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Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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:
The study was performed between 23 November 2011 and 15 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to GLP and guideline
Reason / purpose:
reference to same study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection 2011-06-27 to 2011-07-21; Date of signature 2011-08-15
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK
- Age at study initiation: (P) approximately 12 weeks
- Weight at study initiation: (P) Males: 308-335 g Females: 193-219 g
- Fasting period before study: no
- 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 pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful 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.
- Diet (e.g. ad libitum): (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories UK Ltd, Oxon, UK)
- Water (e.g. ad libitum): free access to mains drinking water supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 7 days during which health status of each animal was assessed

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 ºC
- Humidity: 55 ± 15 %
- Air changes: at least 15 per hour
- Photoperiod: 12 hours continuous light and 12 hours dark via low intensity fluorescent lighting

IN-LIFE DATES: From: 29 December 2011 (first day of treatment) To: 11 February 2012 (final necropsy)
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF TEST ITEM
- Test material 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 twice monthly and stored at +4ºC in the dark.
Details on mating procedure:
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the 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) and the males were subsequently returned to their original holding cages
(unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.






Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of 9-decenoic acid, methyl ester (9DAME) in the test item formulations was determined by gas chromatography (GC) using an external standard technique.

The standard and sample solutions were analysed by GC using the following conditions:
GC system : Agilent Technologies 5890, incorporating autosampler and workstation
Column : ZB-5 (30 m x 0.53 mm id x 5 μm film)
Oven temperature program : initial 150 ºC for 1 mins
rate 10 ºC/min
final 340 ºC for 6 mins
Injection temperature : 300 ºC
Flame ionisation detector temperature: 300 ºC
Injection volume : 1 μl
Retention time : ~ 5.4 mins
Duration of treatment / exposure:
Up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females).
Frequency of treatment:
Test item was administered daily
Remarks:
Doses / Concentrations:
0, 30, 300 and 1000 mg/kg bw/day. Treatment volume (4 ml/kg) corresponding to concentrations of 7.5, 75 and 250 mg/ml, respectively.
Basis:

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 work (Harlan Laboratories Ltd., Project Number 41103600). See IUCLID section 7.5.1 for study summary.
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes 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). All observations were recorded.


FUNCTIONAL OBSERVATIONS:
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. 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 ASSESSMENTS:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: Gait, Tremors, Twitches, Convulsions, Bizarre/Abnormal/Stereotypic behaviour, Salivation, Pilo-erection, Exophthalmia, Lachrymation, Hyper/Hypothermia, Skin colour, Respiration, Palpebral closure, Urination, Defecation, Transfer arousal, Tail elevation.

MOTOR ACTIVITY:
Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The Tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).

FORELIMB/HINDLIMB GRIP STRENGTH
An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).


SENSORY REACTIVITY:
Each tested animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The following parameters were observed: Grasp response, Vocalisation, Toe pinch, Tail pinch, Finger approach, Touch escape, Pupil reflex, Blink reflex, Startle reflex.

BODY WEIGHT:
Individual body weights were recorded 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
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. 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 on Days 1 and 4 post partum.

FOOD EFFICIENCY (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.

WATER CONSUMPTION
Water intake was measured daily throughout the pre-pairing phase of the study

LABORATORY INVESTIGATIONS
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

HAEMATOLOGY
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), Erythrocyte indices, Total leucocyte count (WBC), Differential leucocyte count, Platelet count (PLT), Reticulocyte count (Retic), Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).

BLOOD CHEMISTRY
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: Urea, Glucose, Total protein (Tot.Prot.), Albumin, Albumin/Globulin (A/G) ratio (by calculation), Sodium (Na+), Potassium (K+), Calcium (Ca++), Inorganic phosphorus (P), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (AP), Creatinine (Creat), Total cholesterol (Chol), Chloride (Cl-), Triglycerides, Total bilirubin (Bili), Bile acids.

PATHOLOGY
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.

ORGAN WEIGHTS
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles, Spleen, Testes, Thymus, Thyroid (weighed post-fixation with Parathyroid), Uterus (weighed with Cervix).
Litter observations:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

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):
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.

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 (Salewski 1964).

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.

HISTOPATHOLOGY:
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals, Ovaries, Aorta, (thoracic) Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary Bone & bone marrow (sternum), Prostate, Brain, (including cerebrum, cerebellum and pons), Oesophagus, Caecum, Rectum, Coagulating gland, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides♦, Skin (hind limb), Eyes* Spinal cord (cervical, mid-thoracic and lumbar), Gross lesions, Heart, Spleen,
Ileum (including peyer’s patches), Stomach, Jejunum, Thyroid/parathyroid, Kidneys, Trachea, Liver, Testes♦, Lungs (with bronchi)#, Thymus, Lymph nodes (cervical and mesenteric), Urinary bladder, Mammary gland, Uterus/Cervix, Muscle (skeletal) and Vagina

* =E yes fixed in Davidsons fluid
♦ = preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later
# = = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

All tissues were despatched to the Test Site (Propath UK Ltd., Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). The tissues from five selected control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or 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.






Postmortem examinations (offspring):
All 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. Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight- Relative Organ Weights
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any 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 shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was
performed using the Student t-test (parametric) or the Mann-Whitney U test (nonparametric). Data not analysed by the Provantis data capture system were assessed separately using the SPSS statistical package.

Due to the preponderance of non-normally distributed data, reproductive parameters (implantation losses, offspring sex ratio and offspring surface righting) were analysed using non-parametric analyses.

Probability values (p) were calculated as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p0.05 (not significant)
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
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: estrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
MORTALITY:
There were no treatment related deaths.
One female treated with 300 mg/kg bw/day was killed in extremis on Day 41 due to difficulties encountered during parturition. Macroscopic examination revealed a total of 13 dead foetuses in the uterus and histopathological evaluations revealed incidental lesions which were not also present in females treated with 1000 mg/kg bw/day. Therefore in isolation this interim death was considered not to be related to test item toxicity.

CLINICAL OBSERVATIONS:
There were no toxicologically significant clinical signs detected in treated animals.
Clinical signs were confined to transient instances of increased salivation detected in animals of either sex treated with 1000 mg/kg bw/day.

A swollen ano-genital region, pilo-erection and pallor of the extremities was recorded for the female that was killed in extremis on Day 41 following the start of parturition.

BEHAVIOURAL ASSESSMENTS:
Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls All inter and intra group differences in behavioural scores were considered to be a result of normal variation for rats of the strain and age used, and the differences were of no toxicological importance.

FUNCTIONAL PERFORMANCE TESTS:
There were no treatment related changes in functional performance. Statistical analysis of the data did not reveal any significant intergroup differences.

SENSORY REACTIVITY ASSESSMENTS:
There were no treatment-related changes in sensory reactivity.

BODY WEIGHT:
There were no toxicologically significant effects detected in body weight development. Males treated with 1000 and 300 mg/kg bw/day showed a statistically significant reduction in body weight gain during the third week of treatment whilst females from these treatment groups showed a statistically significant reduction in cumulative body weight gain between Days 1 and 14 of gestation. Recovery was evident thereafter (identified as a statistically significant increase in males at 1000 mg/kg bw/day during Week 4) and in the absence of any significant effect on overall body weight development, the intergroup differences were considered not to be of toxicological significance.

FOOD CONSUMPTION:
There were no treatment related effects detected on mean food consumption or mean food efficiency during this study, when compared to the control. Females treated with 300 mg/kg bw/day showed a statistically significant reduction in food consumption during Days 0 - 7 of gestation. In the absence of a similar effect in 1000 mg/kg bw/day females the intergroup difference was considered not to be of toxicological importance.

WATER CONSUMPTION:
There were no adverse effects detected in water consumption for treated animals, when compared to control animals.

HAEMATOLOGY
There were no toxicologically significant effects detected in the haematological parameters examined. Males treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in erythrocyte count. In the absence of a true dose related response the intergroup difference was considered not to be of toxicological importance.

BLOOD CHEMISTRY:
There were no toxicologically significant effects detected in the blood chemical parameters examined. Females treated with 1000 mg/kg bw/day showed a statistically significant reduction in cholesterol. The majority of individual values were within the normal range for rats of the strain and age used, and in the absence of any histology correlates the intergroup difference was considered not to be of toxicological importance.

ORGAN WEIGHTS:
There were no toxicologically significant effects detected in the organ weights measured.
Males from all treatment groups showed a non-dose related statistical significant reduction in thyroid weight both absolute and relative to terminal body weight. In the absence of a true dose related response or any histology correlates the intergroup difference was considered not to be of toxicological importance.


REPRODUCTIVE PERFORMANCE

MATING:
There were no treatment-related effects on mating performance

FERTILITY:
No treatment-related effects on fertility were detected for treated animals, when compared to controls.
One female treated with 300 and 30 mg/kg bw/day did not achieve pregnancy following evidence of mating. No histopathological correlates were evident in the female reproductive organs. The male partner for the female treated with 30 mg/kg bw/day showed bilateral extensive tubular degeneration/atrophy in the testes. These lesions were considered to be the reason for the non pregnancy, but both findings are naturally occurring background changes in rats and in the absence of a dose-relationship in the incidence or severity it was considered not to be of toxicological significance in this
study. The male partner for the female treated with 300 mg/kg bw/day had unilateral tubular degeneration/atrophy however the relation between this lesion and the infertility could not be determined due to there being no differences in severity compared to the lesion recorded in one control male.


GESTATION LENGTH:
No treatment-related effects were detected in the length of gestation for treated females when compared to controls. All animals showed gestation lengths of 22 to 23½ days.







Dose descriptor:
NOAEL
Remarks:
Sytemic toxicity
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Dose descriptor:
NOEL
Remarks:
Reproductive toxicity
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
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
LITTER RESPONSES
In total ten females from the control and 1000 mg/kg bw/day dose group, nine females from the 30 mg/kg bw/day and eight females from the 300 mg/kg bw/day dose group gave birth to a live litter and successfully reared young to Day 5 age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

OFFSPRING LITTER SIZE, SEX RATION AND VIABILITY
No toxicologically significant effects were detected.
No significant differences were detected for corpora lutea, implantation counts, litter size or litter viability for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

OFFSPRING GROWTH AND DEVELOPMENT
There were no toxicologically significant effects detected.
Statistical analysis of the litter, offspring weights or surface righting reflex data did not reveal any significant intergroup differences

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, found dead or missing, were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

PATHOLOGY
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.



Reproductive effects observed:
not specified
Conclusions:
The 'No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day. The NOAEL for systemic toxicity was also considered to be 1000 mg/kg bw/day.
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
Additional information

A 28 day repeat dose oral toxicity study combined with a reproduction/ developmental toxicity screening test was performed in the rat in accordance with GLP and OECD Guideline 422 (Harlan Laboratories Ltd, 2012). The test item, 9-decenoic acid, methyl ester 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. There were no treatment related effects observed on mating, fertility or gestation length at any dose level. The offspring litter size, sex ratio, viability, growth and development were all comparable to controls and no adverse effects were noted. Since no treatment-related effects were observed for reproduction, a NOEL for reproductive toxicity was considered to be 1000 mg/kg bw/day.


Short description of key information:
9-decenoic acid, methyl ester (9-DAME) showed no signs of reproduction/developmental toxicity in an OECD 422 reproduction/developmental screening toxicity test at doses up to 1000 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
The study has been conducted according to OECD Guideline 422 and to GLP and is adequately reported. As such the study as been assigned a reliability 1.

Effects on developmental toxicity

Description of key information
9DAME is not expected to be a developmental toxicant
Additional information

There are no definitive developmental toxicity studies available on 9-decenoic acid, methyl ester (9-DAME). However, the in vitro genotoxicity studies were all negative and the results of a 28 day repeat dose oral toxicity study combined with a reproduction/ developmental toxicity screening test indicated no reproductive or developmental treatment related effects. Furthermore, acute oral and dermal toxicity studies and skin and eye irritation studies indicate that 9-decenoic acid, methyl ester (9-DAME) is not systemically toxic.

Justification for classification or non-classification

Under the conditions of the OECD TG 422 study, 9-decenoic acid, methyl ester (9-DAME) administered to male and female rats up to a dose level of 1000 mg/kg bw/day for a period of up to 8 weeks is not toxic to reproduction and has no effect on fertility or development. No treatment-related effects were observed for reproduction; hence, a NOEL for reproductive toxicity was considered to be 1000 mg/kg bw/day.

Based on these results, 9-decenoic acid, methyl ester (9-DAME) does not warrant classification according to the criteria described in Regulation (EC) No 1272/2008.