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Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproduction/developmental toxicity screening test (OECD TG 421, GLP, K):

NOAEL Fertility and development (foetal growth until day 4) = 1000 mg/kg bw/day (no adverse effects observed at the highest dose tested).

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 16, 2011 to March 01, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 421 guideline study complying with GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP (inspection date 19-21 July 2011)
Limit test:
no
Specific details on test material used for the study:
- Physical state: clear colourless liquid
- Storage condition of test material: approximately +4ºC in the dark over nitrogen
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS: Wistar Han™:RccHan™:WIST strain rats
- Source: Wistar Han™:RccHan™:WIST strain rats
- Age at study initiation: (P) x 12 wks
- Weight at study initiation: (P) Males: 319-360 g; Females: 186-231 g
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages.
- Diet (e.g. ad libitum): certified pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) ad libitum.
- Water (e.g. ad libitum): main drinking water ad libitum
The diet
and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for mated females during gestation and lactation.
- Acclimation period: eight days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2011-08-31 To: 2011-10-24
Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
BP
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.

VEHICLE
- Concentration in vehicle: 0, 7.5, 75 or 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: a maximum of fourteen days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least twenty one days. Formulations were therefore prepared at least fortnightly and stored at approximately +4ºC in the dark.
Samples of each test item formulation were taken and analysed for concentration of the registered substance (EC 456-350-3) at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The concentration of the registered substance (EC 456-350-3) in the test item formulations was determined by gas chromatography (GC) using an external standard technique. The results indicate that the prepared formulations were within acceptable ranges for the purpose of this study (± 15% of nominal concentration).
Duration of treatment / exposure:
Males: during the two week pre-pairing phase, the pairing, up to Day 43.
Females: during the two week pre-pairing phase, pairing, gestation and early lactation, up to Day 5 post partum.
Frequency of treatment:
once daily
Details on study schedule:
Not applicable
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of previous toxicity work (SafePharm Laboratories Limited, Project Number 0161-0494). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item.
Positive control:
none
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, soon after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable).

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION and FOOD EFFICIENCY: Yes
During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period.
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-mating phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated during gestation and lactation

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: was observed daily by visual inspection of water bottles for any overt changes.
Oestrous cyclicity (parental animals):
The stage of oestrus was recorded daily using a vaginal smear.
Sperm parameters (parental animals):
Parameters examined in P male: testis weight, epididymis weight
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Number of offspring born, number of offspring alive recorded daily and reported on Days 1 and 4 post partum, sex of offspring on Days 1 and 4 post partum, clinical condition of offspring from birth to Day 5 post partum, individual offspring weights on Days 1 and 4 (or Day 5) post partum (litter weights were calculated retrospectively from this data).
All live offspring were assessed for surface righting reflex on Day 1 post partum.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on Day 43
- Maternal animals: All surviving animals at Day 5 post partum

GROSS NECROPSY
- Gross necropsy consisted a full external and internal examination, and any macroscopic abnormalities were recorded.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution

HISTOPATHOLOGY
The following tissues were prepared for microscopic examination: Coagulating gland, Prostate, Epididymides, Seminal vesicles, Ovaries, Testes,
Mammary gland (females only), Uterus/Cervix, Pituitary, Vagina.
The tissues from control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Haematoxylin and Eosin for subsequent microscopic examination. The testes and epididymides from the male which failed to mate were also processed. In addition, sections of testes and epididymides from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

ORGAN WEIGHTS
The epididymides and testes were removed from terminal kill adult males dissected free from fat and weighed before fixation.

COLLECTION OF BLOOD PLASMA
From the blood retained during the exsanguination procedure, plasma was extracted and stored at approximately -20°C.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 5 days of age.
- These animals were subjected were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
The following parameters were subjected to statistical analysis: Body weight and body weight change, Food consumption for females during gestation and lactation, Pre-coital interval and gestation length, Litter size and litter weights, Sex ratio, Corpora lutea and implantation sites, Implantation losses and viability indices, Offspring body weight and body weight change, Offspring surface righting, Adult absolute and body weight-relative organ weights (Males).
Data for males and females prior to pairing, where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Bartletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed nonhomogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Data for females during gestation and lactation, and offspring data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney U test.
Non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
Reproductive indices:
Mating Performance and Fertility
-Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
- Fertility Indices:
Mating Index (%) = Number of animals mated / Number of animals paired x 100
Pregnancy Index (%) = Number of pregnant females / Number of animals mated x 100

Gestation and Parturition Data
- Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
- Parturition Index (%) = Number of females delivering live offspring / Number of pregnant females x 100
Offspring viability indices:
Implantation Losses (%)
- % pre–implantation loss = (Number of corpora lutea - number of implantation sites) / Number of Corpora Lutea x100
- % post–implantation loss = (Number of implantation sites - Total number of offspring born) / Number of implantation sites x 100
Live Birth and Viability Indices
- Live Birth Index (%) = Number of offspring alive on Day 1 / Number of offspring born x 100
Viability Index (%) = Number of offspring alive on Day 4 / Number of offspring alive on Day 1 x 100
Sex Ratio (% males) = Number of male offspring / Total number of offspring x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs were confined to the presence of post-dose increased salivation for animals of either sex treated with 1000 mg/kg bw/day. Incidences of increased salivation were also evident for two males treated with 300 mg/kg bw/day. This finding is commonly observed following the oral administration of a slightly unpalatable or slightly irritant test item formulation. In the absence of any supporting data to suggest irritancy, this isolated finding was considered not to represent an adverse health effect. No clinical observations were evident at 30 mg/kg bw/day.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the treatment period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No adverse effects on body weight change were detected for males throughout the treatment period or for females during the pre-pairing, gestation or lactation phases of the study. Statistical analysis of the data did not reveal any significant intergroup differences.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effects on dietary intake were detected for males throughout the treatment period or for females during the pre-pairing, gestation or lactation phases of the study. Statistical analysis of the gestation and lactation data for females did not reveal any significant intergroup differences.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences in water intake.
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
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examinations of control and high dose animals did not reveal any treatment-related effects.
Histopathological examination of the male and female pair which failed to produce a pregnancy revealed tubular degeneration of the testes and azoospermia of the epididymides for the male. This was considered to be the contributing factor to the failure of mating and pregnancy in this pair. This was however considered not to be related to treatment with the test item.
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
- MATING: No adverse effects were detected in mating performance for treated animals when compared to controls.
With the exception of one male: female pair from the 300 mg/kg bw/day dose group which failed to show positive evidence of mating, the majority of animals mated within the first five days of pairing. Statistical analysis of the pre-coital interval data did not reveal any significant intergroup differences.
The 300 mg/kg bw/day male and females pairing which did not show any positive evidence of mating, also did not produce a pregnancy. This was an isolated finding occasionally observed in reproductive studies of this type, and considered to be unrelated to treatment with the test item.
- FERTILITY: No treatment-related effects on fertility were detected. All females which showed positive evidence of mating, resulted in a successful pregnancy.
- GESTATION LENGHT: No treatment-related effects were detected in the length of gestation between control and treated groups. The majority of females showed a gestation length of 23 ½ days. Statistical analysis of the gestation length data did not reveal any significant intergroup
differences.
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Minor clinical signs of post-dose increased salivation were observed at 1000 mg/kg bw/d. In isolation, this finding was considered not to represent an adverse health effect
Key result
Dose descriptor:
NOEL
Remarks:
Reproductive toxicity
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects were detected
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinically observable signs of toxicity observed in offspring. Clinical signs consisted of small, cold, weak, pale, physical injury, missing (completely cannibalised) or found dead for isolated offspring within litters from the control and treated groups. These are low incidence findings in reproductive studies of this type are unrelated to test item toxicity.
Mortality / viability:
no mortality observed
Description (incidence and severity):
No significant differences in sex ratio or litter size were evident for offspring from treated litters when compared to those from the controls. No significant differences in the number or corpora lutea or implantation sites were noted for treated females when compared to controls. The percentages of pre- and post-implantation loss for treated females were comparable to controls. Statistical analysis of the data did not reveal any significant intergroup differences.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences in litter weights or mean offspring weights were evident in the treated groups when compared to controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Post-mortem examinations did not reveal any treatment-related findings in offspring from treated litters. For the two interim death offspring, no milk was present in the stomach. This is commonly observed in offspring found dead soon after parturition and is unrelated to treatment. At terminal kill, one male offspring from a 300 mg/kg bw/day litter showed a reddened left testis. This was an isolated finding and considered to be unrelated to treatment. One litter from the 1000 mg/kg bw/day consisted of two small offspring. Another litter showed one small male. These are low incidence findings in reproductive studies of this type and are considered to be unrelated to treatment with the test item.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Description (incidence and severity):
Surface righting assessments did not reveal any significant differences between litters from treated groups when compared to controls.
Developmental immunotoxicity:
not examined
Statistical analysis of the data did not reveal any significant intergroup differences.
Key result
Dose descriptor:
NOEL
Remarks:
Development
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects were detected
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

None

Conclusions:
The NOAEL for systemic toxicity was considered to be 1000 mg/kg bw/day, i.e. the dose-level resulting in minor and not adverse clinical signs.
Based on the absence of treatment-related effects, the NOEL for reproductive toxicity was considered to be 1000 mg/kg bw/day.
Executive summary:

A reproduction/developmental toxicity screening test was conducted with the test material according to the OECD test guideline No 421 and in compliance with GLP.

The test material was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 30, 300 and 1000 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post partum. Any females which failed to mate or did not produce a pregnancy were terminated following the fourteen day pairing phase of the study. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

There were no unscheduled deaths. No clinical signs of toxicity, no adverse effects on body weight change, and no adverse effects on dietary intake were detected. Daily visual inspection of water bottles did not reveal any overt intergroup differences in water intake.

No treatment-related effects were detected in mating performance, on fertility and in the length of gestation between control and treated groups.

No differences in litter size, sex ratio or viability parameters were detected for litters from treated animals when compared to those from controls.

No significant differences in litter weights were evident in the treated groups when compared to controls. There were no clinically observable signs of toxicity observed in offspring.

No treatment-related macroscopic abnormalities were detected for adults or offspring.

No treatment-related changes were detected for testes and epididymis weights for treated males when compared to controls.

No treatment-related histopathology effects were detected.

The oral administration of the test material to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day, resulted in minor clinical signs which were considered to be unrelated to test item toxicity. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.

No treatment-related effects were detected for the reproductive parameters detected, including offspring development. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Based on this study, the test material not classified for developmental or reproductive toxicity according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is acceptable and satisfies the requirement for a reproduction/developmental toxicity screening test in rats.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Only one study available, GLP-compliant and of high quality (Klimisch score = 1)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A key study was identified (Harlan, 2012, Rel.1). In this reproduction/developmental toxicity screening test conducted in accordance with OECD test guideline No 421 and in compliance with GLP, rats were administered the test material at dose levels of 0, 30, 300 and 1000 mg/kg bw/day for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females).

There were no unscheduled deaths. No clinical signs of toxicity, no adverse effects on body weight change, and no adverse effects on dietary intake were detected.Daily visual inspection of water bottles did not reveal any overt intergroup differences in water intake.

No treatment-related effects were detected in mating performance, on fertility and in the length of gestation between control and treated groups.

No differences in litter size, sex ratio or viability parameters were detected for litters from treated animals when compared to those from controls.

No significant differences in litter weights were evident in the treated groups when compared to controls. There were no clinically observable signs of toxicity observed in offspring.

No treatment-related macroscopic abnormalities were detected for adults or offspring.

No treatment-related changes were detected for testes and epididymis weights for treated males when compared to controls.

No treatment-related histopathology effects were detected.

 

The oral administration of the test material to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day, resulted in minor clinical signs of post-dose increasivation which were considered to be unrelated to test item toxicity. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.

No treatment-related effects were detected for the reproductive parameters detected, including offspring development. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

Since no alerts for developmental toxicity were observed in the screening study, no additional study is proposed at this tonnage level.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Only one study available, GLP-compliant and of high quality (Klimisch score = 1)
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

There are no developmental toxicity / teratogenicity studies on the test material. The reproductive/development toxicity screening test described above showed no-observed-effect-level for offspring developmental toxicity at 1000 mg/kg bw/day, the highest concentration level tested.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data on the source substance, no additional classification is proposed for the target substance according to the Regulation (EC) No 1272/2008 and to the GHS based on the absence of adverse effects observed during the OECD 421 screening study.

Additional information

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