Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
Lipase is not a reproductive toxicant.
Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
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 .
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. certificate)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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
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
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
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Dose descriptor:
NOAEL
Effect level:
>= 5 other: % SP 225 by weight in the diet
Sex:
male/female
Remarks on result:
other: Generation: F0 and F1 (migrated information)
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
Reproductive effects observed:
not specified
Conclusions:
There was no effect of treatment with the test material, SP 225, on either F0 or F1 fertility and general reproductive performance, at exposure levels of up to 5% SP 225 by weight in the diet.
Executive summary:

SP 225, 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 SP 225 :

 

 

 

% SP 225 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% SP 225 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% SP 225 by weight in the diet.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information
Short description of key information:
Lipase does not affect fertility.

Justification for selection of Effect on fertility via inhalation route:
Toxicokinetic data together with the molecular structure of lipase and the weight of evidence from animal studies and human exposure provides no evidence for toxicity on fertility.

Justification for selection of Effect on fertility via dermal route:
Toxicokinetic data together with the molecular structure of lipase and the weight of evidence from animal studies and human exposure provides no evidence for toxicity on fertility.

Effects on developmental toxicity

Description of key information
Lipase is not a teratogen.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
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 .
Qualifier:
no guideline available
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. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Manston, Kent, UK.
- Age at study initiation:8-9 weeks
- Weight at study initiation: 162-219 g
- Housing: 2 per cage.
- Diet (e.g. ad libitum): Rat and Mouse Breeder Diet No. 3 SQC Expanded (ground) ad libitum
- Water : domestic water ad libitum
- Acclimation period: 5 days

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

Route of administration:
oral: feed
Vehicle:
other: Diet without test substance
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: Fresh diet batches were prepared weekly during the study, each dose level being prepared independently.
- Mixing appropriate amounts of lipase with the rat diet to following dose levels, 0, 2, 5 and 10 % in the diet.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were removed from each mixed batch of diet and despatched to the Sponsor for analysis of enzyme activity in lipase units/g.
Details on mating procedure:
108 female rats arrived timed-mated on day 1 of gestation at the research lab.
Duration of treatment / exposure:
12 days (day 6 - day 17 of gestation)

Frequency of treatment:
The diet contained a constant concentration of test material and was available ad libitum.
Duration of test:
The animals had access to treated diet from day 6 - day 17 of gestation (day 0= day of verification of copulatory plug in situ).
Remarks:
Doses / Concentrations:
0, 2, 5 and 10 %
Basis:
nominal in diet
No. of animals per sex per dose:
27 mated females
Control animals:
yes, plain diet
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During each day

BODY WEIGHT: Yes, at day 1, 6, 9, 13, 17 and 20 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each cage determined daily
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


Ovaries and uterine content:
On day 20, the animals were killed by nitrogen asphyxiation. The reproductive tract was dissected out, weighed and then examined.
Fetal examinations:
- External examinations: Yes all per litter
- Soft tissue examinations: Yes, 1/3 of the foetuses by free-hand sectioning (Wilson), while the other 2/3 were fixed for gross visceral abnormalities.
- Skeletal examinations: Yes, 2/3 of the foetuses
- Head examinations: Yes, 2/3 of the foetuses
Number of implants were recorded, if they were alive or dead (late vs early)
Statistics:
Body weight and foetal 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.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Slight reductions in maternal body weight gain and food consumption occurred at the 10% SP 225 dose level only
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
At 5% and 10% SP 225 in the diet, there was a very slight reduction in foetal weight, together with some other evidence of slight immaturity amongst parameters of skeletal ossification state and in growth of the viscera of a small number of fetuses.
Dose descriptor:
NOAEL
Effect level:
other: >= 2 % SP 225 by weight in the diet
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Maternal effect of treatment in this study were slight reductions in weight gain and food consumption at 10% SP 225 in the diet.

At 5% and 10% SP 225 in the diet, there was a very slight reduction in foetal weight, together with some other evidence of slight immaturity amongst parameters of skeletal ossification state and in growth of the viscera of a small number of fetuses.

It was concluded that, under the conditions of this study, dose levels of 5% and 10% SP 225 by weight in the diet caused slight growth retardation of fetuses, while slight maternal toxicity was confined to the 10% SP 225 dose level. No significant effect was considered to have occurred at a dose level of 2% SP 225 by weight in the diet. No teratogenic potential of SP 225 was demonstrated at any dose level up to 10% SP 225 by weight in the diet.

Executive summary:

SP 225, Batch PPW 1798 was accepted from Novo Industri A/S, Denmark for teratogenicity testing in rats.

 

Mated Sprague-Dawley rats were randomized into 3 treatment groups and one control group, each group containing 27 animals. These animals were exposed continuously to test diets from Day 6 to Day 17 of gestation, where Day 0 was the day of verification of mating by detection of a copulatory plug in situ. Dose levels applied were as follows :

 

 

 

% SP 225 by Weight in the Diet

 

Control

Low dose

Intermediate dose

High dose

 

0

2

5

10

 

 

It was concluded that, under the conditions of this study, dose levels of 5% and 10% SP 225 in the diet caused slight growth retardation of fetuses, while slight reductions in maternal body weight gain and food consumption occurred at the 10% SP 225 dose level only. No significant effect was considered to have occurred at the 2% SP 225 level.

No teratogenic potential of SP 225 was demonstrated at any of the dose levels used.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information
Justification for selection of Effect on developmental toxicity: via inhalation route:
Toxicokinetic data together with the molecular structure of lipase and the weight of evidence from animal studies and human exposure provides no evidence for developmental toxicity.

Justification for selection of Effect on developmental toxicity: via dermal route:
Toxicokinetic data together with the molecular structure of lipase and the weight of evidence from animal studies and human exposure provides no evidence for developmental toxicity.

Toxicity to reproduction: other studies

Additional information

From the toxicokinetic information available, it can be concluded that the bioavailability of enzymes is low due to the fact that no significant absorption can be expected through the respiratory and/or gastrointestinal tract and/or through the skin. Exposure to enzymes will be limited because of the DMEL (derived minimum exposure levels) settings for workers, professionals and consumers to prevent respiratory allergy (supported by exposure scenarios and DMEL values) (ref. 58). Apart from the irritation potential of some proteases, respiratory allergy is generally considered to be the only human health hazard of enzymes indicating that this is the most sensitive endpoint considering enzyme toxicity. Concentrations that are not expected to result in respiratory allergy will certainly not result in any other toxic effect (ref. 59). This conclusion is substantiated by the information that follows.

Although endocrine disrupting chemicals are a broad group of chemicals consisting of man-made and natural compounds it is unlikely that enzymes have the potential to cause endocrine disruption. The enzymatic structure is different from any endocrine disrupter known to date (ref 1). Indeed, enzymes are much larger than endocrine disrupters in general excluding mechanisms such as direct action on hormone receptors (EDSTAC (Endocrine Disruptor Screening and Testing Advisory Committee, US EPA), (Ref. 2)). Due to the high biodegradability of enzymes, it is highly unlikely that they could reach target organs or sites to any significant amount or of any significant period of time. Testing of enzymes in currently available screening assays typically based on hormone receptor binding cannot be expected to provide any evidence for endocrine disruption due to the specific features of enzymes.

Data from acute and subchronic oral toxicity studies provide evidence that enzymes are of very low toxicological activity (ref. 3;4-53, 59, 60). Typically, the derived NOAEL values are significantly higher than the maximum doses applied. None of the oral toxicity studies performed by members of the consortium in the past 40 years, as well as published data from other studies revealed any effect that indicates that enzymes could have an adverse effect on the reproduction system in males or females.

Complementing the above information is data from 26 industrial studies (Novozymes, unpublished data) on fertility and/or teratogenicity and/or reproduction studies primarily in rodents but also other species like dogs and rabbits which did not identify any evidence for reproductive toxicity of enzymes. Both proteolytic and non-proteolytic enzymes have been investigated for their teratogenic and reproductive toxicity potential. Several of these studies have been published in peer reviewed articles (ref. 24;29;33;54). Enzymes have been produced and used for many years without any evidence for reproductive potential in humans. OEL for workers is set to be 60 ng/m3 to protect against respiratory sensitization. Considering that endocrine disrupting chemicals in general are a factor of 100 000 less potent than physiologically relevant hormones (ref. 55), the low worker exposure to enzymes due to rigorous application of airborne limit and very low exposure to consumers (below 15 ng/m3, which is the highest known consumer exposure and only the case when using pre-spotters (ref. 56)) and the low bioavailability together with the high biodegradability of enzymes, no reproductive toxicity effect can be expected in humans. Furthermore, enzymes have been used for decades to treat pancreatic insufficiency in both children and adults without any evidence of reproductive toxicity (ref. 57).

In conclusion, toxicokinetic data together with the enzymatic structure and the weight of evidence from animal studies and human exposure provide no evidence for reproductive toxicity of enzymes.  

In the case of lipase, this is confirmed by the two older supporting studies, a 2 -generation study and a teratogenicity study upon oral exposure via the diet in rats: The no adverse effect level in the 2 studies was expressed in % of the test substance by weight in the diet: 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. No teratogenic potential of Lipase was demonstrated at any dose level up to 10% by weight in the diet.

 

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Justification for classification or non-classification

Lipase should not be classified as a reproductive toxicant. (For further justification, please see discussion above)