiation: age of animals at study initiation was about 12 weeks
- Weight at study initiation: males 214.0g, females 169.2g (average body weights at the start of the study)
- Fasting period before study: no
- Housing: in cages (number of animals in cage according period of the study) in a room equipped with central air-conditioning
- Diet: certified laboratory food (MP-OŠ-06 extrudes, Snina, Slovak Republic) was offered in recommended doses
- Water (e.g. ad libitum): tap water for human consumption (ad libitum)
- Acclimation period: acclimatized to the environmental conditions for 5 days prior to the start of the study

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2° C
- Humidity: 55 ± 10%
- Air changes: no data
- Photoperiod: 12-hour light /12-hour dark cycle

IN-LIFE DATES: From 18 April 2011 to 31 October 2011

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: no data

VEHICLE
- Justification for use and choice of vehicle:
- Concentration in vehicle:
- Amount of vehicle (if gavage): dose volume was 5 ml/kg of body weight and afterwards was adjusted individually according to the weight development of the animals
- Lot/batch no. (if required): 100 (Olificio Lucca, Italy)

Details on mating procedure:

- M/F ratio per cage: Normally 1:1 (one male to one female) mating was used.
- Length of cohabitation: the female was placed with the same male until pregnancy occurs or two weeks have elapsed. Each morning the females were examined for the presence of sperm or vaginal plug. Then the females were replaced to the original cage, and in the evening were co-housing against.
- Proof of pregnancy: presence of sperm or vaginal plug referred to as day 0 of pregnancy
- After 2 weeks 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: one female per cage

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Analytical verification was not possible, as the product (an oil) was be mixed with an other oil (not possible to separate by analysis the components of the test substance from those from the vehicle, as both are oils containing similar fatty acids).

Duration of treatment / exposure:

The all animals were treated with the test article
- 14 days pre-mating,
- 14 days mating (maximum)
- 22 days gestation (approximately)
- 4 days lactation

Frequency of treatment:

Every day (about 9:00 am)

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kgbw/d
Basis:
nominal in diet

No. of animals per sex per dose:

12 animals per sex per dose
+ 6 animals per sex per dose in the satellite groups at 0 and 1000 mg/kg bw (recovery groups) (animals of satellite group were not mated and, consequently, were not used for the assessment of reproduction/developmental toxicity)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: doses of test article were determined according to results of Dose Range Finding Assay

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day, at the same time, considering the peak period of anticipated effects after dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first exposure and at once a week thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of dosing, weekly thereafter, and at the study termination. During pregnancy, females were weighed on days 0, 7, 14 and 20, day 1 and day 4 post-partum.

FOOD CONSUMPTION: During pre-mating, pregnancy and lactation measurement of food consumption were made weekly. The measurements of food consumption during mating were made, but were not evaluated.

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

After sacrifice, reproduction indices were calculated for all females:
- Fertility Index
- Gestation (Pregnancy) Index
- Live Birth Index
- Viability (Survival) Index

Sperm parameters (parental animals):

Parameters examined in male parental generation: testis weight, epididymis weight, sperm count in epididymides, sperm motility, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS: not applicable

PARAMETERS EXAMINED
The following parameters were examined in offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, litter weight on day 1 and 4

GROSS EXAMINATION OF DEAD PUPS: yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
After completion of treatment all adult surviving animals which were not designated for posttreatment observation were sacrificed by application of an overdose of anaesthetic.

GROSS NECROPSY
All adult animals were subjected to a full detailed gross necropsy. Examination of the outer surface of the body, all orifices, cranial cavity, thoracic cavity, gastric cavity and their contents were carried out. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded. The counting of corpora lutea was recorded. Terminal body weights were measured in order to calculate relative organ weights.
The weights of following organs were determined in all males sacrificed at the end of treatment and after recovery period: epididymides, testes.
In addition, for six adult males and females randomly selected from each group, the following tissues were weighed: liver, kidneys, adrenals, thymus, spleen, brain, hearth.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs from six adult males and females, randomly selected from each group, were preserved and intended subsequent histopathological examination: all gross lesions, small and large intestines (including Peyer's patches), thyroid glands, thymus, trachea, lungs, heart, lymph nodes, brain, stomach, liver, spleen, kidneys, adrenals, urinary bladder, uterus, peripheral nerve, spinal cord.
Histopathological examinations were performed on organs and tissues of the selected animals from the control and high dose groups.

Postmortem examinations (offspring):

SACRIFICE: The offspring were sacrificed at 4 days of age.

GROSS NECROPSY: Dead pups killed at day 4 post-partum or shortly, were examined externally for gross abnormalities.

HISTOPATHOLOGY / ORGAN WEIGTHS: not performed

Statistics:

Individual results (the body weight, food consumption, haematology, clinical chemistry, relative weights of organs and reproduction parameters) obtained during the study were statistically evaluated using statistical programme Statgraphics. Statistical evaluation was conducted separately for males and females. Descriptive statistics (means and standard deviation) were performed for males and females in all dose groups. Non parametric Kruskal-Wallis test was applied. In the case of statistically significant results the Kruskal-Wallis test was followed by Mann-Whitney W test to determine which medians are significantly different from the one in control group. All statistical tests were carried out at 5% significance level.

Reproductive indices:

- Fertility Index
- Gestation (Pregnancy) Index

Offspring viability indices:

- Live Birth Index
- Viability (Survival) Index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
There were no test article-related deaths of animals during the study. All animals lived through observation period without important visible signs of intoxication. General clinical findings were typical for Wistar rats of this age. Neither change of health nor negative reactions were registered in satellite animals. The test article was very well tolerated by animals of all dose groups.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
No statistically significant differences in body weights between control and all dose groups of males were detected. In high dose females, (1000 mg.kg-1), average body weights were lower against controls after day 20 of pregnancy. This is related to the lower number of pups (or lower weight of litters) in this dose group. All remaining values of this parameter in females were identical with those of control group.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The food consumption in males and females of all dose groups was similar to the control group, with no statistical significance.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The numbers of corpora lutea and implantation sites were observed (apart from other histological evaluations). The mean number of corpora lutea was 13.67, 11.90, 13.46 and 9.40 in the control, low, medium and high dose group, respectively. The mean number of implantation sites was 11.92, 10.80, 12.91 and 7.56 in the control, low, medium and high dose group, respectively. The decrease of corpora lutea numbers in low and high dose and implantation sites in high dose against control group was statistical significant, however the decrease of corpora lutea numbers in low dose females was probably due to the infertile male. All remaining reproduction parameters were similar to our historical results in all dose groups.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
For high dose group, decreased spermatozoa motility and progressive motility was detected. After a culture at 37oC for 1 hour the spermatozoa motility and progressive motility was the same in the control and high dose group suggesting that possible effects spermatozoa motility were transient.
The percentage of morphological abnormal spermatozoa in both groups was under 20% suggesting no considerable difference.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
- Mating Procedure: twelve pairs in each dose group and control group were started. The evidence of the copulation was demonstrated in 12 females of control and high dose, in 11 females of the low and medium dose groups. In 1 female of low dose, the copulation was not observed, and this female was not pregnant. After pathological examinations of the male of this pair, hypoplasia of testes with degenerative changes was observed. The finding was due to the pre-existing condition. In 1 female of medium dose group, the sperm or vaginal plug was not observed, but this female was pregnant. One female from low dose, 1 female from medium dose, 3 females from high dose were not pregnant but the sperms were found.

- Pregnancy: the pregnancy of 22 days were established in7 females of control group, 4 females of low and high dose, 5 females of medium dose. The pregnancy of 23 days was established in 4 females of the control and medium dose, 6 females of low dose and 3 females of high dose group. The length of
pregnancy 24 days was observed in 1 female of medium and 1 female of the high dose group. In summary, the pregnancy was established in 12, 10, 11 and 9 females of control, low dose, medium dose and high dose, respectively. After the statistical evaluation of the pregnancy length, significant differences between control and all doses were not detected. Any signs of intoxication in all pregnant females were observed.

ORGAN WEIGHTS (PARENTAL ANIMALS):
Relative weights of heart were increased significantly in males from low and high dose group in comparison to the control group. No similar finding was seen in females. Relative weights of kidney left were increased significantly in males from high dose group in comparison to control group, however, not in females. Relative liver weights of female rats from high and medium dose groups were decreased significantly in comparison to control. Historically, it is not uncommon to see the relative weight of liver increased in treated animals versus controls. Also, no significant differences were observed in males or between recovery groups (high dose group and control). However, the effect of treatment cannot be excluded.

GROSS PATHOLOGY (PARENTAL ANIMALS):
All animals survived until the ending of the observation period. Occasional macroscopical lesions during necropsy of rats were observed. Hypoplasia of testes and solid dark mass in epididymis in one male (low dose) were found. Histopathological examination was confirmed by degenerative changes of the spermiogenesis. These findings could be due to the pre-existing condition. Detection of testicular hypoplasia is common in Wistar rats. The light colour of liver in two females of low dose and two females of medium dose was observed.

HISTOPATHOLOGY (PARENTAL ANIMALS):
There were no pathological changes that could be related to the effect of Peroxidised Corn Oil.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no test material related effect

2

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: mating index; fertility index; number of implantation sites; pregnancy index; litter size; litter weight

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING):
Viability Index versus control group in low, medium and high dose groups was 92.5, 100 and 100%, respectively.

GROSS PATHOLOGY (OFFSPRING): The health condition of pups was assessed visually; the abnormalities of pups and behaviour of pups were monitored. The health condition of all pups was normal.

In control group 140 live-born pups were observed (no dead-born pups). In low dose 93 live-born and 6 dead-born pups; in medium dose 117 live-born and 2 dead-born pups and in high dose 60 live-born and 1 dead-born pups were observed after the birth. Up to Day 4 post-partum, 7 pups died at low dose but none in the other groups. Based on historical data, sporadic occurrence of deadborn pups in Wistar rats is common. Cause of these deaths is unknown.
After Day 4 post-partum the mean count of live pups /one female was in control group 12 pups, in low dose 9 pups, in medium dose 11pups and high dose 8 live pups. After the statistical evaluation significant lower number of pups in the litter of female’s high dose against the control group was found.
The litters were weighted on Day 1 and Day 4 post-partum. The mean weights of litter after the birth were 71.25, 60.0, 66.33 and 48.75 g in the control, low, medium and high dose group, respectively. The mean weights of litter determined on Day 4 post-partum were 101.67, 85.56, 92.46 and 72.13 g in the control, low, medium and high dose group, respectively.
Significant decrease of the litter’s body weight in high dose against control group was observed after Day 1 and Day 4 post-partum. A similar decrease at the low dose was also detected after 4 days post-partum. The lower litter´s weight was caused by the lower number of pups per litter. No difference in the body weights of pups in control and all dose groups were found.
The offspring were sexed on Day 4 post-partum. The mean sex ratio was 6 males/6 females in control group, 5 males/4 females in low dose, 6 males/5 females in medium dose and 3 males/5 females in high dose group.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no test material related effect in offspring (screening)

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

NOAEL (parental, male) = 1000 mg/kg bw/d
NOAEL (parental, female) = 300 mg/kg bw/d
NOAEL (offspring, screening, external abnormalies) = 1000 mg/kg bw/d

Executive summary:

In a combined repeated toxicity and screening reproductive toxicity study (OECD 422, GLP) peroxidised corn oil (in olive oil as vehicle) was administered to 12 Wistar rats/sex/dose in by gavage at dose levels of 0 (olive oil), 100, 300 and 1000 mg/kg bw.

Females were treated 14 days before mating, 14 days during mating, 22-24 days during gestation and 4 days during lactation. Males were treated 56 days.

There were no test article-related deaths of animals during the study. All animals lived through observation period without important visible signs of intoxication. The test article was very well tolerated by animals of all dose groups and no sign of systemic toxicity was observed.

No differences in the length of gestation were registered. Decrease of pup’s number in the litter, litter’s body weight, corpora lutea and implantation sites in females of dose group 1000 mg/kg against control group were recorded.

No effects were observed in offsprings up to 1000 mg/kg bw.

Based on the reproduction toxicity, the no observed adverse effect level for female fertility was concluded to be 300 mg.kg-1.

The no observed adverse effect level for male fertility was concluded to be 1000 mg/kg.

The NOAEL for systemic toxicity in males and females was concluded to be 1000 mg/kg bw.

 

 This combined repeated toxicity and screening reproductive toxicity study in the rats is acceptable and satisfies the guideline requirement for a Reproduction screening Toxicity Study (OECD 422) in rats.