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Toxicological information

Toxicity to reproduction

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Substance type: UVCB
- Physical state: liquid
- Lot/batch No.: PPQ38584
- Expiration date of the lot/batch: 09 June 2025
- Stability under test conditions: The test material and dilutions in water (10% and 100%) are stable for at least 24 hours at room temperature
- Storage condition of test material: Frozen (-20°C)

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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

Applicant's summary and conclusion

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).