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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From May 01, 1986 to December 24, 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was performed before formal guidelines but according to best practice at that time, and according to GLP .

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report date:
1987

Materials and methods

Principles of method if other than guideline:
The study was perfermed before any formal guidelines but according to best practice at that time.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Active enzyme protein of Lipase, triacylglycerol (EC no. 232-619-9, CAS no. 9001-62-1, EC name: Lipase, triacylglycerol, Enzyme Class No.: 3.1.1.3)
Cas Number:
9001-62-1
Molecular formula:
Not applicable, see remarks
IUPAC Name:
Active enzyme protein of Lipase, triacylglycerol (EC no. 232-619-9, CAS no. 9001-62-1, EC name: Lipase, triacylglycerol, Enzyme Class No.: 3.1.1.3)
Constituent 2
Reference substance name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available
IUPAC Name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Constituent 3
Reference substance name:
Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
Constituent 4
Reference substance name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
Constituent 5
Reference substance name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Test material form:
solid: particulate/powder
Remarks:
powder
Details on test material:
- Lot/batch No.: PPW 1798
- Expiration date of the lot/batch: December 1996

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Manston, Kent, UK.
- Age at study initiation: F0 males 6-7 weeks, F0 females 16-17 weeks ; F1 approx. 10 weeks
- Weight at study initiation: (P) Males: 134-170 g; Females: 224-272 g; (F1) Males: 53-77 g; Females: 48-73 g
- Housing: F0 animals were housed 2 per cage. After mating, they were housed individually. After weaning, F1 animas were group-housed for a few days, until they could be housed 2 per cage
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): Rat and Mouse Breeder Diet No. 3 SQC Expanded (ground) ad libitum
- Water (e.g. ad libitum): domestic water ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 2°C
- Humidity (%): 55% ± 10%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: Food containing no test substance
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh diet batches were prepared weekly during the study, each dose level being prepared independently.
- Mixing appropriate amounts with (Type of food): The rat and mouse diet
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: Either until mating had occured or after 7 nights.
- Proof of pregnancy: Vaginal smears that showed the presence of spern was designated Day 0 of gestation.
- After 7 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): Individual in solid bottomed cages with white wood shavings as bedding.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were removed from each mixed batch of diet at the start of treatment, then in Week 7, 13, 21, 28 and 32 of the study. One sample of 100 grams was taken on each occasion. These were despatched to the Sponsor on each occasion, together with a 25 gram reference sample of the raw test material.
Duration of treatment / exposure:
The F0 males received treated diet for 10 weeks prior to mating, commencing at age 6-7 weeks. The F0 females received treated diets for 2½ weeks prior to mating, commencing at age 16-17 weeks. Treatment continued for both sexes throughout mating, gestation and lactation, following which the F0 animals were killed.

The F1 animals which were selected for rearing to maturity and breeding then received treated diets for approximately 10 weeks, prior to mating, gestation and early lactation. Shortly after Day 4 of lactation, the F1 animals and their F2 litters were killed.
Frequency of treatment:
Dietary treatment.
Details on study schedule:
- F1 parental animals not mated until 13-14 weeks of age.
- Selection of parents from F1 generation when pups were a few days after Day 21 of lactation (Day 0 being the day of littering).
No. of animals per sex per dose:
24
Control animals:
yes

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: F0: One week proir to first exposure to test diet, then weekly thereafter until the start of the mating period and then at termination. The pre-weaning F1 and F2 pups were weighed by the litter, en masse, sexes separate on Days 1 and Day 4 of lactation (Day 0 being the day of birth). F1 pups were weighed individually in Day 21 of lactation. Post-weaning F1 animals were weighed weekly from selection until the start of their mating period, then at termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each group determined and mean weekly diet consumption calculated as g food/animal/week: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Litter observations:
STANDARDISATION OF LITTERS
Within a few days after Day 21 of lactation (Day 0 being the day of littering), selection was made from each available litter of the next generation's animals. Where they were available one male and one female was selected randomly from each litter. Additional animals required to complete the 24 males and 24 females specified for each treatment group were taken from randomly selected litteres amongst those born closest to the median littering date for that group, but ensuring that not more than 2 males and 2 females in total were contributed by any one litter.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
Yes, dead pups older than Day 11 were necropsied for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead. Dead pups younger than Day 12 were examined for externally visible abnormalities and for the presence of milk in the stomach.
Postmortem examinations (parental animals):
SACRIFICE
- Male and maternal animals: All surviving animals were necropsied at termination, which were after selection of F1 generation (a few days after Day 21 of lactation (Day 0 being the day of littering).

GROSS NECROPSY
- Gross necropsy consisted of external examination, followed by macroscopic examination of the tissue and organs of the cranial, thoracic and abdominal cavities in situ. Any gross lesions were described in terms of location and characteristics. Representative samples of abnormal tissues were taken and fixed in neutral buffered 10% formalin. The following organs associated with reproduction were also fixed: ovaries, uterus, cervix, vagina, testes with epididymis, seminal vesicles and coagulation gland, prostate and pituitary.
Postmortem examinations (offspring):
- The F1 offspring not selected as parental animals were sacrificed after the F1 parental generation had been selected. F1 parental and F2 offspring were sacrificed after completion of observation for Day 4 of lactation.

- These animals were subjected to post mortem examinations (macroscopic and/or microscopic examination) as follows: offspring found dead or killed in extremis on or after Day 12 of lactation were necropsied (external examination followed by macroscopic examination of tissues and organs of the thoracic and abdominal cavities in situ. The cranium was opened only if the skull appeared abnormal externally. Offspring found dead or killed in extremis before Day 12 of lactation were examined for externally visible abnormalities and for the presence of mild in the stomach. F1 weanlings not selected for the second generation studies and F2 weanlings were examined for externally visible abnormalities before being killed. Any showing abnormalities were submitted for gross necropsy.
Statistics:
Body weight data were subjected to analysis of variance, using the Normal linear model for a one-way classification. Treatments were then compared using Dunnett’s t test.

Incidence data were analysed using Contingency tables and the Chi-squared test for Fisher’s Exact probability test.

Survival data were analysed using the Kruskal-Wallis rank-based analysis.
Reproductive indices:
Fertility Index = Number of animals pregnant / Number paired

Gestation Index = Number bearing live pups / Number pregnant
Offspring viability indices:
For each litter and group:

Live Birth Index = Number of pups live on Day 0 of lactation / Number born

Viability Index = Number of pups live on Day 4 of lactation / Number live on Day 0

Lactation Index = Number of pups live on Day 21 of lactation / Number live on Day 4

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:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 5 other: % lipase by weight in the diet
Sex:
male/female
Remarks on result:
other: Generation: F0 and F1 (migrated information)

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:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
There was no effect of treatment with the test material, lipase, on either F0 or F1 fertility and general reproductive performance, at exposure levels of up to 5% lipase by weight in the diet.
Executive summary:

Lipase, batch PPW 1798 was tested in rats for effects on fertility and general reproductive performance over 2 generations of animals.

Male and female rats were randomized into 3 treatment groups and one control group, each group containing 24 males and 24 females. The animals received diet containing the following constant concentration of lipase:

 

 

 

% lipase by Weight in the Diet

Control

Low dose

Intermediate dose

High dose

 0

0.5

1.5

5.0

 

 

 

The F0 males received treated diets 10 weeks prior to mating, the F0 females for 2½ weeks prior to mating. Treatment continued for both sexes throughout mating, gestation and lactation. The F0 animals were then killed and necropsied. The F1 animals which were selected for rearing to maturity and breeding then received treated diet for approximately 10 weeks, prior to mating at 13-14 weeks of age. Treatment continued throughout mating, gestation and lactation, following which the F1 animals and their F2 litters were killed, the former being necropsied.

 

Observations for clinical signs for toxicology, body weight performance, food consumption and reproductive performance were collected for the F0 and F1 generations. Body weight and clinical observations, including survival, were collected for the F2 generation until the time of termination just after Day 4 of lactation.

 

At 5% and 1.5% lipase in the diet, there were very slight enhancement of F0 female body weight performance, such that after 9 weeks of treatment, the animals weighed 6-8% more than controls. No such effect was observed during the growth to maturity of the F1 animals.

 

There was no effect of treatment with the test material on either F0 or F1 fertility and general reproductive performance, at exposure levels of up to 5% lipase by weight in the diet.