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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-01-13 to 2011-12-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study and GLP conform
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V. Kreuzelweg 53 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: males 309 to 341 g; females 188 to 225 g
- Fasting period before study: no
- Housing: singly in Makrolon type-3 cages with wire mesh tops and standard softwood bedding
- Diet: pelleted standard diet (Kliba Nafag 3433 rodent maintenance diet) ad libitum
- Water: tap water ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

- Rate of dose formulation preparation: weekly
- Separate formulations were prepared for each dose level
- Homogeneity was maintained during the daily administration period using a magnetic stirrer
- Storage conditions: at room temperature (20 ± 5 °C) in the dark

VEHICLE
- Justification for use and choice of vehicle (if other than water): low water solubility of the test item
- Concentration in vehicle: up to 25, 75, and 250 g/L
- Amount of vehicle (if gavage): 4 mL/kg
- Lot/batch no. (if required): 280160017
- Provider: Carl Roth, Karlsruhe, Germany
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days until evidence of mating was observed
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged singly
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC-FID using calabrication curve; samples were analysed in duplicate. For further details see study report Appendix IV.
Duration of treatment / exposure:
Males: 4 weeks (2 weeks pre-pairing, 2 weeks pairing period)
Females: 7 weeks (2 weeks pre-pairing, 2 weeks pairing period, 3 weeks gestation)
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 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 rangefinding study results
Positive control:
not required

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
--viability/mortaility: twice daily
--clinical signs: once daily
- Cage side observations checked were included (report p 68/614).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first treatment, weekly thereafter (report p 75/614).

BODY WEIGHT: Yes
- Time schedule for examinations: daily from treatment start to necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined (not during pairing): Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Not required

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
males: the day before scheduled necropsy
females: day 5 post partum
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 per sex
- Parameters listed in section 3.10.1 were examined. Covers and exceeds parameters listed in OECD TG 422.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
males: the day before scheduled necropsy
females: day 5 post partum
- Animals fasted: Yes
- How many animals: 5 per sex
- Parameters listed in section 3.10.2 were examined. Covers and exceeds parameters listed in OECD TG 422.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the treatment period.
-- Males: shortly before scheduled sacrifice
-- Females on day 3 or 4 post partum
- Dose groups that were examined: 5 male and female rats from each dose group
- Battery of functions tested: sensory activity / grip strength / motor activity /
- Test parameters listed in report Appendix V, p 424.
Oestrous cyclicity (parental animals):
no
Sperm parameters (parental animals):
Parameters examined in all male parental rats:
- Weight: (p 148 onwards)
-- Testis
-- Epididymides

- Histopathology (p 455 onwards)
TESTES
- - Tubular degeneration
-- Giant cell spermatids
-- tubular vacuolation

EPIDIDYMIDES
- - mononuclear foci
-- oligospermia
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:

number and sex of pups (p 144)
stillbirths, live births, postnatal mortality (p 144-145)
weight gain (p 166 - 167)
presence of gross anomalies, physical or behavioural abnormalities (p 168)

GROSS EXAMINATION OF DEAD PUPS:
yes
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes.
All parent animals and pups were examined (see report section 3.11.1)

HISTOPATHOLOGY: Yes
All organs and tissues of the control and high-dose parent animals were examined. The same applies to all occurring gross lesions or animals that died spontaneously. Tissues preserved are listed in section 3.11.3 of the report and include male (prostate, testes, seminal vesicles, epididymides) and female (ovaries, uterus with vagina) reproductive organs.

Additional examinations:
All dams: corpora lutea count (p 143)
Postmortem examinations (offspring):
GROSS NECROPSY
- Gross necropsy consisted of external examinations
Statistics:
The following methods were used to analyse food consumption, body weights, and reproduction data:
- calculation of means and standard deviations.
- Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
Fertility index (p 36)
Gestation index (p 36)
Implantation loss (p 144)
Offspring viability indices:
Birth index (p 145)
Viability index Day 4 (p 145)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: not applicable: oral gavage study

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
All animals survived, and there were no test item-related clinical signs at any dose level.

BODY WEIGHT AND WEIGHT GAIN
No effects on body weight or body weight gain of males or females which were considered to be related to treatment were noted at any dose level.

FOOD CONSUMPTION
Food consumption was unchanged.

HAEMATOLOGY
There were no effects on haematology parameters in males or females which were considered to be related to treatment.

CLINICAL CHEMISTRY
No effects on clinical biochemistry parameters, which were considered to be test item-related, were noted at any dose level in males or females.

NEUROBEHAVIOUR
FOB (home cage observations; observation in the hand; open filed observations; cf. Appendix V, p 424): No test item-related effects were noted during functional observational battery in males or females at any dose level. The following observations were noted for individual animals or did not follow a dose dependency pattern and therefore were considered not to be test item-related: vocalization in one male (no. 11) at the dose level of 100 mg/kg bw/day, decreased landing foot splay distance in males at the dose level of 100 mg/kg bw/day, lifting and increased rearings in one female (no. 51) at the dose level of 100 mg/kg bw/day, decreased rearings and increased number of faeces balls in one females (no. 66) at the dose level of 300 mg/kg bw/day. Chromodacryorrhea in female no. 64 was confirmed. No further observations were noted in males or females at any dose level.
Grip strength: no changes observed in treated males (p 81) and females (p 85) at any dose.
Locomotor activity of males (p 88) and females (p 90) not significantly different from controls at any dose.

ORGAN WEIGHTS
No significant absolute and relative organ weight changes in males and females noted at any dose. This includes male and female reproductive organs.
Exceptions:
- Liver: absolute and relative liver weight was statistically significantly increased at 1000 mg/kg bw and day. In the absence of findings indicating liver injury this was regarded to be most probably a result of metabolic adaptation and not adverse.
- Heart: statistically significantly higher absolute and relative heart weights were seen in males. However, the difference to the control value was minor. Further, the mean heart weights were in the range of historical control values, and there was no clear dose relationship. Therefore, this effect was not considered to be treatment-related.
- Thymus: in females at 100 mg/kg and day, statistically significantly higher absolute and relative thymus weights were noted. This effect was not seen in higher dose groups, hence this was not considered to be related to treatment.

GROSS PATHOLOGY
No test item-related findings were noted during necropsy of males and females at any dose level.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment with the test item led to an increased incidence of centrilobular to diffuse hepatocellular hypertrophy of minimal severity in males and females at the dose levels of 1000 and 300 mg/kg bw/day. This finding was noted in one male in the control group and 2 males at each dose level 300 and 1000 mg/kg bw/day and one and three females at the dose levels of 300 and 1000 mg/kg bw/day, respectively. In the absence of any further indicators of liver injury, hepatocellular hypertrophy was considered to be of metabolic nature.

Other findings were within the range of normal background lesions which may be recorded in animals of this strain and age. This includes findings in male and female reproductive organs.


REPRODUCTIVE FUNCTION: ESTROUS CYCLE
Not examined

REPRODUCTIVE FUNCTION: SPERM MEASURES
Not examined. No findings noted during histopathology

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Fertility
No effect on mating performance or fertility was observed at any dose level. Fertility indexes (number of females achieving pregnancy as a percentage of females paired) and conception rates (number of females achieving pregnancy as a percentage of females mated) were 90%, 90%, 80% and 80% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively. In the absence of clear dose dependency, lack of pregnancy was considered not to be test item-related.
- Duration of Gestation
No effects on duration of gestation were observed at any dose level. Mean duration of gestation was 21.4, 21.6, 21.4 and 22.1 days, in order of ascending dose level.
- Corpora Lutea count
No effects on corpora lutea count were observed at any dose level. Mean number of corpora lutea per dam was 14.4, 15.3, 13.9 and 13.6 in order of ascending dose levels.
- Implantation rate
No effect noted at any dose. Mean number of implantations per dam was 13.1, 13.2, 13.1 and 12.0 ; cited in order of ascending dose levels.
- Post-implantation loss
No effect noted at any dose. Mean number of post-implantation loss per dam was 1.4, 1.0, 0.6 and 1.8; cited in order of ascending dose levels.
-Litter size
No effect noted at any dose level. Mean numbers of living pups per dam at first litter check were 11.7, 12.2, 12.5 and 10.3.
- Viability at birth
No effect noted at any dose. Birth indexes (number of pups borne alive as a percentage of implantations) were 89.0%, 92.4%, 95.2% and 85.4% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day.
- Post natal loss Days 0-4 post partum
No effect noted at any dose level. Dead pups were seen as follows: 2/2/2/3 in groups at 0/100/300/1000 mg/kg bw and day, respectively.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related adverse effects noted up to 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
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
- Viability at birth
No effect noted at any dose. Birth indexes (number of pups borne alive as a percentage of implantations) were 89.0%, 92.4%, 95.2% and 85.4% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day.
- Post natal loss Days 0-4 post partum
No effect noted at any dose level. Dead pups were seen as follows: 2/2/2/3 in groups at 0/100/300/1000 mg/kg bw and day, respectively.

CLINICAL SIGNS (OFFSPRING)
No test item-related findings were noted in pups at any dose level. No milk in th estomach of 1/2/3 pups in groups at 100/300/1000 mg/kg bw and day was noted. These pups were found dead at first litter check.

SEX RATIOS
No effect at any dose level. At first litter check, percentages of male pups were 50%, 59%, 45% and 59%, in order of ascending dose level.

BODY WEIGHT (OFFSPRING)
No effect at any dose level during days 0 to 4 post partum.

GROSS PATHOLOGY (OFFSPRING)
No abnormal findings at any dose level during external examination.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related adverse effects noted up to highest dose tested

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Treatment had no effect on fertility. The NOAEL value for toxicity to reproduction (fertility) was 1000 mg/kg bw in this screening study.
Executive summary:

n/i-C13 -C15 aldehyde was tested in an OECD TG 422 oral gavage study in rats under GLP conditions. The test item was administered to 10 male and female rats over approximately 28 days suspended in peanut oil as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. The dose volume was 4 mL/kg bw and day throughout the study. The test substance was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.  

All parental animals terminated the study as scheduled without signs of toxicity. The NOAEL for general toxicity was therefore 1000 mg/kg bw and day for male and female rats. Details are reported in section 7.5.1.

 

Regarding fertility, no effects on mating performance, fertility, corpora lutea count, duration of gestation, implantation rate and post-implantation loss as well as litter size and post-natal loss were noted at any dose level. During histopathology no effects on male and female reproduction organs were noted.

No test item-related findings in pups were noted at any dose. Pups body weights, sex ratio, and viability were noted affected, and there were no malformations noted during macroscopical examinations.

Based on the above it is concluded that the NOAEL value for toxicity to reproduction (fertility) was 1000 mg/kg bw and day in this rat OECD TG 422 screening study. The study is considered to be valid and suitable for assessment.

 

Information on developmental toxicity is also reported in section 7.8.2. 

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