fermentation residues of molasses, yeast extract, sucrose and ammonia with bacillus subtilis

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 December 2009 to 15 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD, EPA and EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

In addition, the study procedures described in this report were essentially conform to the following guidelines:

- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Biomass residue (liquid) obtained after B2 production

IUPAC Name:

Biomass residue (liquid) obtained after B2 production

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age F0 at start treatment: Approximately 11 weeks.
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During activity monitoring, animals were housed individually in Macrolon plastic cages (MIII type; height 15 cm) with sterilised sawdust as bedding material. No cage-enrichment was provided during activity monitoring.

- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C (actual range: 20.0 ¿ 22.1°C)
- Humidity (%): 40 - 70% (actual range: 28 - 71%)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity (with a maximum of 4 hours). Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of pupillary reflex tests examinations in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

IN-LIFE DATES: 28 December 2009 to 15 February 2010

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Elix

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for density of the test substance (1.14 g/mL).

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 13 days was allowed for mating. After 13 days of mating, one female of Group 2, who had not shown evidence of mating was separated from her male.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the oestrus cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. Mating of one female of Group 2, appeared to have been overlooked since live offspring was delivered by this animal.
- After successful mating each pregnant female was caged individually in Macrolon plastic cages (MIII type, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42-49 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. A few females (4, 3, 2 in Groups 1, 3 and 4, respectively) were not dosed during littering.
Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Details on study schedule:

- Age at mating of the mated animals in the study: Approximately 13 weeks.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results of the dose range finding study (NOTOX Project 492228), dose levels for the main study were: 100, 300 and 1000 mg/kg bw. Since no clinical signs were observed in the range finding study, clinical observations in the main study were conducted immediately after dosing.

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found
completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that
were littering were left undisturbed.

Identification of pups:
On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous
injection of Indian ink.

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, macroscopic examination (full list), organ weights (full list) and histopathology.
Haematology: 5 males/group and females: 7 in Group 1, 6 in Group 2 and 3, and 5 in Group 4
Clinical chemistry: 5 males/group and females: 8 in Group 1, 6 in Group 2 and 3, and 5 in Group 4
Only females with live offspring were selected.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Yes - Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
Yes - Time schedule: Detailed clinical observations were made in all animals, at least immediately after dosing. Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually. The time of onset, degree and duration was recorded. All symptoms were recorded and graded according to fixed scales.

BODY WEIGHT:
Yes - Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION :
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY:
(Average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION:
No

OPHTHALMOSCOPIC EXAMINATION:
No

HAEMATOLOGY:
Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One, Bad Haller, Austria) prepared with EDTA for haematological parameters (0.5 mL) and with citrate for clotting tests (0.45 mL).
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: yes (with a maximum of 22.5 hours). Water was provided.
- How many animals: 5 males/group (random) and selected females with live pups (7 in Group 1, 6 in Group 2 and 3, and 5 in Group 4).
- Parameters checked were: white blood cells, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY:
Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One, Bad Haller,
Austria) prepared with Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood
sample (0.25 mL) was collected into untreated tubes for determination of bile acids.
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00
and 10.30 a.m
- Animals fasted: Yes (with a maximum of 22.5 hours). Water was provided.
- How many animals: 5 males/group (random) and selected females with live pups (8 in Group 1, 6 in Group 2 and 3, and 5 in Group 4)
- Parameters examined were: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate, bile acids.

URINALYSIS:
No

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity test (recording period: 1-hour for individual animals, using a computerised monitoring system; Pearson Technical Services, Suffolk, Great Britain).

During the motor activity test, males were caged individually and females were caged with their pups.

In order to avoid hypothermia of pups, dams were removed from the pups for not more than 30-40 minutes during the following tests: hearing ability, pupillary reflex, static righting reflex, grip strength.

Oestrous cyclicity (parental animals):

not determined

Sperm parameters (parental animals):

Parameters examined in all male parental animals:
Testes weight, Epididymides weight.
In addition, for 5 males of the control and high dose group, slides of the testes were prepared to examine staging of spermatogenesis.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made in all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: was determined for all pups on Days 1 and 4 of lactation.

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

GROSS PATHOLOGY
All animals were fasted overnight (with a maximum of 22.5 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised using iso-flurane vapor (Abbott Laboratories Ltd., Hoofddorp, The Netherlands) and subsequently exsanguinated. Necropsy was conducted on the following days:
Females which delivered: Lactation Days 5-6.
Females which failed to deliver: Post-coitum Day 27 (one Group 4 female with evidence of mating) or 22 days after the last day of the mating period (one Group 2 female without evidence of mating).
Males: Following completion of the mating period (a minimum of 28 days of dose administration).

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

The number of former implantation sites and corpora lutea were recorded for all paired females.

Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):

- Selected 5 animals/sex/group:
Identification marks (not processed) , Ovaries, Adrenal glands, (Pancreas), (Aorta), Peyer's patches (jejunum, ileum) if detectable, Brain (cerebellum, mid-brain, cortex), Caecum, Pituitary gland, Cervix, Preputial gland, Clitoral gland, Prostate gland, Colon, Rectum, Coagulation gland, (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Epididymides <1>, Seminal vesicles, (Eyes with optic nerve (if detectable) and Harderian gland) <1>, Skeletal muscle, (Skin), Female mammary gland area, Spinal cord -cervical, midthoracic, lumbar-, (Femur including joint), Spleen, Heart, Sternum with bone marrow, Ileum, Stomach, Jejunum, Testes <1>, Kidneys, Thymus, (Larynx), Thyroid including parathyroid (if detectable),(Lacrimal gland, exorbital), (Tongue), Liver, Trachea, Lung (infused with formalin), Urinary bladder, Lymph nodes -mandibular, mesenteric-, Uterus, (Nasopharynx), Vagina, (Oesophagus), all gross lesions.

- All remaining animals and females which failed to deliver:<2>
Identification marks (not processed) Cervix, Prostate gland, Clitoral gland, Seminal vesicles, Coagulation gland, Testes <1>, Epididymides <1>, Uterus, Ovaries, Vagina, Preputial gland, all gross lesions.

<1> Fixed in modified Davidson's solution (prepared at NOTOX using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial) and Milli-Ro water and transferred to formalin after fixation for at least 24 hours.
<2 > In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites (Salewski staining prepared at NOTOX using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

ORGAN WEIGHTS
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:

- Selected 5 animals/sex/group:
Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate<1>, Liver, Seminal vesicles including coagulating glands<1>, Ovaries, Thyroid including parathyroid <1>.

<1> weighed when fixed for at least 24 hours.

- All remaining males:
Epididymides and Testes

HISTOTECHNOLOGY
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). Of the selected 5 males of the control and high dose group, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

HISTOPATHOLOGY
The following slides were examined by a pathologist:
- The preserved organs and tissues of 5 selected animals/sex of Groups 1 and 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs* of all animals that failed to mate, conceive, sire or deliver healthy pups:
Group 2: one male and female that failed to mate.
Group 4: one male and female that failed to conceive/sire.

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

* Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testis, uterus, and vagina.

Postmortem examinations (offspring):

SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5-6.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
no

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. (Dunnett C.W., 1955)
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution. (Miller R.G., 1981)
- The Fisher Exact-test was applied to frequency data. (Fisher R.A., 1950)

The following additional methods of statistical analysis were used:
The numbers of corpora lutea and implantation sites were transformed by using log x and x², respectively, to obtain a normal distribution. This was followed by an ANOVA. The Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

No statistical analysis was performed on histopathology findings.

Reproductive indices:

For each group, the following calculations were performed:

Mating (%): Number of females mated/Number of females paired x 100

Fertility index (%): Number of pregnant females/Number of females paired x 100

Conception index (%): Number of pregnant females/Number of females mated x 100

Gestation index (%): Number of females bearing live pups/Number of pregnant females x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage of postnatal loss Days 0-4 of lactation:
Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100

Viability index (%): Number of live pups on Day 4 of lactation/ Number of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Other effects:

not examined

Details on results (P0)

MORTALITY
No mortality occurred during the study period.

CLINICAL SIGNS
No clinical signs of toxicity were noted up to 1000 mg/kg bw.

Incidental findings that were noted in females only included alopecia (Groups 1 and 4) and scabbing (Group 1). These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the low incidence observed and due the fact that (also) control animals were affected, these findings were considered signs of no toxicological relevance.

FUNCTIONAL OBSERVATIONS
No toxicologically significant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed.

The variation in motor activity did not indicate a relation with treatment.

BODY WEIGHTS
No toxicologically relevant changes in body weights and body weight gain were noted up to 1000 mg/kg bw.

The statistically significantly lower mean body weight recorded for females in Group 2 during the pre-mating period and on Day 1 of the mating period were caused by the relatively low initial mean body weight of this group.

The relatively high body weight and body weight gain recorded for one Group 2 female during mating period was due to the fact that for this female mating was overlooked.

The statistically significantly higher mean body weight gain recorded for Group 4 males on Day 8 of the pre-mating period was within the normal range of biological variation noted for rats of this strain and age. Moreover, a decrease rather than an increase in body weight would have been expected in case of toxicity.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
A statistically significant increase of relative numbers of neutrophils with concurrently reduced relative numbers of lymphocytes was noted in females of the high dose group as compared to control animals.

At the individual level, relatively high values for red blood cell distribution width (RDW) were noted in one Group 1 male, one Group 2 female, two Group 3 females and two Group 3 males. Since treated as well as control animals were affected, this finding was considered to be no sign of toxicity.

A relatively high activated partial thromboplastin time (APTT) was noted for one female in Group 2. No explanation could be given for this finding. As all other haematology parameters for this animal were within the normal range of biological variation seen for animals of this age and strain treated under comparable conditions, the relatively high APTT was considered to have occurred by chance.

CLINICAL BIOCHEMISTRY
Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment up to 1000 mg/kg bw.

At the individual level, a relatively high activity of alkaline phosphatase (ALP) was note in one female of Group 2. No explanation could be given for this finding.

Any statistically significant changes at 100, 300 and 1000 mg/kg bw were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain.

MACROSCOPIC EXAMINATION
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

Incidental findings among control and treated animals included focus/foci on the glandular mucosa of the stomach (reddish), right cranial, medial and caudal lobe of the lung (reddish), thymus (reddish) or clitoral glands (tan), pale discolouration of the adrenal glands, reddish discolouration of the glandular mucosa of the stomach, enlarged size or dark red discolouration of the mandibular lymph nodes, and alopecia. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological significance.

ORGAN WEIGHTS
No toxicologically significant changes were noted in organ weights and organ to body weight ratios up to 1000 mg/kg bw.

MICROSCOPIC EXAMINATION
All microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar (Han) rats of this age and strain.

No abnormalities were seen in the reproductive organs of the Group 2 female and male that failed to mate and the Group 4 female and male that failed to conceive/sire, which could account for infertility.

The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

REPRODUCTIVE DATA
No toxicologically relevant effects on reproductive parameters were noted.
Mating, fertility and conception index, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

DEVELOPMENTAL DATA
No toxicologically relevant effects on gestation index and duration, parturition, maternal care were observed.

PARTURITION/MATERNAL CARE
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Results: F1 generation

General toxicity (F1)

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings:

not examined

Details on results (F1)

- Early postnatal pup development
No toxicologically relevant effects on early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed. Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

- Mortality
One pup in a litter of Group 2 and five pups in a litter of Group 4 were found dead at first litter check. Necropsy findings for these dead pups included autolysis, cannibalism and/or no milk in the stomach. No toxicological relevance was attributed to these dead pups since the mortality incidence remained within the range considered normal for pups of this age and the surviving pups of both litters developed normally.

- Clinical signs
Incidental clinical symptoms of pups consisted of small size, pale appearance and blue spot on the neck or back. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

- Body weights
Body weights of pups were considered to have been unaffected by treatment.

- Macroscopy
Incidental macroscopic findings among surviving pups included small size and wound tail apex. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Treatment with Biomass residue obtained after B2 production by oral gavage in male and female Wistar (Han) rats at dose levels of 100, 300 and 1000 mg/kg bw/day revealed no parental toxicity up to 1000 mg/kg bw/day. No reproduction and developmental toxicity was observed for treatment up to 1000 mg/kg bw/day.

Based on these results, a parental, reproductive and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day was derived