2-furaldehyde

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 April 2012 (initiation of dosing to F0 generation) to 28 December 2012 (final necrosy of F1 generation)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

22 January 2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

To evaluate potential adverse effects on reproduction in a 2-generation study, to include effects on gonadal function, oestrus cyclicity, mating behaviour, conception, gestation, parturition, lactation, weaning and growth and development of offspring. Crl:CD(SD) rats are an appropriate species/strain for use in reproduction studies and the laboratory has historical control data on this species/strain. The number of animals was based on the OECD 416 and OPPTS 870.3800 guidelines.

Test material

Test material information

Constituent 1

Reference substance name:

2-furaldehyde

EC Number:

202-627-7

EC Name:

2-furaldehyde

Cas Number:

98-01-1

Molecular formula:

C5H4O2

IUPAC Name:

2-furaldehyde

Details on test material:

[carbonyl-14C] Furfural, specific activity 135.75 microCi /mg (radiochemical purity 98.1 % by radio-gas chromatography) was a custom synthesis by Amersham International pic (Amersham. Bucks UK) and was diluted to constant specific activity 29.5 pCi/mg with unlabelled furfural (Aldrich Chemical Co., Gillingham. Dorset. UK) and stored in dichloromethane in a glass vial at a temperature of -20°C. Radiochemical purity was more than 99% by radio-HPLC.

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

Sexually mature males and sexually mature virgin females

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: (F0) approximately 6 wks
- Weight at study initiation: (F0) Males: 185-278 g
- Age / weight at start of cohabitation : (F0) Approximately 16 weeks / males 446-695 g, females 232-440 g. (F1) Approximately 14 weeks / males 405-662 g, females 232-400 g
- Fasting period before study: None
- Housing: Until cohabitation, F0 and F1 parents housed individually in suspended stainless steel, wire mesh cages. Rats were paired for mating in the home cage of the male. After positive evidence of mating, males remained individually housed in their cages until scheduled necropsy and females were transferred to plastic maternity cages with nesting material. Following weaning on lactation day 21, dams were housed individually in suspended stainless steel, wire mesh cages and the weaned F1 pups were housed together by litter for 1 week. On post natal day (PND) 28, the F1 offspring were individually housed in suspended wire-mesh caging until pairing. Following pairing, F1 females and males were housed as described for F0 males and females.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum
- Water: Reverse osmosis purified drinking water ad libitum
- Acclimation period: 14 days
ENVIRONMENTAL CONDITIONS
- Mean daily temperature: 20.8-22.0°C:
- Mean daily humidity: 33.1-59.5%
- Air changes: Minimum of 10 per hr
- Photoperiod: 12 hrs dark / 12 hrs light
IN-LIFE DATES: From: 10 April 2012 To: 28 December 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionised water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared by dissolving the appropriate quantity of test substance in deionised water to give the following concentrations: 4, 8 and 12 mg/mL (for dose levels of 20, 40 and 60 mg/kg bw, respectively). Dosing formulations were prepared approximately weekly as single formulations for each dose level, divided into aliquots and stored at room temperature, protected from light. Formulations were stirred continuously throughout preparation, sampling and dosing.

VEHICLE: Deionised water

Details on mating procedure:

- M/F ratio per cage: 1:1, randomly selected for cohabitation, avoiding sibling matings.
- Length of cohabitation: Up to 14 days, until evidence of mating seen.
- Proof of pregnancy: Sperm in vaginal lavage or presence of vaginal copulatory plug [gestational day 0 ]
- After successful mating each pregnant female was caged (how): Plastic maternity cages with nesting material.
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Solubility in water, homogeneity, resuspension homogeneity and stability (over 8 days) were previously established at concentrations spanning the range of concentrations used. For reconfirmation of test substance stability in dosing formulations, samples were collected from the top and bottom strata of the last dosing aliquot of the first batch of each test substance formulation. Samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group). For each generation (F0 and F1), samples from the first and second preparation were analyzed. In addition, monthly samples and samples from the second to last preparation were analyzed. One set of samples was subjected to the appropriate analyses. The remaining set of samples was stored at -20°C. All analyses used a validated spectrophotometric method using ultraviolet absorbance detection. The analyzed dosing formulations were within 85% to 115% of nominal and were stable for 7 days of room temperature storage. The test substance was not detected in the vehicle formulation that was administered to the control group.

Duration of treatment / exposure:

Animals were dosed once daily for at least 70 consecutive days prior to mating. Dose administration for the F0 and F1 males continued throughout mating and through the day prior to euthanasia, for a total of 127-132 doses and 135-149 doses, respectively. The F0 and F1 females continued to be dosed continuously throughout mating, gestation and lactation until the day prior to euthanasia for a total of 127-132 and 134-149 doses, respectively.

Frequency of treatment:

Continuous by daily oral gavage

Details on study schedule:

- Selection of parents from F1 generation when pups were 21 days of age.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

60 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Determined from the results of previous studies including a historical prenatal developmental toxicity study conducted by WIL Research (Nemec, 1997, WIL-12378) and a 2-year carcinogenicity study conducted by the National Toxicology Program (Irwin, 1990, NTP TR 382). The oral administration of furfural appears to be associated with a steep dose response curve,
• the oral LD50 detemined in the oral acute toxicity study in rats are 100, 105 and 108 mg/kg bw/day for male rats, female rats and combined sexes, respectively. (Mital, 2002)
• in the developmental study moribundity and mortality were reported at 100 mg/kg and 150 mg/kg. In the developmental study, some of the surviving dams exhibited tremors at 50 mg/kg and although these signs subsided they were considered to be related to treatment with furfural and 50 mg/kg/day was considered to be the LOAEL for maternal toxicity.
• In the two-year study, survivability was adversely affected, notably among female rats in the 60 mg/kg/day (highest) dose group and increased mortality in this group was believed to be associated with a treatment related reflex regurgitation. Additionally, in a 13-week subchronic oral toxicity study of furfural in rats (NTP, 1990), liver effects were reported at 45 mg/kg/day and treatment-related mortality occurred at 90 mg/kg/day.
Hence based on this data 60 mg/kg/day was chosen as the high dose level and was expected to show some evidence of maternal toxicity without unacceptable mortality.

- Rationale for animal assignment:
Computerised randomisation procedure based on body weight stratification randomised in a block design.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes f
- Time schedule: Once in morning and once in afternoon for mortality and moribundity plus approximately 2 hours after dosing for signs of toxicity. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labour) or other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for F0 and F1 males until scheduled termination.
Weekly until evidence of copulation was observed for F0 and F1 females, gestation days 0, 4, 7, 11, 14, 17 and 20, lactation days 0 (when possible), 1, 4, 7, 14 and 21, weekly thereafter until scheduled termination.

FOOD CONSUMPTION, FOOD EFFICIENCY AND COMPOUND INTAKE (if feeding study):
- Time schedule: Individual F0 and F1 male and female food consumption weekly until cohabitation. For F1 males and females to constitute the parental generation, food consumption was measured beginning on PND 28. Food intake was not recorded during the mating period. Following mating, food consumption for males and females with no evidence of mating was measured on a weekly basis until the scheduled necropsy. Following evidence of mating, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17 and 20 and lactation days 1, 4, 7, 14 and 21. Food efficiency calculated over same periods.

F0 and F1 PARTURITION: All females were allowed to deliver naturally and rear their young to weaning (PND 21). During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. Beginning on the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery was first observed.

Oestrous cyclicity (parental animals):

Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the estrous cycle of each F0 and F1 female for 21 days prior to cohabitation and continuing until evidence of mating was observed or until the end of the mating period. Vaginal lavages were also performed on the day of necropsy to determine the stage of the oestrous cycle.

Sperm parameters (parental animals):

Parameters examined in F0 and F1 male parents: Right testis and epididymis from all treated F0 and F1 males were weighed, sperm motility and sperm morphology determined (minimum of 200 spermatozoa per animal). Left testis and epididymis from all treated F0 and F1 groups were weighed, stored frozen, homogenised, and analysed for determination of homogenisation resistant spermatid count and calculation of sperm production rate (a minimum of 200 cells, if possible, counted for each sample).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes (4/sex/litter as nearly as possible); excess pups were weighed, killed and discarded.

PARAMETERS EXAMINED
- Clinical observations, body weights, and sex. Developmental landmarks (balanopreputial separation from day 35 and vaginal patency from day 25) were evaluated for the F1 rats selected to constitute the F1 generation. Non-selected F1 pups were necropsied on PND 21.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
- All surviving F0 adults were killed following the selection of the F1 generation and completion of a clinical observation and all surviving F1 adults were killed following weaning of the F2 pups. Animals were killed by carbon dioxide inhalation.

GROSS NECROPSY
- A complete necropsy was conducted on all parental animals (F0 and F1). The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. For females that delivered or had macroscopic evidence of implantation, the numbers of former implantation sites were recorded. Numbers of corpora lutea were also recorded for females with macroscopic evidence of implantation and for females necropsied during gestation through lactation day 4. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathological findings that may have interfered with pregnancy.

ORGAN WEIGHTS
- The following organs from F0 and F1 parents were weighed: adrenal glands, seminal vesicles with coagulating glands (with accessory fluids), brain, epididymidesa (total and cauda), spleen, kidneys, testes, liver, thyroid gland, ovaries, thymus gland, pituitary gland, uterus with oviducts and cervix, prostate gland.

HISTOPATHOLOGY
- Microscopic evaluations were performed on the following tissues for all F0 and F1 parental animals from the control and high-dose groups and for all adult animals found dead or killed in extremis: brain, pituitary gland, cervix, prostate gland, coagulating gland, seminal vesicles, right epididymis (caput, corpus and cauda), right testis, uterus, kidneys, vagina, liver, vas deferens, mammary gland (females only), ovaries, all gross (internal) lesions (all groups).
Note: PAS and hematoxylin staining were used for the right testis and epididymis. Transverse sections of 2-4 microns of the testes and longitudinal sections of 2-4 microns of the epididymides were made. If the left testis or epididymis was noted with a gross lesion, the left testis and epididymis was fixed in modified Davidson’s solution and the right testis and epididymis were homogenised. 5 sections were taken approximately 100 μm apart from the inner third of each ovary from the F0 and F1 females in the control and high-dosage groups at scheduled termination and for any females suspected of reduced fertility. Qualitative histopathological evaluation of a single section of each ovary was performed on the F0 and F1 females from the control and high-dose groups. For all F1 females in the control and high-dose groups at the scheduled termination and for any F1 females suspected of reduced fertility, a quantitative histopathological evaluation of multiple sections was conducted; this examination included enumeration of the total number of primordial follicles. For females that were found dead or killed in extremis, a single section from each ovary was qualitatively evaluated. In addition, reproductive organs of all animals suspected of reduced fertility (i.e. failure to mate,
conceive, sire or deliver healthy offspring, or animals with effects on oestrous cyclicity or sperm number, motility, or morphology) were subjected to histopathological evaluation.

Postmortem examinations (offspring):

SACRIFICE
- The F1 and F2 offspring not selected as parental animals were killed by carbon dioxide inhalation on post natal day 21.

GROSS NECROPSY
- Gross necropsies with emphasis on developmental morphology and organs of the reproductive system were performed on all non-selected offspring.

ORGAN WEIGTHS
- Brain, spleen, and thymus were weighed from 1 male and 1 female weanling from each F1 and F2 litter at the scheduled necropsies.

HISTOPATHOLOGY
- Not examined.

Statistics:

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex. Parental mating, fertility, copulation, and conception indices were analyzed using the Chi-square test with Yates’ correction factor. Mean parental (weekly, gestation, and lactation) and offspring body weights and body weight changes, parental food consumption, and food efficiency data, estrous cycle lengths, pre-coital intervals, gestation lengths, former implantation sites, live litter sizes, unaccounted-for sites, numbers of pups born, balanopreputial separation data (day of attainment and body weight), vaginal patency data (day of attainment and body weight), absolute and relative organ weights, sperm production rates, epididymal and testicular sperm numbers, and ovarian primordial follicle counts were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of postnatal pup survival and pup sexes at birth (percentage of males per litter), percentages of motile and progressively motile sperm, and percentages of sperm with normal morphology were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group. Histopathological findings in the test substance-treated groups were compared to the control group using a two-tailed Fisher’s Exact test.

Reproductive indices:

The following were determined: Male Mating Index (%), Female Mating Index (%), Male Fertility Index (%), Female Fertility Index (%), Male Copulation Index (%), Female Conception Index (%), Oestrous Cycle Length (days), Pre-Coital Interval (days)

Offspring viability indices:

Mean live litter size, postnatal survival between birth and PND0 or PND4 (pre-selection) as % per litter, postnatal survival for all other intervals as % per litter, total litter loss

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related clinical findings included red material around the nose, noted in a dose-related manner at approximately 2 hours post-dosing. In addition, red material around the mouth was noted for males in the 40 and 60 mg/kg/day groups and females in the 60 mg/kg/day group at approximately 2 hours post-dosing. Due to the nature and sporadic incidences of these findings, they were not considered adverse. No other test substance-related clinical findings were noted at the detailed physical examinations or 2 hours post-dosing.

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

No test substance-related effects were noted at any dose level in the F0 generation on survival, body weights, food consumption and reproduction. No test substance-related macroscopic findings or changes in organ weights were noted. F0 male spermatogenesis was unaffected by test substance administration at all dose levels. Nonadverse, test substance-related clinical findings, including red material around the nose and/or mouth, were noted in the F0 generation at approximately 2 hours following dose administration. Red material around the nose was noted in a dose-related manner for F0 males and females in the 20, 40, and 60 mg/kg/day groups. Red material around the mouth was noted for F0 males in the 40 and 60 mg/kg/day groups and for F0 females in the 60 mg/kg/day group. The apparent dose-related observation of red material around the mouth was attributable to the presence of a residual amount of dosing solution in the area following administration; the test substance is known to take on a characteristic reddish-brown color upon exposure to air and/or light.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

parental systemic toxicity

Effect level:

60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

dermal irritation

body weight and weight gain

food consumption and compound intake

food efficiency

water consumption and compound intake

ophthalmological examination

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

neuropathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

other:

Remarks on result:

other: No test substance-related effects were noted at any dose level

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related clinical findings, including red material around the nose and/or mouth, were noted at approximately 2 hours following dose administration. Red material around the mouth was noted for F1 males and females in the 60 mg/kg/day group. The apparent dose-related observation of red material around the mouth was attributable to the presence of a residual amount of dosing solution in the area following administration; the test substance is known to take on a characteristic reddish-brown color upon exposure to air and/or light.

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

No test substance-related effects were noted at any dose level in the F1 generation on survival, body weights, food consumption, and reproduction. No test substance-related macroscopic findings or changes in organ weights were noted. F1 male spermatogenesis was unaffected by test substance administration at all dose levels. Nonadverse, test substance-related clinical findings, including red material around the nose and/or mouth, were noted in the F1 generation at approximately 2 hours following dose administration. Red material around the mouth was noted for F1 males in the 40 and 60 mg/kg/day groups and for F1 females in the 60 mg/kg/day group. The apparent dose-related observation of red material around the mouth was attributable to the presence of a residual amount of dosing solution in the area following administration; the test substance is known to take on a characteristic reddish-brown color upon exposure to air and/or light. Nonadverse, test substance-related microscopic findings were noted for F1 males in the 60 mg/kg/day group. Hepatocellular vacuolation was noted for 7 F1 males in the 60 mg/kg/day group. This was likely due to glycogen accumulation, and the finding was considered nonadverse and of no toxicological significance due to the low incidence (7 of 30 males) and severity (2 minimal, 5 mild) and the lack of any changes in liver weight.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

The mean oestrus cycle length at 60 mg/kg/day was significantly lower than the control value (4.5, 6.8 days, respectively). This difference was attributed to a high control group value as the value at 60 mg/kg/day was equal to the mean value in the WIL historical control data.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P1)

Test substance-related clinical findings, including red material around the nose and/or mouth, were noted in the F1 generations at approximately 2 hours following dose administration. Red material around the mouth was noted for F1 males and females in the 60 mg/kg/day group. The apparent dose-related observation of red material around the mouth was attributable to the presence of a residual amount of dosing solution in the area following administration; the test substance is known to take on a characteristic reddish-brown color upon exposure to air and/or light. Nonadverse, test substance-related microscopic findings were noted for F1 males in the 60 mg/kg/day group. Hepatocellular vacuolation was noted for 7 F1 males in the 60 mg/kg/day group. This was likely due to glycogen accumulation, and the finding was considered nonadverse and of no toxicologic significance due to the low incidence (7 of 30 males) and severity (2 minimal, 5 mild) and the lack of any changes in liver weight.

Effect levels (P1)

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: No effects at highest dose tested

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Mean ages of attainment of balanopreputial separation, mean ages of vaginal patency and mean body weights were unaffected by test substance administration.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

sexual maturation

mortality

body weight and weight gain

Remarks on result:

other: No effects on F1 pups

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

Remarks on result:

other: No effects on F2 pups at highest dose tested

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 2: Intergroup comparison of reproductive performance –F0 females

	Dose level (mg/kg/day)
Observation	0	20	40	60
Mean oestrus cycle (days)	4.3	4.3	4.8	4.7
Number mated	30	30	30	30
Number with positive signs of mating	27	28	30	27
Number females died or killed	1a	1b	0	0
Females delivered	23	25	25	26
Females with total litter loss	0	0	0	1
Females with viable pups	23	25	25	25
Total females pregnant	24a	27	25	26
Male mating index (%)	93.3	96.7	100.0	96.7
Female mating index (%)	93.3	96.7	100.0	96.7
Male fertility index (%)	80.0	90.0	83.3	86.7
Female fertility index (%)	80.0	90.0	83.3	86.7
Male copulation index (%)	85.7	93.1	83.3	89.7
Female copulation index (%)	85.7	93.1	83.3	89.7
Mean pre-coital interval ±SD	3.2 ± 2.97	3.4 ± 3.48	2.5 ± 2.10	2.4 ± 1.05
Gestation length ±SD (days)	22.1 ± 0.42	21.8 ± 0.48	21.8 ± 0.37	22.0 ± 0.59
Mean no. implantation sites	12.7	14.1	14.7	13.3
Mean number pups born	12.0	13.2	14.2	12.6

a killed in extremis during gestation; included in total numbers of “females pregnant” only

b killed in extremis following breeding period; included in all calculations

No statistically significant differences from control values

 

Table 3: Intergroup comparison of sperm analysis –F0 males

	Dose level (mg/kg/day)
Observation	0	20	40	60
Sperm production rate (millions/g/day)	18.6	18.3	18.1	17.5
Normal morphology (%)	99.8	99.7	99.8	99.8

No statistically significant differences from control values

 

Table 4: Intergroup comparison of reproductive performance –F1 females

	Dose level (mg/kg/day)
Observation	0	20	40	60
Number mated	30	30	30	30
Number with positive signs of mating	30	30	29	28
Number females died or killed	0	1a	0	1b
Females delivered	25	25	28	26
Females with total litter loss	0	0	0	0
Females with viable pups	25	25	28	26
Total females pregnant	25	25	28	26
Male mating index (%)	100.0	100.0	96.6	96.6
Female mating index (%)	100.0	100.0	96.6	96.6
Male fertility index (%)	83.3	86.2	93.1	89.7
Female fertility index (%)	83.3	83.3	93.3	89.7
Male copulation index (%)	83.3	86.2	96.4	92.9
Female copulation index (%)	83.3	83.3	96.6	92.9
Mean pre-coital interval ±SD	3.2 ± 2.86	3.0 ± 1.47	3.1 ± 2.81	2.7 ± 1.67
Gestation length ±SD (days)	21.8 ± 0.47	21.0 ± 0.49	22.0 ± 0.64	22.1 ± 0.39
Mean no. implantation sites	14.3	13.9	14.0	14.5
Mean number pups born	13.5	13.4	13.5	13.7

a killed in extremis following delivery of 1 pup on lactation day 0 with foetuses retained in uterus; included in calculations

b found dead prior to breeding period; excluded from calculations

No statistically significant differences from control values

 

Table 5: Intergroup comparison of sperm analysis –F1 males

	Dose level (mg/kg/day)
Observation	0	20	40	60
Sperm production rate (millions/g/day)	16.8	17.5	15.9	17.1
Normal morphology (%)	99.5	99.6	99.4	99.5

No statistically significant differences from control values

 

Table 6: Intergroup comparison of animal developmental parameters –F1

	Dose level (mg/kg/day)
Observation	0	20	40	60
Balanopreputial separation (PND)	46.6	47.5	47.4	46.5
Male mean body weight (g)	251.8	255.6	250.8	255.8
Vaginal patency (PND)	32.4	32.8	33.0	33.0
Female mean body weight (g)	111.7	113.1	115.8	117.0

 Table 7: Litter parameters for F1 pups

	Dose level (mg/kg/day)
Observation	0	20	40	60
Mean number born	12.0	13.2	14.2	12.6
Males per litter (%)	51.4	46.6	47.9	47.4
Mean live litter size (PND 0)	11.7	12.9	14.0	12.6
Mean survival (% per litter)
- PND 0 (relative to number born)	97.6	97.3	98.3	99.7
- PND 0-1	99.3	100.0	99.1	99.5
- PND 1-4 (pre-selection)	98.6	97.8	98.6	93.9
- PND 4-7 (post-selection)	99.5	99.0	98.5	93.8
- PND 7-14	100.0	100.0	100.0	100.0
- PND 14-21	100.0	100.0	100.0	98.0
- birth to PND 4 (pre-selection)	95.6	95.1	96.0	93.1
- PND 4-21 (post-selection)	99.5	99.0	98.5	91.8
Mean male pup weight (g)
- PND 1	7.5	7.0	6.9	7.4
- PND 4 (before selection)	10.3	9.8	9.6	10.2
- PND 7	16.0	15.6	15.7	16.1
- PND 14	31.2	31.6	32.2	32.3
- PND 21	51.6	50.0	52.0	53.6
Mean female pup weight (g)
- PND 1	6.9	6.6	6.6	7.0
- PND 4 (before selection)	9.6	9.3	9.1	9.7
- PND 7	15.2	14.9	15.0	15.4
- PND 14	30.2	30.5	31.1	30.9
- PND 21	49.9	48.5	49.9	50.8

No statistically significant differences from control values

 Table 8: Litter parameters for F2 pups

	Dose level (mg/kg/day)
Observation	0	20	40	60
F1 Generation
Mean number born	13.5	13.4	13.5	13.7
Males per litter (%)	52.2	52.1	45.3	49.2
Mean live litter size (PND 0)	13.4	13.1	13.3	13.2
Mean survival (% per litter)
- PND 0 (relative to number born)	99.2	97.5	98.3	96.2
- PND 0-1	98.5	99.2	98.8	98.4
- PND 1-4 (pre-selection)	98.8	98.1	97.8	97.3
- PND 4-7 (post-selection)	99.5	95.8	96.0	100.0
- PND 7-14	99.5	98.1	99.6	100.0
- PND 14-21	100.0	100.0	98.7	100.0
- birth to PND 4 (pre-selection)	96.5	94.9	95.1	92.0
- PND 4-21 (post-selection)	99.0	94.3	94.2	100.0
Mean male pup weight (g)
- PND 1	7.0	6.9	7.2	7.0
- PND 4 (before selection)	9.8	9.4	9.9	10.1
- PND 7	15.8	14.9	15.6	15.9
- PND 14	33.3	31.6	32.6	32.8
- PND 21	54.6	51.1	53.1	53.0
Mean female pup weight (g)
- PND 1	6.6	6.5	6.8	6.7
- PND 4 (before selection)	9.3	8.9	9.4	9.7
- PND 7	15.2	14.4	14.7	15.4
- PND 14	32.7	31.2	31.4	31.9
- PND 21	52.7	49.9	50.3	51.6

No statistically significant differences from control values

Applicant's summary and conclusion

Conclusions:

Due to the lack of any adverse effects, a dosage level of 60 mg/kg/day, the highest dose level evaluated, was considered to be the NOAEL (no-observed-adverse-effect level) for parental systemic toxicity, reproductive toxicity, and neonatal toxicity of the test substance when administered orally by gavage to Crl:CD(SD) rats.

Executive summary:

This study was conducted to evaluate the potential adverse effects of the test substance on reproduction in a 2-generation study. This included determining the effects of the test substance on male and female reproductive processes including gonadal function, oestrous cyclicity, mating behaviour, conception, gestation, parturition, lactation, weaning and growth and development of the offspring. One litter per dam was produced in each generation.

 

Three groups of Crl:CD(SD) rats (30/sex/group) were administered the test substance daily, by oral gavage, for at least 70 consecutive days prior to mating. Dose levels were 20, 40, and 60 mg/kg/day for the F0 and F1 generations. A concurrent control group of 30 rats/sex/group received the vehicle (deionised water). F0 animals were approximately 6 weeks of age at the initiation of test substance administration. The test substance was administered to offspring selected to become the F1 parental generation following weaning (beginning on PND 21). The F0 and F1 males received test substance throughout mating and until the day prior to euthanasia. The F0 and F1 females received the test substance throughout mating, gestation, and lactation until the day prior to euthanasia. For both generations (F1 and F2), 8 pups per litter (4 per sex, when possible) were selected on PND 4 to reduce the variability among the litters. Offspring (1/sex/litter, if possible) from the pairing of the F0 animals were selected prior to PND 21 to constitute the F1 generation. F0 males and females were dosed for 127-132 consecutive days, and F1 males and females were exposed for 135-149 and 134-149 consecutive days, respectively. All F0 and F1 females were allowed to deliver and rear their pups until weaning on lactation day 21. Clinical observations, body weights, and sexes for F1 and F2 pups were recorded at appropriate intervals. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the F1 rats selected to constitute the F1 generation. Nonselected F1 pups were necropsied on PND 21, and F2 pups were necropsied on PND 21. Selected organs were weighed from both F1 and F2 pups necropsied on PND 21. Each surviving F0 and F1 parental animal received a complete detailed gross necropsy following completion of weaning of the F1 and F2 pups; selected organs were weighed. Spermatogenic endpoints (sperm motility [including progressive motility]), morphology, and numbers) were recorded for all F0 and F1 males, and ovarian primordial follicle counts were recorded for all F1 females in the control and high-dose groups at the scheduled necropsy and from all F1 females in the 20 and 40 mg/kg/day groups suspected of reduced fertility. Designated tissues from all F0 and F1 parental animals were examined microscopically.

 

No test substance-related effects were noted at any dosage level in the F0 and F1generations on survival, body weights, food consumption, and reproduction or on the survival, growth, and development of F1and F2 offspring. In addition, no test substance-related macroscopic findings or changes in organ weights were noted. F0 and F1 male spermatogenesis was unaffected by test substance administration at all dose levels.

 

Nonadverse, test substance-related clinical findings, including red material around the nose and/or mouth, were noted in the F0 and F1 generations at approximately 2 hours following dose administration. Red material around the nose was noted in a dose-related manner for F0 males and females in the 20, 40, and 60 mg/kg/day groups. Red material around the mouth was noted for F0 and F1 males in the 40 (F0 only) and 60 mg/kg/day groups and for F0 and F1 females in the 60 mg/kg/day group. The apparent dose-related observation of red material around the mouth was attributable to the presence of a residual amount of dosing solution in the area following administration; the test substance is known to take on a characteristic reddish-brown colour upon exposure to air and/or light.

 

Nonadverse, test substance-related microscopic findings were noted for F1 males in the 60 mg/kg/day group. Hepatocellular vacuolation was noted for seven F1 males in the 60 mg/kg/day group. This was likely due to glycogen accumulation, and the finding was considered nonadverse and of no toxicological significance due to the low incidence (7 of 30 males) and severity (2 minimal, 5 mild) and the lack of any changes in liver weight.

 

Due to the lack of any adverse effects, a dose level of 60 mg/kg/day, the highest dose level evaluated, was considered to be the NOAEL (no-observed-adverse-effect level) for parental systemic toxicity, reproductive toxicity, and neonatal toxicity of furfural when administered orally by gavage to Crl:CD(SD) rats.