Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Xylanase is not an reproductive toxicant.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
21 July 2015 to 08 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed according to OECD test guideline no. 422 (1996), and in compliance with GLP. The purpose of the study was to satisfy regulatory demands because the enzyme is also used for production of food in EU.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: ca. 77 days (males); ca. 70 days (females)
- Weight on initiation of study: ca. 354-439 g (males); 213-255 g (females)
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatisation, gestation, littering, lactation and maturation periods. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing. Solid bottom cages contained softwood based bark-free fibre bedding, which was changed at appropriate intervals each week. Number of animals per cage: two to five animals (pre-pairing); one male and one female (pairing ); two to five animals (males after mating); one female (gestation) and one female + litter (lactation). A soft white untreated wood block and a plastic shelter were provided to each cage throughout the study (except during pairing and lactation).
- Diet: ad libitum
- Water: domestic mains water, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 40- 70 %
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
In advance of the formulation period the required amount of containers of test material were transferred from the freezer to refrigerated (2-8oC) storage to ensure thawing. The thawed containers were inverted ten times and gently magnetically stirred whilst aliquoting. On the day prior to formulation the required amount of aliquots was removed from frozen storage to refrigerated (2-8oC) storage to ensure thawing. On the day of formulation, the required volume of vehicle was added to the premeasured aliquot of test material. This was gently mixed using a magnetic stirrer and gentle inversion. Dosing formulations were prepared daily and administered within four hours of preparation.

ORAL ADMINISTRATION
The animals were dosed by oral administration, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth. The dosing volume was 10 mL/kg body weight, which was calculated from the most recently recorded scheduled body weight. The control animals were dosed with vehicle at the same volume dose as the treated groups. All animals were dosed once daily, at approximately the same time each day.
Details on mating procedure:
Details on mating:
Pairing commenced after a minimum of two weeks of treatment. The male/female ratio was 1:1 from within the same treatment groups. Duration of pairing was up to two weeks and the animals were checked daily for evidence of mating by observation for ejected copulation plugs in cage tray and sperm in the vaginal smear. Day 0 of gestation was defined when positive evidence of mating was detected and the male and female rats were separated when mating evidence was detected. The pre-coital intervals were calculated for each female (as the time between first pairing and evidence of mating).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The formulated samples were analysed with regard to total Nitrogen content, N-total %. The absence of nitrogen (below LOQ, i.e. <0.05% w/w) in the control samples was confirmed, demonstrating that no inadvertent cross contamination had occurred.
The total nitrogen % values for all the dose groups were within a satisfactory range to expected values, demonstrating correct preparation of the dosing formulations.
Duration of treatment / exposure:
The males were dosed from two weeks before pairing until termination.
The females were dosed daily for two weeks before pairing, throughout pairing, gestation and until Day 6 of lactation.
Frequency of treatment:
Daily during treatment period.
Dose / conc.:
0 other: mg TOS/kg bw/day
Dose / conc.:
110.1 other: mg TOS/kg bw/day
Dose / conc.:
4 363.4 other: mg TOS/kg bw/day
Dose / conc.:
1 101.3 other: mg TOS/kg bw/day
No. of animals per sex per dose:
10 males and 10 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected following evaluation of existing toxicological data.
Positive control:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

BODY WEIGHT: Yes
- Time schedule for examinations: F0 males were weighed before dosing on the day that treatment commenced (Week 0), weekly thereafter and on the day of necropsy. F0 females were weighed before dosing on the day that treatment commenced (Week 0), weekly before pairing, on Days 0, 6, 13 and 20 after mating, on Day 1, 4, and 7 of lactation and on the day of necropsy.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: For F0 animals, the food consumption was recorded weekly, from the day that treatment commenced, but not during the period when paired for mating (Week 3). It was recommenced for males in Week 4. For females after mating food consumption was recorded for the following periods: Days 0-5, 6-12, 13-19 after mating and Days 1-3 and 4-6 of lactation. From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.


Postmortem examinations (parental animals):
SACRIFICE
All adult animals were euthanized by carbon dioxide asphyxiation with subsequent exsanguination.

GROSS NECROPSY
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Necropsy of F0 males were performed after the Week 5 investigations were completed. Necropsy of the F0 females were performed on Day 7 of lactation.

HISTOPATHOLOGY
Tissue samples for histology were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required. For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was reported.

For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.
Postmortem examinations (offspring):
SACRIFICE
Offspring was euthanized by intraperitoneal injection of sodium pentobarbitone.

GROSS NECROPSY
Necropsy of the F1 offspring was performed on Day 7 of age.
Reproductive indices:
The following indices of mating performance and fertility were evaluated for each group:
- Percentage mating (%) = (Number animals mating/ Animals paired) x 100
- Conception rate (%) = (Number animals achieving pregnancy/Animal mated) x 100
- Fertility Index (%) = (Number animals achieving pregnancy/Animals pairing) x 100
Offspring viability indices:
The following were calculated for each litter:
- Post-implantation survival index (%) = (Total number of offspring born/Total number of uterine implantation sites) x 100
- Live birth index (%) = (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
- Viability index (%) = (Number of live offspring on Day 7/Number of live offspring on Day 1 after littering) x 100
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
The haematological investigation in Week 2 of treatment (i.e. before pairing) revealed, when compared with the controls, a small reduction of lymphocyte and large unstained cell counts in males receiving the high dose of the Xylanase. There was no similar finding in females. These findings were not adverse. The bone marrow composition was unaffected.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The biochemical examination of the blood plasma in Week 2 of treatment (i.e. before pairing) revealed, when compared with the controls, slightly but statistically significantly low creatinine concentration in males and females receiving the high dose of the Xylanase and in the females this associated in the same group with a small reduction of plasma urea concentration, though statistical significance was not attained. For creatinine concentration, only one male receiving the high dose of the Xylanase had an individual value below the concurrent control range and for females the majority of individual values for high dose animals were within the concurrent control range. For animals receiving the high dose of the Xylanase, all individual creatinine concentrations for males and females and all individual female urea concentrations were within the historic control range (90- percentile control range for creatinine was 21-37 µmol/L for males (n=100) and 28-46 µmol/L for females (n=100) and for urea was 4.2-9.0 mmol/L in females (n=100)).
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive performance:
no effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
(reproductive toxicity)
Effect level:
1 101.3 other: TOS/kg bw/day
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive performance
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Conclusions:
It was concluded that oral administration of Xylanase to Sprague-Dawley rats at doses up to 1101.3 mg total organic substance (TOS)/kg body weight/day for 5 weeks to males and for 2 weeks before and during pairing throughout gestation and to Day 6 of lactation for females was well tolerated and did not cause any adverse change. The no observed adverse-effect level (NOAEL) for systemic and reproductive/developmental toxicity was therefore considered to be the highest (equivalent to 1101.3 mg TOS/kg body weight/day).
Executive summary:

The objective of this study was the assessment of general systemic toxic potential of Xylanase in rats, including a screen for reproductive/development effects, after once daily by oral (gavage) administration for at least five weeks.

Three groups, each comprising ten male and ten female Sprague-Dawley rats received three different doses of Xylanase. Males were treated daily for two weeks before pairing, throughout pairing and up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 6 of lactation. Females were allowed to litter, rear their offspring and both females and F1 offspring were killed on Day 7 of lactation. The F1 generation received no direct administration of the test substance, meaning that any exposure was in utero or via the milk. A similarly constituted control group of both sexes received the vehicle (reverse osmosis water) at the same volume dose as treated groups, 10 mL/kg body weight.

 

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology (peripheral blood and bone marrow), blood chemistry, oestrous cycles, precoital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology investigations were undertaken. Histopathology investigations were undertaken for the first five males and first five females from the control and high dose groups. The clinical condition, litter size and survival, sex ratio, body weight and macropathology for all offspring were also assessed. 

 

General appearance and behavior, sensory reactivity responses, grip strength and motor activity were not affected by treatment, there were no deaths during the treatment period and there was no effect of treatment on bodyweight gain or on food consumption.

 

The haematological investigation in Week 2 indicated a small reduction of lymphocyte and large unstained cell count in males given 100% of Xylanase, batch PPQ38584, and the biochemical examination of the blood plasma indicated, at 100% Xylanase, batch PPQ38584, a small reduction of creatinine concentration in both sexes and low urea and potassium concentrations in females; these findings were not adverse. The bone marrow composition was unaffected.

 

There were no treatment related organ weight changes and there were no macroscopic or histopathological findings that were attributable to treatment.

 

Reproductive assessment demonstrated that there was no adverse effect of Xylanase on oestrous cycles, mating performance, fertility or gestation length. There was no adverse effect observed on litter size, and sex ratio and offspring survival and body weight up to Day 7 of age. There were also no treatment related macroscopic findings in the offspring. 

 

It was concluded that oral administration of Xylanase to Sprague-Dawley rats at doses up to 1101.3 mg TOS/kg body weight/day for 5 weeks to males and for 2 weeks before and during pairing throughout gestation and to Day 6 of lactation for females was well tolerated and did not cause any adverse change. The no-observed-adverse-effect-level (NOAEL) for systemic and reproductive/developmental toxicity was therefore considered to be the highest dose tested (equivalent to 1101.3 mg TOS/kg body weight/day).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 101.3 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Toxicological data has been generated within the enzyme producing industry during the last 40 years. Substantial documentation on the safety of the production strains has been generated, and the enzyme test materials are thoroughly characterized. High quality studies for all relevant endpoints for in vivo studies as well as in vitro studies show that industrial enzymes from well-known and well characterized production strains have very similar safety profiles across the catalytic activities. Read-across can therefore be applied for the majority of toxicological endpoints. The database can therefore be considered of high quality.
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:
Xylanase does not affect fertility.

Justification for selection of Effect on fertility via oral route:

Toxicokinetic data together with the molecular structure of xylanase and the weight of evidence from animal studies and human exposure provides no evidence for toxicity on fertility. Further, oral administration of Xylanase to rats at doses up to 1101.3 mg TOS/kg body weight/day for 5 weeks to males and for 2 weeks before and during pairing throughout gestation and to Day 6 of lactation for females was well tolerated and did not cause any adverse change.



Justification for selection of Effect on fertility via inhalation route:
Toxicokinetic data together with the molecular structure of xylanase 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 xylanase 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

Xylanase is not a teratogen.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 101.3 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Toxicological data has been generated within the enzyme producing industry during the last 40 years. Substantial documentation on the safety of the production strains has been generated, and the enzyme test materials are thoroughly characterized. High quality studies for all relevant endpoints for in vivo studies as well as in vitro studies show that industrial enzymes from well-known and well characterized production strains have very similar safety profiles across the catalytic activities. Read-across can therefore be applied for the majority of toxicological endpoints. The database can therefore be considered of high quality.
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 oral route: 

Toxicokinetic data together with the molecular structure of xylanase and the weight of evidence from animal studies and human exposure provides no evidence for developmental toxicity. Further, oral administration of Xylanase to rats at doses up to 1101.3 mg TOS/kg body weight/day for 5 weeks to males and for 2 weeks before and during pairing throughout gestation and to Day 6 of lactation for females was well tolerated and did not cause any adverse change. There was no adverse effect observed on litter size, and sex ratio and offspring survival and body weight up to Day 7 of age. There were also no treatment related macroscopic findings in the offspring. 

 

Justification for selection of Effect on developmental toxicity: via inhalation route: 

Toxicokinetic data together with the molecular structure of xylanase 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 xylanase 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 27 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.  

 

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Justification for classification or non-classification

Xylanase is not classified as it is not a reproductive toxicant (for further justification see discussion above).