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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:
key study
Study period:
22 August 2013 - 17 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is GLP compliant and follow the OECD 422 guideline. Acceptable with no restrictions.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Cross-reference
Reason / purpose:
read-across: supporting information

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Clear colorless liquid
Details on test material:
- Name of test material: Nonene, branched
- CAS No.:97280-95-0
- Substance type: UVCB
- Physical state: Clear colorless liquid
- Analytical purity: 100%
- Lot/batch No.: TQ-12-2003-23042013
- Data received: 29 April 2013
- Label: Nonene Sampling Date Apr 22.2013
- Expiration date of the lot/batch: 23 April2014
Specific details on test material used for the study:
- Name of test material: Nonene, branched
- CAS No.:97280-95-0
- Substance type: UVCB
- Physical state: Clear colorless liquid
- Analytical purity: 100%
- Lot/batch No.: TQ-12-2003-23042013
- Data received: 29 April 2013
- Label: Nonene Sampling Date Apr 22.2013
- Expiration date of the lot/batch: 23 April2014

Test animals

Species:
rat
Strain:
other: Wistar Han™:RccHan™:WIST strain rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: 12 weeks old
- Weight at study initiation: (P) Males: 311 to 375 g; Females: 194 to 222 g.
- Housing: Pre-mating: groups of four animals were housed in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). Pairing phase: polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Post mating: the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.)
- Water (e.g. ad libitum): free access to food and water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/-2°C
- Humidity (%): 55 =/- 15%
- Air changes (per hr): at least 15 air changes per hour. The low intensity fluorescent lighting was controlled to give twelve hours continuous light
- Photoperiod (hrs dark / hrs light): 12 hr dark and 12 hr light

The study was performed between 10 June 2013 and 10 June 2014. The in-life phase of the study was conducted between 22 August 2013 (first day of treatment) and 17 October 2013 (final day of necropsy).



Administration / exposure

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

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Arachis oil BP
- Storage temperature of food: 4 C

VEHICLE
- Amount of vehicle (if gavage): 4 ml/kg
- Concentration in vehicle:
- Control: 0 ml/kg
- Low: 25 mg/ml
- Intermediate: 75 mg/ml
- High: 250 mg/ml
- Lot/batch no. (if required): 2018C-020713MA
- The volume of the test and control item administrated to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation:15 days
- Proof of pregnancy referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually during gestation and lactation in solid floor polypropylene cages.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
6 weeks
Frequency of treatment:
The test item was administrated daily by gavage using a stainless cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 ml/kg of Arachis oil BP.
Details on study schedule:
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii. At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43.
ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:

Basis: actual ingested 100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:

Basis: actual ingested 300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:

Basis: actual ingested 1000 mg/kg bw/day
No. of animals per sex per dose:
Three groups, each of twelve males and twelve females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose selected during the Range-finding experiment - Study Number 41301414

Examinations

Parental animals: Observations and examinations:
Clinical Observations:
Signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one hour after dosing throughout the treatment period.

Functional Observations:
All animals were observed for signs of functional/behavioural toxicity prior to the start of treatment and at weekly intervals thereafter.
Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
- behavioural assessment
- functional Performance Tests
- Sensory Reactivity

Body Weight:
Individual body weights: Day 1 (prior to dosing), weekly for surviving males until termination and weekly for females until mating was evident.
Female: Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 post partum and terminal kill.

Food Consumption:
Pre-pairing period and after (male): weekly
Females showing evidence of mating: post coitum Days 0-7, 7-14 and 14-20.
Females with live litters: Days 1 and 4 post partum.

Food efficiency:
Males (throughout the study period-with the exception of the mating phase, and females during the pre-pairing phase

Water Consumption:
Daily- visual inspection of the water bottles

Reproductive Performance:
Presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina.
A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded.
The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).

Pregnancy and Parturition:
Pregnant females: observations at approximately 0830, 1230 and 1630 hours and around the period of expected parturition (08.30 and 12.30 hr at weekends and public holidays)

Female data collections:

i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Laboratory Investigations:
- Haematology
- Blood Chemistry
Litter observations:
Number of live and dead offspring
For each litter the following was recorded:
i. Number of offspring born
ii.Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

All live offspring were assessed for surface righting reflex on Day 1 post partum.
Postmortem examinations (parental animals):
Pathology Females: Uterus (for signs of implantation and the number of uterine implantations in each horn), the corpora lutea. All adult animals and offspring: full external and internal examination. All macroscopic abnormalities were recorded.
- Organ Weights- Histopathology: Tissues from 5 selected male and female animals/group were preserved. Histopathological examination was undertaken on tissues from control and high dost animals.
To clarify possible treatment-related changes, histopathological examination was extended to include similarly prepared sections of the liver, kidneys (males only), stomach, thyroid and pituitary from animals in the low and intermediate groups. In addition, male kidney tissue was subject to immunohistochemical examination to confirm the presence of alph-2-microglobulin.
Postmortem examinations (offspring):
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05.
Where appropriate, data transformations were performed using the most suitable method: Bartlett’s test, ANOVA, or if required, ANCOVA, Williams Test, Shirley Test, Dunnett’s (parametric) or Steel (non-parametric) test, Student t-test (parametric) or the MannWhitney U test (non-parametric).
Reproductive indices:
- Pre-coital Interval
- Fertility Indices
- Gestation length
- Parturition Index

Offspring viability indices:
- Implantation Losses (%)
- Live Birth and Viability Indices
- Sex Ratio (% males)

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 and 300 mg/kg bw/day: Increasing salivation in males and females. No effects in animals of either sex treated with 100 mg/kg bw/day.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day (males): statistically significant reduction in weeks 1 and 3.
300 mg/kg bw/day (female): statistically significant increase in cumulative body weight gain during the final week of gestation. This is not considered an adverse effect.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day (males): statistically significant reduction in weeks 1 and 3.
300 mg/kg bw/day (female): statistically significant increase in cumulative body weight gain during the final week of gestation. This is not considered an adverse effect.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: Nine males had enlarged kidneys, eight of which had pale kidneys; four showed a mottled appearance to the kidneys and five of these males also had a dark liver at necropsy.
300 mg/kg bw/day: one male had enlarged kidneys.
100 mg/kg bw: no findings

Females of any dose levels: no findings

100 mg/kg bw/day: one make had small testes and epididymides.
300 mg/kg bw/day: one female had reddened lungs
100 mg/kg bw/day: two female had reddened lungs

Histopathological examination of these tissues did reveal associated microscopic findings to the macroscopic abnormalities however they were considered to be of no toxicological importance.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver: hepatocyte hypertrophy was evident in animals of either sex treated with 1000 and 300 mg/kg bw/day and in males treated with 100 mg/kg bw/day. No such effects were detected in females treated with 100 mg/kg bw/day.

Kidneys: proximal tubular basophilia and hyaline droplets were evident in males from all treatment groups. Tubular degeneration/debris was evident in males treated with 1000 and 300 mg/kg bw/day.
These findings were demonstrated by immunohistochemical staining to be due to alpha-2-microglobulin nephropathy. No such effects were detected in any treated female.

Stomach: epithelial hyperplasia was evident in the fore stomach of animals of either sex treated with 1000 mg/kg bw/day and in males treated with 300 mg/kg bw/day. No such effects weredetected in females treated with 300 mg/kg bw/day or animals of either sex treated with 100 mg/kg bw/day.

Thyroid: follicular cell hypertrophy and hyperplasia was evident in animals of either sex treated with 1000 and 300 mg/kg bw/day and in males treated with 100 mg/kg bw/day. No such effects were detected in females treated with 100 mg/kg bw/day.

Pituitary: increased incidence and severity of hypertrophic/vacuolated cells in pars anterior was evident in males treated with 1000 and 300 mg/kg bw/day. No such effects were detected in any treated female or in males treated with 100 mg/kg bw/day.
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No unscheduled deaths
1000 and 300 mg/kg bw/day: Increasing salivation in males and females. No effects in animals of either sex treated with 100 mg/kg bw/day.

FUNCTIONALE OBSERVATIONS: No treatment-related effects

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
1000 mg/kg bw/day (males): statistically significant reduction in weeks 1 and 3.
300 mg/kg bw/day (female): statistically significant increase in cumulative body weight gain during the final week of gestation. This is not considered an adverse effect.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
No adverse effect on food consumption or food efficiency.

WATER CONSUMPTION (PARENTAL ANIMALS)
1000 mg/kg bw/day: marked increase in water consumption in males
300 or 100 mg/kg: slight increase in males during the treatment period.
Females from all treatment groups: increased water consumption throughout the treatment period with statistical significance being achieved throughout gestation and lactation at 1000 mg/kg bw/day.

REPRODUCTIVE FUNCTION: MATING (PARENTAL ANIMALS)
No treatment-related effects on mating performance.

REPRODUCTIVE FUNCTION: FERTILITY (PARENTAL ANIMALS)
No treatment-related effects on fertility performance.

REPRODUCTIVE PERFORMANCE: GESTATION LENGHT (PARENTAL ANIMALS)
No differences in gestation length.

BLOOD CHEMISTRY: No toxicologically significant effects.

NECROPSY
1000 mg/kg bw/day: Nine males had enlarged kidneys, eight of which had pale kidneys; four showed a mottled appearance to the kidneys and five of these males also had a dark liver at necropsy.
300 mg/kg bw/day: one male had enlarged kidneys.
100 mg/kg bw: no findings

Females of any dose levels: no findings

100 mg/kg bw/day: one make had small testes and epididymides.
300 mg/kg bw/day: one female had reddened lungs
100 mg/kg bw/day: two female had reddened lungs

Histopathological examination of these tissues did reveal associated microscopic findings to the macroscopic abnormalities however they were considered to be of no toxicological importance.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Increase in absolute and relative liver weight: males and females treated with 1000 and 300 mg/kg bw/day and females treated with 100 mg/kg bw/day
Increase in kidney weight both absolute and relative to terminal body weight: Males treated with 1000 mg/kg bw/day
Increase in absolute and relative thyroid weight: males from all treatment groups
Reduction in absolute and relative thyroid weight: females treated with 1000 mg/kg bw/day
The microscopic thyroid changes observed in thyroid weights cannot be excluded as an effect of treatment.
Reduction in pituitary weight and an increase in kidney weight both absolute and relative to terminal body weight: females treated with 1000 mg/kg bw/day. Fot the changes detected in the kidneys or pituitary of females the intergroup differences were considered not to be of toxicological importance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Liver: hepatocyte hypertrophy was evident in animals of either sex treated with 1000 and 300 mg/kg bw/day and in males treated with 100 mg/kg bw/day. No such effects were detected in females treated with 100 mg/kg bw/day.

Kidneys: proximal tubular basophilia and hyaline droplets were evident in males from all treatment groups. Tubular degeneration/debris was evident in males treated with 1000 and 300 mg/kg bw/day.
These findings were demonstrated by immunohistochemical staining to be due to alpha-2-microglobulin nephropathy. No such effects were detected in any treated female.

Stomach: epithelial hyperplasia was evident in the fore stomach of animals of either sex treated with 1000 mg/kg bw/day and in males treated with 300 mg/kg bw/day. No such effects weredetected in females treated with 300 mg/kg bw/day or animals of either sex treated with 100 mg/kg bw/day.

Thyroid: follicular cell hypertrophy and hyperplasia was evident in animals of either sex treated with 1000 and 300 mg/kg bw/day and in males treated with 100 mg/kg bw/day. No such effects were detected in females treated with 100 mg/kg bw/day.

Pituitary: increased incidence and severity of hypertrophic/vacuolated cells in pars anterior was evident in males treated with 1000 and 300 mg/kg bw/day. No such effects were detected in any treated female or in males treated with 100 mg/kg bw/day.

Effect levels (P0)

open allclose all
Dose descriptor:
LOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Based on hepatocellular and thyroid follicular cell hypertrophy and kidney effects (mediated by alpha-2-microglobulin).
Dose descriptor:
LOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on hepatocellular and thyroid follicular cell hypertrophy.
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: Based on adaptive findings in liver, thyroid and pituitary – not considered toxicologically significant. Also male rat specific kidney findings that are not relevant for human health
Dose descriptor:
NOEL
Remarks:
(reproductive toxicity)
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on post-natal effects on offspring viability, litter size and weight at 1000 mg/kg/day

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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING): No toxicological significant effects detected. Offspring viability on Day 4 post partum was statistically significantly reduced in these litters. A total litter loss was also observed for one female treated with 1000 mg/kg bw/day. No such effects were
detected in litters from females treated with 300 or 100 mg/kg bw/day.

CLINICAL SIGNS (OFFSPRING): No toxicological significant effects detected. The incidental clinical signs detected in the control and treated groups (small size, cold, weak, pale, no milk in stomach, physical injury, found dead or missing) were considered unrelated to test item toxicity.

PATHOLOGY: No treatment-related macroscopic abnormalities were detected.


Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced post natal offspring viability, offspring body weight gain and litter size at 1000 mg/kg bw/day.

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
not specified
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Oral administration of Nonene, branched to rats for 8 weeks, at levels of 100, 300 and 1000 mgk/kg/day, resulted in treatment related effects in both sexes and at all treatment levels. The LOEL for both males and females was 100 mg/kg/day. The effects seen were however either adaptive in nature or were not considered toxicologically relevant for man. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 300 mg/kg bw/day based on reduced offspring viability, offspring body weight gain, litter size and litter weigh at 1000 mg/kg bw/day.
Executive summary:

The test material Nonene, branched with CAS no. 97280-95-0 was administrated by gavage, at dose levels of 100, 300, 1000 mg/Kg bw day for 8 weeks (pre-pairing, gestation and early lactation for female). Treatment related effects were reported in animals of either sex from all treatment groups, therefore a No Observed Effect Level (NOEL) was not established. The LOEL was therefore 100 mg/kg/day.

The results showed stomach changes in animals treated with 1000 mg/kg bw/day and the reduction in body weight gain in males. These effects are considered to be a result of local irritation of the test item rather than a true effect of systemic toxicity. The organ weight changes detected in all female treated groups and microscopic liver and thyroid changes in females at 1000 and 300 mg/kg bw/day were considered to be an adaptive response to treatment, of no toxicological significance. The No Observed Adverse Effect Level (NOAEL) can be therefore be established at 1000 mg/kg bw/day for females.

The organ weight changes in all male treatment groups and the microscopic liver, thyroid and pituitary changes in treated males were also considered to be an adaptive response to treatment. The male kidney findings were considered to be associated with alpha-2-microglobulin mediated male rat nephropathy, a species and sex specific effect which is not relevant for human health. Therefore, a No Observed Adverse Effect Level (NOAEL) can be established at 1000 mg/kg bw/day for males.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 300 mg/kg bw/day due to the reduced post natal offspring viability, offspring body weight gain and litter size at 1000 mg/kg bw/day.