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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
4 August 2004 to 15 February 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

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)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Physical state: Brown slightly viscous liquid
- Storage conditions: Room temperature in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: approximately 8 weeks old
- Weight at study initiation: 201 - 266g (males); 177 - 238g (females)
- Housing: initially in groups of 4, during the mating phase animals were housed in pairs (one of each sex), following mating females where housed individually and males in groups. Cages were polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. Mated females were housed in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes (Datesand Ltd. Cheshire, UK).
- Diet (e.g. ad libitum): pelleted diet (Rodent PMI 5002 (Certified) diet, BCM IPS Limited, London, UK), ad libitum
- Acclimation period: Up to 16 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The appropriate concentrations were prepared in the vehicle weekly and stored at approximately 4 ºC in the dark
- The test material was administered daily by gavage using a plastic catheter attached to a disposable plastic syringe
- Control animals were treated in an identical manner with 4 mL/kg/day of the vehicle
- The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at regular intervals
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 the oestrous cycle 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 and the males were subsequently returned to their original holding cages. 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:
Samples from each preparation were analysed using gas chromatography to verify the concentration, stability and homogeneity of each formulation.

Method:
- Gas chromatography.
- Samples and standards were prepared in methanol to give a final concentration of 0.1 mg/mL
- Samples were analysed within two days of preparation
- The method was validated using a range of standard solutions covering the range 0 to 0.1527 mg/mL. The results of which have been considered to be sufficiently accurate for the purpose of this study

Conditions:
- GC system: Agilent Technologies 5890, incorporating autosampler and workstation
- Column: DB-1 (30 m x 0.32 mm id x 0.25 µm film)
- Oven temperature program: initial 50 ºC for 0 mins, rate 5 ºC/min, final 200 ºC for 0 mins.
- Injection temperature: 150 ºC
- Flame ionisation detector temperature: 250 ºC
- Injection volume: 1 µL
- Retention time: ~14.7 mins.

Results:
- The results indicate that the prepared formulations were within ± 9 % of the nominal concentration
- Analytical verification of the stability and homogeneity of the test preparations was also performed and the formulation was found to be stable for at least fourteen days
Duration of treatment / exposure:
54 days
Frequency of treatment:
Daily
Details on study schedule:
Study Sequence:
- Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable).
- Prior to the start of treatment and once weekly, all animals were observed for signs of functional/behavioural toxicity.
- One day prior to pairing (Day 14), blood samples were taken from five males and five females, randomly selected from each dose group and analysed for haematological and blood chemical assessment.
- On Day 15, all animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
- Following evidence of mating, the males were returned to their original cages and females were transferred to individual cages.
- On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
- Pregnant females were allowed to give birth and maintain their offspring until Day 4 post partum. Evaluation of each litter size, litter weight, mean pup weight, clinical observations and landmark developmental signs were also performed during this period.
- At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
- Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically.
- At Day 5 post partum, all surviving females and offspring were killed and examined macroscopically.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
other: nominal ingested dose ± 9%
No. of animals per sex per dose:
10 males and 10 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
Preliminary Study:
- A fourteen day oral range finding study was conducted to provide information for the selection of dose levels for the definitive study. Animals were dosed and observed for adverse effects resulting from toxicity including; clinical observations, bodyweight and gross pathology.
- Animals were kept under the same conditions as in the definitive study.
- The test material was prepared in the same manner as the definitive study and verified analytically.
- The test material was administered in a limit test daily, for up to 14 consecutive days. Animals were administered 1000 mg/kg/day via oral gavage, a volume of 4mL/kg and a concentration of 250 mg/mL. Control animals were treated in an identical manner with 4 mL/kg/day of polyethylene glycol.
Positive control:
None reported

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before and after dosing, then at 1 and 5 hours after dosing. The 5 hour observation was omitted at weekends, except for females during parturition where applicable.
- Observations recorded: overt signs of toxicity, ill-health and behavioural changes.

BODY WEIGHT: Yes
- Time schedule for examinations: males were weighed on day 0, then weekly until termination. Females were weighed on day 0 and then weekly until evidence of mating was evident. Bodyweights were recorded on day 0, 7, 14 and 20 post coitum, and on day 1 and 4 post partum.

FOOD AND WATER CONSUMPTION: Yes
- Weekly food consumption was recorded for each caged group of adults, which was continued for males after mating. Food consumption for females showing evidence of mating was recorded for the periods covering days 1-7, 7-14 and 14-21. For females with lie litters, consumption was recorded during the lactation period.
- Water consumption was observed daily by visual inspection of water bottles for any overt change.

FUNCTIONAL OBSERVATIONS: YES
- Time schedule: prior to the start of treatment and at weekly intervals.
- Observations recorded: signs of functional behavioural toxicity (except for non-pregnant female number 71). Functional performance tests were also performed on five selected males and females per dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

BEHAVIOURAL ASSESSMENTS: Yes
- Observations recorded: 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.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 14, prior to pairing.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: five males and five females per dosing group.
- Parameters examined on blood collected into tubes containing potassium EDTA anti-coagulant: haemoglobin, erythrocyte count, haematocrit, erythrocyte indices, total leucocyte count, differential leucocyte count, platelet count, prothrombin time and activated thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 14, prior to pairing.
- Animals fasted: No
- How many animals: five males and five females per dosing group.
- Parameters examined on blood collected into tubes containing lithium heparin anti-coagulant: urea, glucose, total protein, albumin, albumin/globulin ratio (by calculation), sodium, pptassium, chloride, calcium, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, Alkaline phosphatise, creatinine, total cholesterol and total bilirubin.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity, forelimb/hind limb grip strength and motor activity

Sensory reactivity: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed: grasp response, vocalisation, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, startle reflex and blink reflex.

Grips strength: An automated grip strength 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.

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).

PREGNANCY AND PARTURITION:
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female: date of mating, date and time of observed start of parturition, date and time of observed completion of parturition and duration of gestation.
Oestrous cyclicity (parental animals):
Not performed.
Sperm parameters (parental animals):
Not performed.
Litter observations:
On completion of parturition, the number of live and dead offspring was recorded.
For each litter the following was recorded: number of pups born, number and sex of pups alive recorded daily and reported on Day 1 and 4 post partum, clinical condition of pups from birth to Day 4 post partum, individual pup and litter weights on Day 1 and 4 post partum.

All live offspring were observed for the detachment of pinna and assessed for reflexological response to stimuli by assessing surface righting reflex on Day 1 post partum.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
- Maternal animals: The interim death female was killed by carbon dioxide asphyxiation followed by cervical dislocation. The remaining surviving adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum.

GROSS NECROPSY
- All adult animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
- In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy. The uterine implantation sites were counted. The procedure was enhanced by staining the uteri with a 1 % ammonium polysulphide solution.

HISTOPATHOLOGY
- Samples of the following tissues were preserved from five males and five females, from each dose group: adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, coagulating gland, colon, duodenum, epididymides, eyes, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), mammary gland, muscle (skeletal), ovaries, pancreas, pituitary, prostate, oesophagus, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical), spleen, stomach, thyroidparathyroid, trachea, testes, thymus, urinary bladder, uterus/cervix and the vagina.

ORGAN WEIGHTS
- The following organs were dissected free from fat and weighed at the end of the study; adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes and the thymus.
Postmortem examinations (offspring):
GROSS NECROPSY
All surviving offspring were terminated, including those dying during the study, via intracardiac overdose of sodium pentobarbitone. They were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate. Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain and quantitative functional performance 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, pair-wise 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.

The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.

Probability values (p) are presented as follows:
p < 0.001 ***
p < 0.01 **
p < 0.05 *
p 20.05 (not significant)
Reproductive indices:
MATING PERFORMANCE AND FERTILITY

The following parameters were calculated from the individual data during the mating period of the parental generation.

-Pre-coital interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

-Fertility Indices, for each group the following were calculated:
Mating Index (%) = [No. of animal mated/No. of animals paired] x 100
Pregnancy Index (%) = [No. of pregnant females/ No. of animals mated] x 100

GESTATION AND PARTURATION DATA

The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.

-Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition. Where the start of parturition occurred overnight, the total was adjusted by subtracting half a day.

-Parturition Index, the following was calculated for each group:
Parturition Index (%) = [No. of females delivering live offspring/No. of pregnant females] x 100
Offspring viability indices:
LACTATION DATA

-Live birth and viability indices, the following indices were calculated for each group form individual data:
Live Birth Index (%) = [No. of offspring alive on day 1/No. of offspring born] x 100
Viability Index (%) = [No. of offspring alive on day 4/No.of offspring alive on day 1] x 100

-Sex ratio (% males), group mean values calculated from each litter value on day 1 and 4 using the following formula:
Sex ratio = [No. of male offspring/No. of offspring of determined sex] x 100

-Implantation losses (%), group mean percentile pre-implantation and post implantation loss were calculated as follows:
% pre-implantation loss = [(No. of Corpora Lutea – No. of implantation sites)/No. of corpora lutea] x 100
% post-implantation loss = [(Group no. of implantation sites – No. of offspring)/No. of implantation sites] x 100

Results and discussion

Results: P0 (first parental animals)

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
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

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)

MORTALITY:

No treatment related deaths were observed during the course of the study. One female treated with 1000 mg/kg/day was killed in extremis during parturition. This was attributed to dystocia and of no toxicological significance.

The interim deaths of offspring from the control, 100 and 300 mg/kg/day treatment groups during the lactation phase of the study are normal low incidence findings and considered unrelated to treatment.

CLINICAL OBSERVATIONS:

Increased salivation was detected prior to dosing and up to one hour after dosing fix animals of either sex treated with 1000 mg/kg/day from Day 3 onwards. An isolated incident of increased salivation was also detected up to one hour after dosing for one female treated with 300mg/kg/day on Day 17 only. This observation is often recorded following the oral administration of an unpalatable or irritant test material. Isolated incidents of noisy respiration were also observed at 1000 mg/kg/day.

No such effects were detected at 100 mg/kg/day. Incidents of scab formation, generalised fur loss and staining of the external body surface were observed throughout the treatment and control groups. These are occasionally seen in laboratory maintained rodents and are considered to be incidental and not indicative of toxicity.

Isolated incidents of noisy respiration were detected for one male and one female treated with 1000 mg/kg/day during the Week 1 assessments. One 1000 mg/kg/day male also showed noisy respiration during Week 6.

BODYWEIGHT:

There were no adverse effects on bodyweights for males throughout the study. Bodyweight gains for treated females throughout the maturation, gestation and lactation phases of the study were comparable to controls.

FOOD COMSUMPTION:

Food consumption: no adverse effects were observed on dietary intake and food efficiency during the course of the study.
The statistically significant reductions in dietary intake detected for all male treatment groups during the final week of treatment were minimal (p<0.05) and attributable to a slightly higher dietary intake for the controls and considered unrelated to treatment.

REPRODUCTIVE PERFORMANCE:

No adverse effects were detected on mating performance or fertility.

One female treated with 1000 mg/kg/day showed positive evidence of mating after one day of pairing, however, this animal failed to achieve pregnancy. This is a normal low incidence finding on reproductive studies of this type and in isolation, considered unrelated to treatment.

A reduced parturition index was evident at 1000 mg/kg/day, but this was attributed to the termination of one female due to dystocia.

ORGAN WEIGHTS:

No toxicologically significant effects of treatment were detected.

Elevated liver weights, relative to terminal bodyweights were detected for animals (of either sex treated with 1000 mg/kg/day, with statistical significance achieved for males (p<0.01). Elevated adrenal weights, both absolute and relative to terminal bodyweight was detected for females treated with 1000 mg/kg/day. In the absence of histopathological correlates, these effects were most probably attributed to xenobiotic induction and of no toxicological importance.

The statistically significant reduction in absolute adrenal and heart weights detected for males treated with 1000 mg/kg/day were not reflected in the relative weights and therefore considered unrelated to treatment.

The statistically significant increase in relative liver weight detected for 100 mg/kg/day males was minimal (p<0.05) and in the absence of a convincing dose related response, was considered incidental and unrelated to treatment.

NECROPSY/GROSS PATHOLOGY:

No treatment-related macroscopic abnormalities were detected for offspring at terminal kill. The macroscopic findings observed for interim and terminal kill offspring throughout the dose groups, were consistent with normally expected low incidence findings in pups of the age examined and were of no toxicological importance.

The interim death pregnant female treated with 1000 mg/kg/day showed four placentae and four dead foetuses in the right uterine horn. The left uterine horn was blood filled. This animal was terminated due to difficulties in birthing. Dystocia is a common low incidence finding in reproductive studies of this type and in isolation, this death was considered unrelated to test material toxicity.

No treatment-related macroscopic abnormalities were detected for adult animals at terminal kill.

One male showed dorsal scab formation at termination but this isolated incident was unrelated to treatment.
No treatment-related changes were detected in corpora lutea and implantation site counts. Statistical analysis of the data did not reveal any significant differences.

HISTOPATHOLOGY
The following treatment-related changes were observed:

Kidneys: Globular accumulations of eosinophilic material were observed in the tubular epithelium of males treated at all treatment levels. One control male was similarly affected. There was no indication of associated degenerative tubular changes and no dose relationship. This finding is consistent with the presence of hydrocarbon nephropathy following treatment with petroleum based test materials. This results from the excessive accumulation of alpha2-microglobulin in renal proximal tubular epithelial cells, found only in the proximal tubular epithelium of adult male rats.

Thyroids: A marginal effect was observed in respect of the prevalence and severity of follicular cell hypertrophy in relation to treatment for animals of either sex treated with 1000 mg/kg/day. A similar effect was also seen for males treated with 300 mg/kg/day, extending into the 100 mg/kg/day dose group. Although not convincing due to similar findings in the controls, the possibility that treatment with the test material has affected the thyroid follicular epithelium cannot be entirely excluded; this can, however, be generally regarded as an adaptive change.

Stomach: Minimal acanthosis, occasionally with associated hyperkeratosis, was observed in relation to treatment for two male rats and for three females treated with 1000 mg/kg/day, but not at any other treatment level. These are adaptive responses, secondary to oral gavage administration of a highly concentrated epithelial irritant.

All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

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: Concentration was verified to be ± 9 %

Target system / organ toxicity (P0)

Critical effects observed:
no

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:
- There were no treatment-related intergroup differences in litter sizes, sex ratio, live birth indices or post partum viability.

CLINICAL SIGNS:
- No toxicologically significant clinical findings were observed. The clinical observations recorded for offspring throughout the dose groups were consistent with normally expected incidence findings in offspring of the age examined and were of no toxicological significance.

BODY WEIGHT:
- There were no significant intergroup differences in mean litter weights or offspring bodyweights. Surface righting or pinna unfolding assessments for treated offspring were comparable to controls.

GROSS PATHOLOGY:
- No treatment-related macroscopic abnormalities were detected for offspring at terminal kill. The macroscopic findings observed for interim and terminal kill offspring throughout the dose groups, were consistent with normally expected low incidence findings in pups of the age examined and were of no toxicological significance.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
maternal dose
Sex:
male/female
Basis for effect level:
other: Concentration was verified to be ± 9 %

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Results of Preliminary toxicity Study:

- Increased salivation was detected up to ten minutes after dosing for animals of either sex treated with 1000 mg/kg/day from Day 2 onwards. This observation is often recorded following the oral administration of an unpleasant tasting and/or locally irritant test material formulation and in isolation, is not considered to be indicative of systemic toxicity.

- No mortalities were observed or systemic signs of toxicity.

Applicant's summary and conclusion

Conclusions:
There was no evidence of reproductive toxicity in this study. One high dose female failed to achieve pregnancy and another female from this dose group was sacrificed at parturition due to dystocia. These are normal low incidental findings observed on reproductive studies and considered unrelated to treatment with the test material.

No effect of treatment was detected in reproduction, therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive toxicity was considered to be 1000 mg/kg/day.
Executive summary:

In a GLP compliant study the reproductive toxicity of the test material was determined in a repeat dose toxicity study, performed according to the standardised guideline OECD 422.

Sprague-Dawley rats (10 per sex per dose) were given 54 consecutive doses of the test material, receiving 100, 300 or 1000 mg/kg/day. Both the adults and the F1 generation were observed during 54 day study and subjected to necropsy at termination. Examination of reproductive performance offspring litter sizes, sex ratios, reproductive and viability indices show that there is no evidence of reproductive toxicity in this study. One high dose female failed to achieve pregnancy and another female from this dose group was sacrificed at parturition due to dystocia. These are normal low incidental findings observed on reproductive studies and considered unrelated to treatment with the test material. No treatment related effects were detected in reproduction, therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive toxicity was considered to be 1000 mg/kg/day.