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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

None available.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 September 2013 to 01 July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with OECD, EU & US EPA test guidelines in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
See "Principles of method if other than guideline" below.
Qualifier:
according to guideline
Guideline:
EU Method B.35 (Two-Generation Reproduction Toxicity Test)
Deviations:
yes
Remarks:
See "Principles of method if other than guideline" below.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
yes
Remarks:
See "Principles of method if other than guideline" below.
Principles of method if other than guideline:
List of protocol deviations
1. Temporary Deviations from the daily mean relative humidity occurred.
Evaluation: Laboratory historical data do not indicate an effect of the deviations.
2. On 18 and 20 March and 24 May 2014 clinical observations were not performed online and on 28 October 2014 no food consumption was determined for cage 25 (Group 1 F0 females; Days 8-15 pre-mating). Additionally, no clinical observations were registered on paper for the pups of litter
107 (Group 1) and 125 (Group 2) over several days.
Evaluation: Sufficient data is available for a thorough evaluation.
3. Pups from a few litters (distributed across all treatment groups) were weighed on Day 36 instead of on Day 35. Details are listed in the raw data.
Evaluation: One day difference at this age was only slight and still provides representative information.
4. From litter 98 the organ weights of 2 female pups were determined instead of from one male and one female pup. Inadvertently, for a few litters (distributed across treatment groups) the visceral examination was omitted for individual pups.
Evaluation: Sufficient data for male organ weights were available; weights for both females are reported. An external macroscopic examination was performed, and thus some evaluation was conducted. In both cases, sufficient data were available for a thorough evaluation.
5. Progressive motility was assessed from all sperm samples and the number of corpora lutea were assessed for non-pregnant and non-mated females during the Salewski staining procedure.
Evaluation: This only provides additional information for evaluation and has no negative impact on the study.
6. Inadvertently, no estrous smear was performed for F0 females prior to necropsy and a few organs were not weighed at necropsy and were not available for histopathology. Tissues were not discernible at necropsy or trimming, or were erroneously not collected at necropsy. Missing tissues are listed in raw data and pathology report.
Evaluation: Sufficient data were available for a thorough evaluation.
7. The mean accuracy of the Group 2 samples, measured on 13 March 2014 (Week 22) was 75% instead 80-120%.
Evaluation: Since the deviation <80% was small it was considered to have no impact on the study.

The study integrity was not adversely affected by the deviations.
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test System Rat: Crl:WI(Han) (outbred, SPF-Quality). Females were nulliparous and non-pregnant at initiation of the study.
Rationale: This species and strain of rat has been recognized as appropriate for reproduction studies. WIL Research Europe BV has reproductive historical control data in this species from the same strain and source.
This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Source F0: Charles River Deutschland, Sulzfeld, Germany.
Number of animals: F0-generation: 96 males and 96 females; F1-generation: 970 pups; F2-generation: 1060 pups.
Age at start treatment F0: Approximately 5-6 weeks.
Acclimatization F0: At least 5 days prior to start of treatment.
Health inspection F0: At least upon receipt of the animals.
Randomization F0: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification F0 and F1: Earmark and tattoo.

Selection of F1-generation: Since fertility rates were seldom equal between groups, the following procedure were used for selection of the F1-generation. A minimum of one male and one female per litter were selected when litters attained an age of 21 days. An additional male or female was selected from a litter, if necessary, to obtain 24 males and 24 females for each group. Only pups not expected to survive due to physical limitations were not available for selection.
Identification pups: On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet.

Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 room air changes/hour, and a 12-hour light/12-hour dark cycle (lights are on 07:00-19:00). Any variations to these conditions were recorded in the raw data, were evaluated and were considered to have no effect on the outcome of the study.

Accommodation
Pre-mating: Animals were housed in groups of 4 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 4 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material ((Litalabo, S.P.P.S., Argenteuil, France) or (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United= Kingdom) (until 27 November 2013) were supplied.
Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During the acclimatization period, animals had free access to the same diet (without the test substance) received from the supplier in pelleted form (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water: Free access to tap-water.
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could be considered likely to interfere with the study.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Diet: Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Method: The test substance was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Water (approximately 15% in total) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35°C before storage. The control animals received similarly prepared pellets but without the test substance.
On 27 January 2014, control animals were supplemented with pelleted diet from the supplier instead of pelleted diet prepared in-house.

Frequency of preparation: Diets were prepared at least once every 29 days when kept at room temperature. The same diets were used for the pilot and main rat teratology studies (Projects 503364 and 503365).

Storage conditions: Diets were kept at room temperature in the diet store room in the animal house, or stored at ≤-15°C when not used within 29 days after preparation. Diets were kept in the freezer for a maximum of 18 days.
Details on mating procedure:
Mating procedures: Following a minimum of 70 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
A maximum of 15 days was allowed for mating. If no evidence of copulation was obtained after 10 days, the female was placed with another male (for an additional five days) of the same treatment group who has successfully mated, again avoiding sibling mating.

Parturition: The females was allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.

Culling: To reduce variability among the litters, on Day 4 of lactation or shortly thereafter, eight pups from each litter of equal sex distribution (if possible) were selected. For litters consisting of fewer than eight pups, adjustments for litter size were not performed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analysis of diet preparations
Analyses were conducted on seven occasions during the treatment phase, according to a validated method (Project 503368) as specified below. (Reported in table form – see Any other information).

In addition, random samples from all diet preparations were taken and stored at ≤-15°C for possible future analysis. Any remaining samples were discarded after approval by the sponsor, or at finalization of the study report.
The accuracy of diet preparations was considered acceptable if the mean measured concentrations were 80-120% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Diet preparations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
F0-generation: A minimum of 70 days prior to mating and continuing until euthanasia.
F1-generation (F0-pups): The F1-generation were potentially exposed to the test substance in utero, through nursing during lactation and directly when they begin eating solid food. After weaning, pups were treated for a minimum of 70 days prior to mating and continuing until euthanasia.
F2-generation (F1-pups): The F2-generation were potentially exposed to the test substance in utero and through nursing during lactation.
Frequency of treatment:
Ad libitum.
Details on study schedule:
F0 males and F0 females were exposed to the test substance from 10 weeks prior to mating and exposure was continued until euthanasia (males) or one day before euthanasia (females). F0 females were allowed to produce and rear a litter until Day 21 of lactation. On Day 4 of lactation or shortly thereafter, litters were reduced in size to eight pups by random culling of F1 pups. After weaning, one F1 male and one F1 female of each litter of Groups 1-4 were selected for mating with a pup of another litter of the same dose group to produce a F2 generation. F1 females were allowed to produce and rear a litter until Day 21 of lactation.
Remarks:
Doses / Concentrations:
1500, 5000 and 15000 ppm (approximately equivalent to 81-127, 278-440, 955-1366 mg/kg)
Basis:
nominal in diet
No. of animals per sex per dose:
F0 Generation: 4 groups of 24 males & 24 females
F1 Generation: 4 groups of 24 males & 24 females
Control animals:
yes, plain diet
Details on study design:
Based on the results of a 14-day dose range finding study, the dose levels for this two-generation reproduction toxicity study were 1500, 5000 and 15000 ppm by dietary administration, approximately equivalent to 81-127, 278-440, 955-1366 mg/kg.
Positive control:
Postive control not required for this study.
Parental animals: Observations and examinations:
Mortality / Viability: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstance of any death was recorded in detail.

Clinical signs: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Body weights: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1, 4, 7, 14 and 21.

Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and during lactation on Days 1, 4, 7, 14 and 21.

Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
Oestrous cyclicity (parental animals):
Estrous cycle determination: Daily vaginal lavage were performed to determine the stage of estrous beginning 21 days prior to initiation of the mating period and until evidence of copulation is observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.

General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation was used to aid in confirmation of pregnancy. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Sperm parameters (parental animals):
Sperm samples were taken from the proximal part of the vas deferens (right):
1. Sperm motility and progressive motility were assessed from all samples.
2. Sperm smears for morphological evaluation was fixed from all samples and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal were recorded.
Evaluation was performed for all samples of the control and high dose group.
Litter observations:
Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).

Clinical signs: At least once daily, detailed clinical observations were made for all animals.

Body weights: Live pups were weighed on Days 1, 4 and 7 of lactation and weekly thereafter.

Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Balanopreputial separation: Each selected male F1-pup (24 rats/group) was observed for balanopreputial separation (prepuce opening) beginning on postnatal Day 35. Examination of the males was continued daily until balanopreputial separation was present. The body weight of each male was recorded on the day of acquisition of balanopreputial separation.

Vaginal perforation: Each selected female F1-pup (24 rats/group) was observed for vaginal perforation (vaginal opening) beginning on postnatal Day 25. Examination of the females was continued daily until vaginal perforation is present. The body weight of each female was recorded on the day of acquisition of vaginal perforation.

Anogenital distance: As there was no treatment related effect in the F1-sex ratio or sexual maturation of the F1-generation, anogenital distance was not measured for F2-pups on Day 1 of lactation.
Postmortem examinations (parental animals):
Termination
All animals surviving to the end of the observation period and all moribund animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

Condition Day of necropsy
Parental Males: As soon as possible after delivery of the litters.
Females which delivered: Lactation Day 21-23.
Females which failed to deliver1: Post-coitum Day 25-30 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).
Dead pups: Within 24 hours of death.
Females with no evidence of mating1: Approximately 21 days after the last day of the mating period.
Euthanized in extremis2: When pain, distress or discomfort was considered not transient in nature or was likely to become more severe.
1 In case a female was not pregnant, the uterus was stained using the Salewski technique in order to determine any former implantation sites (Salewski staining prepared at WIL Research Europe using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).
2 Recognizable fetuses were examined externally, sexed (if possible), and euthanized by decapitation (if necessary).

Macroscopic examination
After sacrifice or death all animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs.
Descriptions of all macroscopic abnormalities were recorded.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):
Identification marks: not processed (Pituitary gland)
(Adrenal glands) Preputial gland
(Brain (cerebellum, mid-brain, cortex)) Prostate gland
Cervix Seminal vesicles
Clitoral gland (Spleen)
Coagulation gland Testis (1) a
Epididymidis (1) a (Thyroid including parathyroid (if detectable))
Female mammary gland area Uterus
(Kidneys) Vagina
(Liver) All gross lesions
Ovaries
a Testes and epididymis (right only) were fixed in modified Davidson's solution (prepared at WIL Research Europe using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial) (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

Examination of reproductive organs
For all paired females, the number of former implantation sites in the uterus was assessed. These numbers were listed in a footnote for non-pregnant and non-mated females. At the time of necropsy, a vaginal lavage was taken to determine the stage of estrous.
For all surviving males, the following assessments were performed:
-Sperm samples were taken from the proximal part of the vas deferens (right):
1. Sperm motility and progressive motility were assessed from all samples.
2. Sperm smears for morphological evaluation was fixed from all samples and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal were recorded.
Evaluation was performed for all samples of the control and high dose group.
-One testis and one epididymis (left) were removed, placed in labelled bags, and kept in the freezer at ≤-15°C. After thawing the left testis and epididymis were weighed, homogenized and evaluated for sperm numbers.
Evaluation was performed for all samples of the control and high dose group.

Organ weights
The following organ weights and terminal body weight were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands Prostate 1
Brain Seminal vesicles with coagulating glands1
Epididymides Spleen
Kidneys Testes
Liver Thyroid including parathyroid
Ovaries Uterus (including cervix)
Pituitary gland1
1 Weighed after fixation

Histotechnology
All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).
HE stained step sections of ovaries at a thickness of 6 micrometers (4 step sections in total, including the routine section) were prepared from 10 control and 10 high dose females from the F1-generation for quantitative evaluation of follicles.
Postmortem examinations (offspring):
Termination
Pups, younger than 7 days were killed by decapitation.
All remaining pups were sacrificed using Euthasol®20% (AST Farma B.V., Oudewater, The Netherlands) by ip injection.
Pups found dead during the weekend were fixed in identified containers containing 70% ethanol (Klinipath, Duiven, The Netherlands) if not necropsied on the same day.
Culling was performed on Day 4 of lactation or shortly thereafter. The remaining pups (excluding F1- pups selected for mating) were killed on Day 21 of lactation or shortly thereafter.
For training purposes, culled pups were used for collection of blood. This was done after determination of sex and external examination as stated in the protocol. Details were documented in the raw data but were not reported.

Macroscopic examination
Stillborn pups and pups dying between birth and Day 4 of lactation were sexed and dissected (including the heart and the brain examined by a mid-coronal slice) by a technique described by Stuckhardt and Poppe. If a skeletal anomaly was suspected, the pups were eviscerated, cleared and stained with Alizarin Red S as described by Dawson and examined. These examinations were only performed when practically possible (e.g. not in the case of cannibalism, autolysis).
For pups found dead or killed in extremis from Day 5 of lactation to weaning a gross necropsy was performed (including sex determination) and gross lesions were saved for possible future histopathological examination.
Culled pups were sexed and externally examined with emphasis on developmental morphology.
All nonselected F1- and F2-weanlings were sexed and subjected to external examination of the cranium, and macroscopic examination of the thoracic and abdominal tissues and organs with emphasis on developmental morphology. Descriptions of all macroscopic abnormalities were recorded. All gross lesions were collected and placed in 10% buffered formalin.

Organ weights
The following organ weights (and terminal body weight) were recorded from one selected pup/sex/litter (both generations) on the scheduled day of necropsy:
Brain
Spleen
Thymus
After weighing, samples of these organs were fixed in 10% buffered formalin for possible future analysis.
Statistics:
The following statistical methods was used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may be rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
For each group, the following calculations were performed:

Number of males mated
Percentage mating males = ------------------------- x 100
Number of males paired

Number of females mated
Percentage mating females = --------------------------- x 100
Number of females paired

Number of pregnant females
Fertility index males = ------------------------------- x 100
Number of males paired

Number of pregnant females
Fertility index females = ------------------------------- x 100
Number of female paired

Number of pregnant females
Conception rate = ------------------------------- x 100
Number of female mated

Number of females bearing live pups
Gestation index = ------------------------------------- x 100
Number of pregnant females

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Foe each group, the following calculations were performed:
Number of live male pups at First Litter Check
Percentage live males at First Litter Check = -------------------------------------------- x 100
Number of live pups at First Litter Check

Number of live female pups at First Litter Check
Percentage live females at First Litter Check = --------------------------------------------- x 100
Number of live pups at First Litter Check

Number of dead pups on Day 4 of lactation
Percentage of postnatal Loss lactation Days 0-4 = -------------------------------------- x 100
Number of live pups at First Litter Check

Number of dead pups between Days 5 and 21 of lactation
Percentage of breeding loss Day 5 until weaning = ----------------------------------------------------- x 100
Number of live pups on Day 4 of lactation

Number of live male pups on Day 21 of lactation
Percentage live males at weaning = --------------------------------------------- x 100
Number of live pups on Day 21 of lactation
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower body weights and body weight gains during the post coitum period and lower body weight gains on Day 64 of the premating period. Absolute body weights were decreased during lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Lower body weights and body weight gains during the post coitum period and lower body weight gains on Day 64 of the premating period. Absolute body weights were decreased during lactation.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
F0-generation Mortality
There were no treatment related deaths noted up to 15000 ppm.
One female at 5000 ppm (no.147) was euthanized in extremis after 14 days of treatment. The animal was noted with flat posture, abdominal swelling, piloerection, pale appearance and hypothermia prior to her death. At the macroscopic examination, this animal was emaciated and found with a hard, grey-white nodule on the cervix, an enlarged spleen and iliac lymph node, and a large, reddish, hard nodule (46 x 41 mm) in the abdominal cavity. This female also had a higher body weight gain that was attributable to the large mass found at necropsy, which was also the cause of her poor condition. A malignant mesenchymoma tumour was found at the microscopic examination. This was not considered to be treatment related because the animal was only exposed to the test substance for a very brief time prior to her death.
One control female, no. 103 was killed in extremis after she was observed with pale appearance, had delivered 4 dead pups and was struggling with parturition. At the macroscopic examination she was found with pale discoloration of the liver and thyroid glands, reddish discoloration of the mandibular lymph nodes, an enlarged spleen, and one fetus was found in the left uterine horn. There were no relevant microscopic findings noted for this female at the histological examination.

F0-generation Clinical signs
No clinical signs of toxicity were noted during the observation period.
Incidental findings seen for control and/or treated animals that were noted included alopecia, wound, scabbing on various areas of the body, chromodacryorrhoea, and swelling and redness of the ear. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. There was no dose- dependent distribution and at the incidence observed, these were considered signs of no toxicological relevance.

F0-generation Body weights
At 15000 ppm, females had statistically significantly lower body weights and body weight gains during the post coitum period (not always statistically significant). These females also had significantly lower body weight gains on Day 64 of the premating period (-8.9%). Absolute body weights were decreased during lactation. In contrast, body weight gains were statistically significantly higher on Day 21 of the lactation period.
No other statistically significant differences in body weights or body weight gains were seen for F0 animals.

F0-generation Food consumption
In general, F0 animals in all treated groups had a dose-dependent increase in absolute and relative food consumption compared to controls (differences from controls were not always statistically significant). This effect was most pronounced for animals at 15000 ppm with absolute and relative food consumption higher for both sexes during the entire treatment period.
Absolute and relative food consumption were also higher for animals of both sexes at 5000 ppm during the majority of the treatment period (not always statistically significant).
Relative food consumption was higher for males at 1500 ppm on Days 7-14 post mating. Females at this dose level had higher relative food consumption during the complete premating period and on Days 0-4 post coitum (not always statistically significant). Absolute food consumption was statistically significantly higher than controls Days 43-50 of the premating period only.
The dose-dependent increase in food consumption over all dose levels may reflect that animals had to eat a greater volume of food to meet their nutritional needs as a larger proportion of the diet was composed of (non-nutritive) test item with each subsequent test group, from the low to the high dose level.

F0-generation Test article intake
Reported in table form - see Any other information.

F0-generation Macroscopic examination
Macroscopic observations of surviving animals at necropsy did not reveal any alterations that were considered attributable to treatment.
Incidental findings noted for control and/or treated animals included alopecia and/or scabbing on various body areas, pelvic dilation of one or both kidneys, reddish discoloration or many/several reddish foci on the thymus, thickened or discoloration (yellow or reddish) of the liver papillary process, reduced size of the liver lobe, papillary process or left lateral lobe grown together with the stomach, uterus contains fluid, yellowish, hard nodule on the liver, enlarged clitoral glands, tan focus on the preputial glands, greenish, hard nodule on the epididymal adipose tissue, diaphragmatic hernia, many red-brown foci on the kidneys, tan focus on the preputial gland, enlarged liver, a gray-white, hard nodule on the cervix, thickened skin and reddish discoloration of the ear region, reduced size of the thyroid, and a red-brown or tan hard nodule on the uterine adipose tissue. These findings are commonly seen for animals of this age and strain and occurred in the absence of a treatment related distribution. Taken together, these were not considered to be toxicologically relevant.

F0-generation Organ weights
At 15000 ppm, kidney weights were higher for males (7.4% absolute and 10.8% relative) and females (8.1% relative only). Females also had statistically significantly lower terminal body weights (6.3% lower than controls). While the differences from controls was only slight, it was considered to be treatment related.
All other statistically significant changes were not considered to be treatment related or toxicologically relevant as values remained within the range considered normal, the differences from controls were only slight, the changes were considered secondary to their lower terminal body weights, occurred in the absence of a dose-dependent distribution and/or were not supported by any relevant microscopic findings.

F0-generation Microscopic examination
There were no test item-related microscopic observations in the reproductive organs of the 10 selected rats of each sex treated at 15000 ppm of the F0 generation.
Examination of infertile animals revealed moderate to marked inflammation in the prostates of male nos. 28 (1500 ppm) and 57 (5000 ppm) which could have contributed to the lack of offspring. This finding was considered to be unrelated to treatment.
Findings in the remaining animals were regarded as incidental and not responsible for the suspected infertility. Furthermore, spermatogenic staging profiles were normal for all males examined.
All remaining microscopic findings recorded were considered to be within the range of background pathology encountered in Wistar (Han) rats of these ages.

F0-generation Sperm motility, concentration and morphology
There were no treatment related effects on sperm motility, concentration and morphology noted up to 15000 ppm.

F0-generation Estrous cycle
Estrous cycle length was unaffected by treatment up to 15000 ppm.
The percentage of females per group that were classified as having a ‘regular’ cycle were 100.0, 91.7, 100.0 and 100.0% at 0, 1500, 5000 and 15000 ppm, respectively. In these animals, the percentage of irregularities was 0%, 8.3% 0%, and 0%, respectively. All animals had individual cycle lengths of 4 days. The only exceptions were two females at 1500 ppm; female no. 130 had one 8 day cycle and no. 131 had two 2-day cycles. The rest were all 4 days for these females. One control female and three, two and one females in the 1500, 5000 and 15000 ppm groups had cycles of 4 days and one extended di-estrus cycle.

F0-generation Reproduction
No toxicologically relevant effects on reproductive parameters were noted.
The mating, fertility and conception indices, precoital time, the gestation index or duration, and the number of implantation sites were unaffected by treatment.
Aside from female no: 103. No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth and no deficiencies in maternal care were observed.
There were 21, 20, 19 and 24 litters available for evaluation in the control, 1500, 5000 and 15000 ppm groups, respectively.
Dose descriptor:
NOAEL
Effect level:
5 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
15 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive/developmental toxicity No developmental or reproductive toxicity was observed with treatment up to 15000 ppm for any generation.
Remarks on result:
other: Generation: P, F1, F2 (migrated information)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 15000 ppm pup body weights (both sexes) were statistically significantly lower from Days 14-21 of lactation.
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Terminal body weights were statistically significantly lower for males and females at 15000 ppm and for females at 5000 ppm
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
F1-pup development
The number of dead and living pups at first litter check, sex ratio, postnatal and breeding loss, viability and weaning indices, pup clinical signs and macroscopy did not reveal treatment-related findings.
The viability index was statistically significantly lower for females at 5000 ppm. No toxicological relevance was attributed to this as no dose response effect was evident, and the low viability index was secondary to higher postnatal loss at this dose level (5 pups in 4 litters). In the absence of a dose related distribution this was not considered to be toxicologically relevant. Moreover, all values remained within normal limits.

F1-pup mortality
There were no treatment related or toxicologically relevant effects on pup mortality seen up to 15000 ppm.
First litter check: Four pups of the control group and eight and four pups of the 5000 and 15000 ppm groups were dead at the first litter check. There were no dead pups at the first litter check in the 1500 ppm group.
At 5000 ppm, female no.154 had 6 dead pups at the first litter check. Discounting her litter, the remaining number of dead pups remained within the range of the other dose groups.
PND 1 – PND 4: No pups of the control group, three, five and three pups in the 1500, 5000 and 15000 ppm groups were found dead, went missing or was euthanized in extremis (one pup at 1500 ppm) between the first litter check and culling on Day 4. Missing pups were most likely cannibalised.
The postnatal loss was statistically significantly higher for pups at 5000 ppm, however, this was not considered to be treatment related as it occurred in the absence of a dose-dependent distribution and remained within the range considered normal.
The pup at 1500 ppm was euthanized on Day 1 because of a large wound on the right leg. At this single occurrence, it was not considered treatment related.
PND 5 – PND 21: One pup of the control group went missing on Day 7 and one pup at 15000 ppm went was missing on PND 21. Missing pups were most likely cannibalised.
None of these pup deaths were considered toxicologically relevant as all remained within the range seen for animals used in this type of study and no dose response effect was seen.

F1-pup Clinical signs
There were no treatment related clinical signs noted with treatment up to 15000 ppm.
One pup at 1500 ppm was euthanized on Day 1 of lactation due to a wound on the leg. Pale appearance (1 pup at 1500 ppm) and a wound on the chin (1 pup at 15000 ppm) were additional clinical signs noted for pups that later went missing or were found dead. These were not related to treatment.
Incidental clinical signs of pups consisted of pale appearance, black discoloration or discoloration of the tail, missing tail apex, alopecia, scabs on the nose, agenesis of the left eye and chromodacryorrhoea. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

F1-pup Body weights
At 15000 ppm pup body weights (both sexes) were statistically significantly lower from Days 14-21 of lactation.

F1-pup Macroscopic findings
There were no treatment related or toxicologically relevant macroscopic findings noted with treatment up to 15000 ppm.
Incidental macroscopic findings of pups that were found dead included autolysis, cannibalism, no milk in the stomach and wound on the lower jaw.
Macroscopic findings seen for animal surviving to the scheduled necropsy included a missing tail apex and discoloration of the tail, scabs on the nose, agenesis of the left eye, pelvic dilation of the kidney, and reddish discoloration of the thymus. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were not considered to be of toxicologically relevant.

F1-pup Organ weights
There were not toxicologically relevant effects on pup organ weights with treatment up to 15000 ppm.
A statistically significant increase in relative thymus weights was observed for female pups at 5000 and 15000 ppm and higher spleen to body weight ratios were observed for male pups at 5000 ppm.
These differences were not considered to be toxicologically relevant, however, as the differences from controls were only slight, values remained within the range considered normal, and/or no dose dependent effect was seen.
All other organ weights and organ to body weight ratios were unaffected by treatment up to 15000 ppm.

F1-pup Vaginal opening and balanopreputial separation
The day of vaginal opening (VO) and balanopreputial separation (BPS) was similar for control and treated pups. Male pups in all treated groups had lower body weights on day of BPS compared to controls (185, 178, 178 and 174 grams for controls, 1500, 5000 and 15000 ppm, respectively). This was not considered to be toxicologically relevant as the difference from controls was only slight and the day of reaching balanopreputial separation was unaffected.

F1-generation Mortality
No mortality occurred.

F1-generation Clinical signs
No treatment-related clinical signs were observed.
Incidental findings consisted of alopecia, wound and/or scabs on different parts of the body, broken tail apex, and chromodacryorrhea. These findings are commonly seen among rats used in these types of studies. At the incidence observed or in the absence of a treatment-related distribution these were not considered to be toxicologically relevant.

F1-generation Body weights
Body weights were statistically significantly lower for males at 15000 ppm than controls beginning Day 29 of the premating period and persisted through the post mating period. Female body weights were similarly affected, with statistically significant differences from controls seen beginning on Day 22 of the premating period. Lower body weights for females persisted through the post coitum and lactation periods, but the differences from controls were not always statistically significant. There were no statistically significant differences in body weight gains between treated animals and controls.
Females at 1500 ppm had statistically significantly higher body weight gains on Day 17 of the post coitum period only. These differences were not considered to be toxicologically relevant as they were only slight and were transient.

F1-generation Food consumption
In general, F1 animals in all treated groups had a dose-dependent increase in food consumption compared to controls (differences from controls were not always statistically significant).
Males and females at 15000 ppm had higher absolute and relative food consumption throughout the premating and mating periods (not always statistically significant for females). Females also had statistically significantly increased absolute food consumption on Days 11-17 of the post coitum period. Relative food consumption was increased during the post coitum and lactation periods (not always statistically significant).
At 5000 ppm relative food consumption was higher for males during most of the premating, mating and post mating periods (not always statistically significant). Absolute food consumption was only statistically significantly different from controls on Days 8-15 of the mating period. Females had higher absolute food consumption on Days 1-29 of the premating period (not always statistically significant) and over Days 1-14 of the lactation period. Relative food consumption was higher for females at this dose level during most of the premating period, though the differences from controls were greater in the first half of the premating period (not always statistically significant). Relative food consumption for females was higher than controls during most of the treatment period and statistically significant increases were seen on Days 11-20 post coitum and lactation Days 1-14.
Males and females at 1500 ppm had higher relative food consumption during the entire treatment period (not always statistically significant). Absolute food consumption was only statistically significantly higher for females Days 15-29 of the premating period, Days 11-14 post coitum and Days 7-14 of the lactation period.
As with the F0-generation, the dose-dependent increase in food consumption over all dose levels may reflect that animals had to eat a greater volume of food to meet their nutritional needs as a larger proportion of the diet was composed of (non-nutritive) test item with each subsequent test group.

F1-generation Test article intake
Reported in table form see Any other information.

F1-generation Macroscopic findings
There were no treatment related or toxicologically relevant macroscopic findings noted with treatment up to 15000 ppm.
Incidental findings noted for control and/or treated animals included pelvic dilation of the kidneys, reduced size of the seminal vesicle, pituitary gland or prostate gland, bent tail (apex), isolated, tan focus on the preputial glands or clitoral glands, several dark red foci on the thymus, a watery-clear cyst on the uterine horn, greenish discoloration of the clitoral glands, diaphragmatic hernia of the liver, uterus contains fluid, and alopecia. These findings did not show a treatment related distribution and are commonly seen for animals of this age and strain. As such, the macroscopic findings were not considered to be toxicologically relevant.

F1-generation Organ weights
Terminal body weights were statistically significantly lower for males and females at 15000 ppm (- 7.3% males, -6.3% females) and for females at 5000 ppm (-5.3%).
The statistically significant changes for organ weights were not considered to be treatment related or toxicologically relevant as values remained within the range considered normal, the differences from controls were only slight, the changes were considered secondary to their lower terminal body weights, occurred in the absence of a dose-dependent distribution and/or were not supported by any relevant microscopic findings.

F1-generation Microscopic examination
There were no test item-related microscopic observations in the reproductive organs of the 10 selected rats of each sex treated at 15000 ppm of the F1 generation. The numbers of primordial and primary follicles quantitatively assessed were within background ranges.
Examination of infertile animal no. 276 (15000 ppm) revealed moderate inflammation with slight decreased prostatic fluid in the prostate, which could have attributed to the lack offspring. This finding was considered to be unrelated to treatment.
Findings in the remaining animals were regarded as incidental and not responsible for the suspected infertility. Furthermore, spermatogenic staging profiles were normal for all males examined.

F1-generation Sperm motility, concentration and morphology
No toxicologically relevant effects on sperm motility, concentration and morphology were noted up to 15000 ppm.
The epididymal sperm count was statistically significantly lower for males at 15000 ppm while the number of coiled tails seen in sperm samples were statistically significantly higher. These were not considered to be treatment related or toxicologically relevant, as all values remained within the range considered normal and were considered to be secondary to slightly high or low values obtained for controls.

F1-generation Estrous cycle
The regularity and duration of the estrous cycle were unaffected by treatment.
The percentage of females per group classified as having a ‘regular’ cycle of 4-5 days were 100, 95.8, 87.5 and 95.8% at 0, 1500, 5000 and 15000 ppm, respectively. In these females, the percentage of irregularities was 0, 4.2, 12.5 and 4.2%, respectively. Of the females with irregular cycles, one at 1500 ppm had extended di-estrus, one at 5000 ppm was acyclic and two others at this dose level had irregular cycles of 6-7 days and 3-6 days, respectively. One female at 15000 ppm was acyclic. All other animals had regular cycles. Two females at 1500 ppm and two females at 15000 ppm had extended di-estrus, but were still classified as having a regular cycle.

F1-generation Reproduction
No toxicologically relevant effects on reproductive parameters were noted with treatment up to 15000 ppm.
The mating, fertility and conception indices, precoital time, gestation index or duration and number of implantation sites were unaffected by treatment.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth and no deficiencies in maternal care were observed.
There were 24, 24, 23 and 22 litters available for evaluation in the control, 1500, 5000 and 15000 ppm groups, respectively.

F2-pup Development
There were no treatment related or toxicologically relevant effects on pup development seen up to 15000 ppm.
The number of living and dead pups at first litter check, postnatal loss and the viability index were similar between control and treated animals and no differences in breeding loss, living pups at Day 21 or in the weaning index were seen. In addition, pup clinical signs and macroscopic examination did not reveal treatment-related findings.
Females at 5000 ppm had a lower male:female ratio of pups at the first litter check compared to controls. This was not considered treatment related as there was no dose dependent distribution and the difference from controls was only slight.

F2-pup Mortality
There were no treatment related or toxicologically relevant effects on pup mortality seen up to 15000 ppm.
First litter check: There was one pup in the control group and two pups at 1500 ppm that were found dead at the first litter check. There were no dead pups at the first litter check in the 5000 and 15000 ppm groups.
PND 1 – PND 4: There were two, one, two and two pups in the control, 1500, 5000 and 15000 ppm groups, respectively, that died or went missing in the first 1-4 days of lactation. Missing pups were most likely cannibalized.
PND 5 – PND 21: One pup at 1500 ppm died spontaneously on Day 7 of lactation. All other pups in each group survived between Days 5-21.
No toxicological relevance was attributed to any dead or missing pups as no treatment related distribution was evident and the number remained within the range considered normal for pups of this age.

F2-pup Clinical signs
There were no treatment related clinical signs noted for pups up to 15000 ppm.
Clinical signs noted for pups that were later found dead or went missing included blue spots on several body areas, lean appearance, cold to the touch and no milk in the stomach.
Incidental clinical signs seen for surviving control and treated pups included less milk in the stomach, alopecia, blue spots or scabbing over various body regions, missing tail or tail apex, black discoloration of the tail apex, and pale appearance. These observations remained within the range considered normal for animals of this age and strain and occurred in the absence of a treatment related distribution. As such, they were not considered to be treatment related or toxicologically relevant.

F2-pup Body weights
There were no differences in F2 pup body weights with treatment up to 15000 ppm.

F2-pup Macroscopic findings
There were no treatment related macroscopic findings noted for pups up to 15000 ppm.
Macroscopic findings seen for pups that were found dead included beginning or moderate autolysis and no milk in the stomach.
Macroscopic findings noted for pups surviving to the schedule necropsy included control and treated pups included, red discoloration of the thymus, pale appearance and missing tail or tail apex. These were seen in the absence of a dose dependent distribution and were considered to be incidental in nature.

F2-pup Organ weights
There were no treatment related effects on pup organ weights up to 15000 ppm.
Relative brain weights were significantly higher for male pups at 5000 ppm. This was secondary to their slightly lower terminal body weights and were not considered to be treatment related or toxicologically relevant.
There were no other differences in organ weights or organ to body weight ratios between control and treated pups.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
5 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Reproductive effects observed:
not specified

F0-generation Test article intake

The mean test article intake values (mg substance/kg boy weight/day) are summarized in the following table.

Mean test article intake based on nominal dose level of the diet:

Nominal dose level

Group 2

1500 ppm

Group 3

5000 ppm

Group 4

15000 ppm

MALES

Pre mating

Mating

Post mating

 

116

90

87

 

399

296

297

 

1254

943

989

FEMALES

Pre mating

Post coitum

Lactation

 

128

118

112

 

434

392

372

 

1365

1141

1308

 

F1-generation Test article intake

The mean test article intake values (mg substance/kg body weight/day) are summarized in the following table.

Mean test article intake based on the nominal dose level of diet:

Nominal dose level

Group 2

1500 ppm

Group 3

5000 ppm

Group 4

15000 ppm

MALES

Pre mating

Mating

Post mating

 

113

87

81

 

381

298

278

 

1241

992

955

FEMALES

Pre mating

Post coitum

Lactation

 

127

114

126

 

421

383

440

 

1366

1227

1264

 

F0-GENERATION ORGAN WEIGHTS (GRAM) SUMMARY - MALES

 

GROUP 1

CONTROL

GROUP 2

1500 PPM

GROUP 3

5000 PPM

GROUP 4

15000 PPM

END OF TREATMENT

BODY W. (GRAM)

MEAN

ST.DEV

N

442

27

24

435

32

24

442

36

24

428

27

24

BRAIN

(GRAM)

MEAN

ST. DEV

N

2.11

0.09

24

2.12

0.09

24

2.11

0.07

24

2.12

0.08

24

PITUITARY

(GRAM)

MEAN

ST. DEV

N

0.013

0.001

24

0.013

0.001

24

0.013

0.001

24

0.013

0.001

24

LIVER

(GRAM)

MEAN

ST. DEV

N

13.14

2.01

24

12.44

1.29

24

12.72

1.00

24

12.47

1.09

24

THYROID

(GRAM)

MEAN

ST. DEV

N

0.016

0.005

24

0.017

0.005

24

0.0018

0.005

24

0.017

0.004

24

KIDNEYS

(GRAM)

MEAN

ST. DEV

N

2.85

0.21

24

2.85

0.19

24

2.91

0.24

24

3.06**

0.25

24

ADRENALS

(GRAM)

MEAN

ST. DEV

N

0.048

0.005

24

0.052

0.008

24

0.060

0.007

24

0.045

0.005

24

SPLEEN

(GRAM)

MEAN

ST. DEV

N

0.647

0.076

24

0.653

0.087

24

0.682

0.079

24

0.686

0.072

24

TESTES

(GRAM)

MEAN

ST. DEV

N

3.84

0.28

24

3.78

0.30

24

3.66

0.25

24

3.57**

0.28

24

PROSTATE

(GRAM)

MEAN

ST. DEV

N

0.751

0.159

24

0.690

0.145

24

0.730

0.169

24

0.726

0.143

23

EPIDIDYMIDES

(GRAM)

MEAN

ST. DEV

N

1.245

0.121

24

1.230

0.087

24

1.234

0.120

23

1.184

0.101

24

SEMINAL VES

(GRAM)

MEAN

ST. DEV

N

1.688

0.218

24

1.720

0.238

24

1.793

0.283

24

1.714

0.213

23

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

F0-GENERATION ORGAN/BODY WEIGHT RATIOS (&) SUMMARY - MALES

 

GROUP 1

CONTROL

GROUP 2

1500 PPM

GROUP 3

5000 PPM

GROUP 4

15000 PPM

END OF TREATMENT

BODY W. (GRAM)

MEAN

ST.DEV

N

442

27

24

435

32

24

442

36

24

428

27

24

BRAIN

(&)

MEAN

ST. DEV

N

0.48

0.03

24

0.49

0.04

24

0.48

0.04

24

0.50

0.03

24

PITUITARY

(%)

MEAN

ST. DEV

N

0.003

0.000

24

0.003

0.0000

24

0.003

0.000

24

0.003

0.0000

24

LIVER

(%)

MEAN

ST. DEV

N

2.97

0.44

24

2.85

0.14

24

2.88

0.13

24

2.91

0.12

24

THYROID

(%)

MEAN

ST. DEV

N

0.004

0.001

24

0.004

0.001

24

0.004

0.001

24

0.004

0.001

24

KIDNEYS

(%)

MEAN

ST. DEV

N

0.65

0.04

24

0.66

0.04

24

0.66

0.05

24

0.72**

0.04

24

ADRENALS

(%)

MEAN

ST. DEV

N

0.011

0.001

24

0.012*

0.002

24

0.011

0.001

24

0.011

0.001

24

SPLEEN

(%)

MEAN

ST. DEV

N

0.146

0.015

24

0.150

0.015

24

0.155

0.017

24

0.160**

0.014

24

TESTES

(%)

MEAN

ST. DEV

N

0.87

0.07

24

0.87

0.08

24

0.83

0.08

24

0.84

0.07

24

PROSTATE

(%)

MEAN

ST. DEV

N

0.170

0.032

24

0.159

0.036

24

0.165

0.038

24

0.170

0.033

23

EPIDIDYMIDES

(%)

MEAN

ST. DEV

N

0.282

0.030

24

0.283

0.020

24

0.279

0.031

23

0.277

0.023

24

SEMINAL VES

(%)

MEAN

ST. DEV

N

0.382

0.046

24

0.397

0.064

24

0.406

0.065

24

0.403

0.053

23

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

F0-GENERATION ORGAN WEIGHTS (GRAM) SUMMARY - FEMALES

 

GROUP 1

CONTROL

GROUP 2

1500 PPM

GROUP 3

5000 PPM

GROUP 4

15000 PPM

END OF TREATMENT

BODY W. (GRAM)

MEAN

ST.DEV

N

287

23

23

278

19

24

276

17

23

269**

17

24

BRAIN

(GRAM)

MEAN

ST. DEV

N

1.89

0.10

23

1.89

0.09

24

1.90

0.09

23

1.92

0.07

24

PITUITARY

(GRAM)

MEAN

ST. DEV

N

0.015

0.002

22

0.015

0.003

24

0.015

0.003

23

0.014

0.002

24

LIVER

(GRAM)

MEAN

ST. DEV

N

11.75

1.68

23

11.25

2.18

24

10.91

1.93

23

11.98

1.161

24

THYROID

(GRAM)

MEAN

ST. DEV

N

0.017

0.004

22

0.014

0.003

24

0.014**

0.004

23

0.014*

0.003

24

KIDNEYS

(GRAM)

MEAN

ST. DEV

N

2.13

0.18

23

2.07

0.23

24

2.14

0.27

23

2.15

0.15

24

ADRENALS

(GRAM)

MEAN

ST. DEV

N

0.071

0.012

23

0.066

0.009

24

0.069

0.010

23

0.069

0.011

24

SPLEEN

(GRAM)

MEAN

ST. DEV

N

0.510

0.071

23

0.524

0.071

24

0.562*

0.070

23

0.069

0.011

24

OVARIES

(GRAM)

MEAN

ST. DEV

N

0.136

0.017

23

0.144

0.023

24

0.140

0.015

23

0.141

0.086

24

UTERUS

(GRAM)

MEAN

ST. DEV

N

0.507

0.119

23

0.523

0.135

23

0.512

0.120

23

0.541

0.173

24

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

F0-GENERATION ORGAN/BODY WEIGHT RATIOS (%) SUMMARY - FEMALES

 

GROUP 1

CONTROL

GROUP 2

1500 PPM

GROUP 3

5000 PPM

GROUP 4

15000 PPM

END OF TREATMENT

BODY W. (GRAM)

MEAN

ST.DEV

N

287

23

23

278

19

24

276

17

23

269**

17

24

BRAIN

(%)

MEAN

ST. DEV

N

0.66

0.05

23

0.68

0.06

24

0.69

0.05

23

0.72**

0.04

24

PITUITARY

(%)

MEAN

ST. DEV

N

0.005

0.001

22

0.005

0.001

24

0.006

0.001

23

0.005

0.001

24

LIVER

(%)

MEAN

ST. DEV

N

4.08

0.43

23

4.02

0.63

24

3.94

0.58

23

4.46*

0.33

24

THYROID

(%)

MEAN

ST. DEV

N

0.006

0.002

22

0.005

0.001

24

0.005*

0.001

23

0.005

0.001

24

KIDNEYS

(%)

MEAN

ST. DEV

N

0.74

0.04

22

0.74

0.05

24

0.77

0.08

23

0.80**

0.06

24

ADRENALS

(%)

MEAN

ST. DEV

N

0.025

0.003

23

0.024

0.003

24

0.025

0.003

23

0.025

0.004

24

SPLEEN

(%)

MEAN

ST. DEV

N

0.178

0.021

23

0.188

0.019

24

0.203**

0.018

23

0.199**

0.024

24

OVARIES

(%)

MEAN

ST. DEV

N

0.048

0.006

23

0.052

0.010

24

0.051

0.005

23

0.053

0.038

24

UTERUS

(%)

MEAN

ST. DEV

N

0.177

0.042

23

0.188

0.051

23

0.186

0.046

23

0.202

0.068

24

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

F0-GENERATION REPRODUCTION DATA SUMMARY

 

GROUP 1

CONTROL

GROUP 2

1500 ppm

GROUP 3

5000 ppm

GROUP 4

15000 ppm

Females paired

24

24

23

24

Females mated

23

23

22

24

Males paired

24

24

23

24

Males mated

23

21

21

23

Pregnant females

22

21

19

24

Females with implantation sites only

0

1

0

0

Females with living pups on Day 1

21

20

19

24

Mating index females (%)

(females mated / females paired) * 100

96

96

96

100

Mating index males (%)

(males mated / males paired) * 100

96

88

91

96

Fertility index females (%)

(pregnant females / females paired) * 100

92

88

83

100

Fertility index males (%)

(pregnant females / males paired) * 100

92

88

83

100

Conception index (%)

(pregnant females / females mated) * 100

96

91

86

100

Gestation index (%)

(females with living pups on Day 1 / pregnant females)*100

95

95

100

100

 

F1-GENERATION ORGAN WEIGHTS (GRAM) SUMMARY - MALES

 

GROUP 1

CONTROL

GROUP 2

1500 PPM

GROUP 3

5000 PPM

GROUP 4

15000 PPM

END OF TREATMENT

BODY W. (GRAM)

MEAN

ST.DEV

N

464

34

24

449

36

24

451

34

23

430**

35

24

BRAIN

(GRAM)

MEAN

ST. DEV

N

2.13

0.09

24

2.14

0.10

24

2.17

0.09

24

2.12

0.11

24

PITUITARY

(GRAM)

MEAN

ST. DEV

N

0.014

0.002

24

0.013

0.003

24

0.014

0.003

24

0.013

0.002

24

LIVER

(GRAM)

MEAN

ST. DEV

N

13.37

1.48

24

12.86

1.70

24

12.80

1.03

24

12.52

1.16

24

THYROID

(GRAM)

MEAN

ST. DEV

N

0.016

0.004

24

0.018

0.004

24

0.019*

0.003

24

0.016

0.004

24

KIDNEYS

(GRAM)

MEAN

ST. DEV

N

2.91

0.27

24

2.83

0.34

24

2.83

0.23

24

2.88

0.27

24

ADRENALS

(GRAM)

MEAN

ST. DEV

N

0.048

0.007

24

0.048

0.007

24

0.049

0.004

24

0.045

0.06

24

SPLEEN

(GRAM)

MEAN

ST. DEV

N

0.643

0.116

24

0.623

0.093

24

0.692

0.091

24

0.657

0.089

24

TESTES

(GRAM)

MEAN

ST. DEV

N

3.98

0.29

24

4.03

0.32

24

3.92

0.29

24

3.57**

0.25

24

PROSTATE

(GRAM)

MEAN

ST. DEV

N

0.747

0.094

24

0.723

0.133

24

0.744

0.124

24

0.697

0.101

24

EPIDIDYMIDES

(GRAM)

MEAN

ST. DEV

N

1.262

0.097

24

1.296

0.096

24

1.271

0.105

24

1.216

0.093

24

SEMINAL VES

(GRAM)

MEAN

ST. DEV

N

1.676

0.297

24

1.801

0.234

24

1.684

0.303

24

1.685

0.234

24

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level 

Conclusions:
Based on these findings, the parental No Observed Adverse Effect Level (NOAEL) was established as being 5000 ppm. The reproduction and developmental NOAELs were established as being at least 15000 ppm.
When corrected for mean test article intake, the NOAEL of 5000 ppm corresponds to 278-399 mg and 372-440 mg DVS 005 (Weston 705T) per kg body weight per day for males and females, respectively.
The NOAEL of 15000 ppm corresponds to 943-1254 and 1141-1366 mg DVS 005 (Weston 705T) per kg body weight per day for males and females, respectively.
Executive summary:

The purpose of this study was to assess the effect of DVS 005 (Weston 705T) on male and female reproductive performance, such as gonadal function, estrous cycle, mating behaviour, conception, parturition, lactation, weaning, and on the growth and development of the pups when administered during two complete reproductive cycles. The study may also provide information of the test substance about developmental toxic effects, such as neonatal morbidity, mortality, behaviour and teratogenesis and to serve as a guide for subsequent tests.

A parental, reproductive (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Level (NOAEL) was evaluated.

 

The study was based on the following guidelines:

- OECD 416, Two-Generation Reproduction Toxicity Study, January 2001.

- OPPTS 870.3800, Reproduction and Fertility Effects, August 1998.

- EC No 440/2008 Part B, "Two-generation Reproduction Toxicity Test ", May 2008.

 

Rationale for dose levels

Based on the results of a 14-day dose range finding study (Project 503366), the dose levels for this two-generation reproduction toxicity study were 1500, 5000 and 15000 ppm by dietary administration, approximately equivalent to 81-127, 278-440, 955-1366 mg/kg.

 

Study outline

After acclimatization, four groups of 24 male and 24 female Wistar Han rats were exposed via dietary administration to the test substance at dose levels of 1500 (Group 2), 5000 (Group 3) and 15000 ppm (Group 4). Rats from the control group (Group 1) received preparations of standard rodent SM R/M-Z diet in pelleted form without DVS 005 (WESTON 705T). Rats from Groups 2-4 were exposed to DVS 005 (Weston 705T) that was included into standard pelleted rodent diet.

F0 males and F0 females were exposed to the test substance from 10 weeks prior to mating and exposure was continued until euthanasia (males) or one day before euthanasia (females). F0 females were allowed to produce and rear a litter until Day 21 of lactation. On Day 4 of lactation or shortly thereafter, litters were reduced in size to eight pups by random culling of F1 pups. After weaning, one F1 male and one F1 female of each litter of Groups 1-4 were selected for mating with a pup of another litter of the same dose group to produce a F2 generation. F1 females were allowed to produce and rear a litter until Day 21 of lactation.

 

Evaluated parameters

The following observations and examinations were evaluated: mortality / viability, clinical signs, body weight and food consumption, macroscopic examination at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio, sexual maturation and postnatal pup development (mortality, clinical signs, body weights, macroscopic examination and organ weights). Chemical analyses of diets were conducted seven times to assess accuracy, homogeneity and stability (up to 29 days at room temperature).

 

RESULTS

Accuracy, homogeneity and stability of diets were demonstrated by the results of the diet analyses.

 

F0- and F1-generation – general toxicity

At 15000 ppm, F0 females had lower absolute body weights during post coitum and lactation. These females also had lower body weight gains on Day 64 (-8.9%) of the premating period and during the post coitum period.

F1 animals of both sexes at 15000 ppm had lower absolute body weights through the complete treatment period.

At 15000 ppm, terminal body weights were lower for F1 males (-7.3%) and for females of both generations (-6.3% F0, -6.3% F1). Relative kidney weights at this dose were higher than controls for both sexes of the F0 generation (10.8% males, 8.1% females), and absolute kidney weights were also increased for F0 males (7.4%). While the differences from controls were slight, they were considered treatment related.

Food consumption was dose-dependently higher than controls for animals of both generations in all treated groups. This was considered treatment related and may have reflected a need to consume larger amounts of test diet (due to its large test article content) to match the same nutrition provided by control rodent diet.

 

F0- and F1-generation - reproductive and developmental toxicity

No developmental or reproductive toxicity was observed with treatment up to 15000 ppm for any generation.

 

CONCLUSION

Based on these findings, the parental No Observed Adverse Effect Level (NOAEL) was established as being 5000 ppm. The reproduction and developmental NOAELs were established as being at least 15000 ppm.

When corrected for mean test article intake, the NOAEL of 5000 ppm corresponds to 278-399 mg and 372-440 mg DVS 005 (Weston 705T) per kg body weight per day for males and females, respectively.

The NOAEL of 15000 ppm corresponds to 943-1254 and 1141-1366 mg DVS 005 (Weston 705T) per kg body weight per day for males and females, respectively.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
278 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
One study with Klimisch score of 1, therefore quality of database is acceptable.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The screening study (Liwska, 2010) was conducted in line with GLP and standardised guidelines with a sufficient level of detail to assess the quality of the study. Wistar rats were exposed to the test material at the following concentrations; 100, 300 and 1000 mg/kg/day in a screening study. Ten animals per sex, per dose were administered the test material via oral gavage. No treatment-related effects, unscheduled mortalities or systemic signs of toxicity were observed in the parental animals or offspring, at any dose. The NOEL for reproductive toxicity was therefore considered to be 1000 mg/kg/day. The study was performed to a good standard and was assigned a reliability score of 1 using the principles for assessing data quality as set out in Klimisch (1997).

The full 2 generation study gave the following results:

F0- and F1-generation – general toxicity

At 15000 ppm, F0 females had lower absolute body weights during post coitum and lactation. These females also had lower body weight gains on Day 64 (-8.9%) of the premating period and during the post coitum period.

F1 animals of both sexes at 15000 ppm had lower absolute body weights through the complete treatment period.

At 15000 ppm, terminal body weights were lower for F1 males (-7.3%) and for females of both generations (-6.3% F0, -6.3% F1). Relative kidney weights at this dose were higher than controls for both sexes of the F0 generation (10.8% males, 8.1% females), and absolute kidney weights were also increased for F0 males (7.4%). While the differences from controls were slight, they were considered treatment related.

Food consumption was dose-dependently higher than controls for animals of both generations in all treated groups. This was considered treatment related and may have reflected a need to consume larger amounts of test diet (due to its large test article content) to match the same nutrition provided by control rodent diet.

 F0- and F1-generation - reproductive and developmental toxicity

No developmental or reproductive toxicity was observed with treatment up to 15000 ppm for any generation.

Based on these findings, the parental No Observed Adverse Effect Level (NOAEL) was established as being 5000 ppm. The reproduction and developmental NOAELs were established as being at least 15000 ppm.

When corrected for mean test article intake, the NOAEL of 5000 ppm corresponds to 278-399 mg and 372-440 mg DVS 005 (Weston 705T) per kg body weight per day for males and females, respectively.

The NOAEL of 15000 ppm corresponds to 943-1254 and 1141-1366 mg DVS 005 (Weston 705T) per kg body weight per day for males and females, respectively.

Short description of key information:

TOXICITY TO REPRODUCTION

DVS005u did not cause any treatment-related effects for reproductive toxicity according to a study performed in line with GLP and to the standardised guideline OECD 421. The NOEL for reproductive toxicity was determined to be 1000 mg/kg/day.

TWO GENERATION REPRODUCTIVE TOXICITY

Based on these findings, the parental No Observed Adverse Effect Level (NOAEL) was established as being 5000 ppm. The reproduction and developmental NOAELs were established as being at least 15000 ppm.

When corrected for mean test article intake, the NOAEL of 5000 ppm corresponds to 278-399 mg and 372-440 mg DVS 005 (Weston 705T) per kg body weight per day for males and females, respectively.

The NOAEL of 15000 ppm corresponds to 943-1254 and 1141-1366 mg DVS 005 (Weston 705T) per kg body weight per day for males and females, respectively.

Justification for selection of Effect on fertility via oral route:

Main 2-generation study available.

Effects on developmental toxicity

Description of key information

DEVELOPMENTAL TOXICITY/TERATOGENICITY SCREENING

DVS005u did not cause any treatment-related effects for developmental toxicity according to a study performed in line with GLP and to the standardised guideline OECD 421. The NOEL for developmental toxicity was determined to be 1000 mg/kg/day.

FURTHER DEVELOPMENTAL TOXICITY STUDIES

Based on the results of the prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for DVS 005 (WESTON 705T) was established as 5000 ppm and the developmental NOAEL was established as at least 15000 ppm. Dose levels of 5000 and 15000 ppm correspond to a mean test article intake of 439 and 1266 mg/kg/day, respectively.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 October 2013 to 05 December 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD, EU & US EPA test guidelines in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See "Principles of method if other than guideline" below.
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See "Principles of method if other than guideline" below.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See "Principles of method if other than guideline" below.
Principles of method if other than guideline:
List of protocol deviations
1. On 16-November-2013 animals were observed but the data was not saved online. One control female (no. 17) had a late resorption (fetus no. 13) where no degree of autolysis,
crown-rump length measurement or external findings were recorded.
Evaluation: Sufficient data were available for a thorough evaluation.
2. From 27 November 2013 onwards, Lignocel S 8-15 (Rettenmaier & Sohne GmbH, Rosenberg, Germany) was used for sterilized sawdust bedding instead of the bedding specified in the protocol.
Evaluation: Both bedding types are suitable for use in this type of study.
3. Only the left ovary was preserved for animal no. 28 (1500 ppm).
Evaluation: Sufficient data were available for a thorough evaluation.
4. Organ weights were collected for all animals and not just from the pregnant animals.
Evaluation: This just represents additional information and does not negatively impact the
study.
The study integrity was not adversely affected by the deviations.
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Test System Rat: Crl:WI(Han) (outbred, SPF-Quality).
Females were nulliparous, non-pregnant and untreated at initiation of the study. Stock male rats Crl:WI(Han) (outbred, SPF-Quality) were used for mating with the females. After mating these males were placed back in their stock and might be used in further studies.

Rationale: This species and strain of rat has been recognized as appropriate for developmental toxicity studies. WIL Research Europe B.V. has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source.
This animal model has been proven to be susceptible to the effects of developmental toxicants.

Source: Charles River Deutschland, Sulzfeld, Germany.
Number of animals: F0-generation: 88 females. F1-generation: 893 fetuses.
Age at delivery: Females were approximately 11 weeks
Acclimatization: At least 5 days prior to start of treatment.
Health inspection: Upon receipt of the animals.

Randomization: Upon detection of mating (Day 0 post-coitum), the females were distributed in a random sequence over the test groups. Females which were mated on the same day were classified in the same subgroup.
Identification: By indelible ink.

Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle (lights on at 0700).
Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation
Pre-mating: During acclimatization, females were housed in groups of maximum 5 animals/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-coitum: Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France, from 06-26 November and Lignocel S 8-15, Rettenmaier & Sohne GmbH, Rosenberg, Germany from 27 November onwards) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied.
Diet: Free access to prepared diets. During acclimatization and pre-treatment (Day 0 to 6 post-coitum), animals had free access to the same diet (without the test substance) received from the supplier in pelleted form (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water: Free access to tap-water.
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Diet: Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Method: When necessary, the test substance was heated up to 70°C for a maximum of 6 hours before mixing with powder feed. The test substance was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Water (approximately 15% in total) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35°C before storage. The control animals received similarly prepared pellets but without the test substance.

Frequency of preparation: Diets were prepared at least once every 15 days. The same diets were used for the two-generation study (Project 503367).
No correction was made for the purity/composition of the test substance.

Stability of prepared diets: Stability of the diets at 1500 and 15000 ppm for at least 29 days at room temperature and for at least 18 days at ≤-15°C was confirmed during the range finding study for the two-generation study (Project 503366).

Storage conditions: Diets were kept at room temperature in the diet store room in the animal house.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed on one day during the treatment period according to a validated method (Project 503368). Samples of diets were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
Random samples from all diet preparations were taken in the two-generation study (Project 503367) and stored at ≤-15°C for possible future analysis. No additional reserve samples were taken separately for the present study. Any remaining samples were discarded at finalization of the study report.
The accuracy of diet preparations was considered acceptable if the mean measured concentrations were 80-120% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Details on mating procedure:
One female was placed on a one-to-one-basis in the cage of a male rat. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the female was separated from the male. When sufficient mated females had been obtained for each dose group, the surplus females were removed from the study.
Duration of treatment / exposure:
Days 6 to 20 post-coitum, inclusive.
Frequency of treatment:
Ad libitum from Days 6 to 20 post-coitum, inclusive.
Duration of test:
Days 6 to 20 post-coitum, inclusive.
Remarks:
Doses / Concentrations:
0, 1500, 5000, 15000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
22 females per dose group
Control animals:
yes, plain diet
Details on study design:
Rationale: This species and strain of rat has been recognized as appropriate for developmental toxicity studies. WIL Research Europe B.V. has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source.
This animal model has been proven to be susceptible to the effects of developmental toxicants.

Rationale for dose levels: Dose levels were selected based on results of the dose range finding study.
Maternal examinations:
Mortality / Viability: At least twice daily.

Clinical signs: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Body weights: Days 0, 3, 6, 9, 12, 15, 17 and 20 post-coitum.

Food consumption: Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum.

Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

Necropsy: All animals were sacrificed on Day 20 post-coitum using an oxygen/carbon dioxide procedure (approximately 40/60% oxygen/carbon dioxide respectively) and subsequently subjected to a limited post mortem examination, including an macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).

Organ weights: The following organ weights and terminal body weight were recorded from all pregnant females on the scheduled day of necropsy:
Liver
Spleen
After weighing, samples of these organs were fixed in 10% buffered formalin for possible future analysis.
Ovaries and uterine content:
Each ovary and uterine horn of animals surviving to planned necropsy was dissected and examined as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal inspections (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.
In case implantations were not macroscopically visible, the uterus was stained using the Salewski technique in order to determine any former implantation sites (Salewski staining prepared at WIL Research Europe using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).
The female genital tract including the placentas was preserved in 10% buffered formalin for possible histopathological examination.
Fetal examinations:
External, visceral and skeletal fetal findings were recorded as developmental variations or malformations.

External: Each viable fetus was examined in detail, weighed and sexed. All live fetuses were euthanized by administration of approximately 0.05 mL (=10mg) of sodium pentobarbital (Euthasol® 20%; AST Farma B.V., Oudewater, The Netherlands) into the oral cavity using a small flexible plastic or metal feeding tube. The crown-rump length of late resorptions was measured, the degree of autolysis recorded, a gross external examination performed (if possible). Late resorptions with malformations were fixed in 10% buffered formalin.

Visceral (Internal): All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by
Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The sex of all fetuses was confirmed by internal examination.
The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution (Klinipath, Duiven, The Netherlands). Tissues were then transferred to a 70% aqueous ethanol solution for subsequent processing and soft-tissue examination using the Wilson sectioning technique. After examination, the tissues were stored in 10% buffered formalin.
All carcasses, including the carcasses without heads, were eviscerated and fixed in identified containers containing 96% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent examination of skeletons.

Skeletal: Each eviscerated fetus, following fixation in 96% aqueous ethanol, was macerated in potassium hydroxide (Merck, Darmstadt, Germany) and stained with Alizarin Red S (Klinipath, Duiven, The Netherlands) by a method similar to that described by Dawson (Ref. 5). The skeletal examination was done following this procedure on the fetuses from all groups.
The specimens were archived in glycerin (Klinipath, Duiven, The Netherlands) with bronopol (Alfa Aesar, Karlsruhe, Germany) as preservative.
A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and study director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.

No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
For each litter the following calculations were performed:

(number of corpora lutea – number of implantation sites)
Pre-implantation loss (%) = ------------------------------------------------------------ x 100
Number of corpora lutea

(number of implantation sites – number of live foetuses)
Post-implanatation loss (%) = ----------------------------------------------------------- x 100
Number of implantation sites

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of foetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected foetuses as a men litter proportion on a total group basis, where:

Number of viable foetuses affected/litter
Viable foetuses affected/liiter (%) = --------------------------------------------- x 100
Number of viable foetuses/litter
Historical control data:
Historical data used for Fetal Morphology.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality: There were no unscheduled deaths during the study.

Clinical signs: No clinical signs were noted for any animal.

Body weights: At 15000 ppm, animals had reduced body weight gain from Day 9 post coitum onwards. Body weight gain corrected for the gravid uterus weight was also lower for these animals.
Females at this dose level also had slightly heavier absolute body weights on Day 0. As treatment commenced on Day 6 post coitum, the difference from controls was attributable to chance.

Food consumption: There were no toxicologically relevant effects on food consumption with treatment. At 15000 ppm, relative food consumption was lower from Days 9-12 of the post coitum period. As this was transient, it was not considered to be adverse.
Food consumption was higher than controls for females at 5000 and 15000 ppm on post coitum Days 12-15 (absolute and relative) and 17-20 (5000 ppm = absolute only, 15000 ppm = relative only). This was not considered to be toxicologically relevant, however, as values remained within normal limits, and a reduction in food consumption would be more likely if a toxicologically relevant effect on food consumption were present.

Test article intake: Dose levels of 1500, 5000 and 15000 ppm corresponded to a mean test article intake of 128, 439 and 1266 mg/kg/day, respectively.

Macroscopic examination: There were no toxicologically relevant effects on macroscopic findings noted with treatment up to 15000 ppm.
At 5000 ppm, an enlarged liver and uterus containing fluid were noted for individual animals. These were incidental and had no relationship with treatment.

Organ weights: Liver and spleen weights were similar between controls and treated animals.

Maternal pregnancy data: There were 21, 18, 18 and 21 pregnant females at 0, 1500, 5000 and 15000 ppm, respectively.
There were no differences between treated animals and controls in the number of corpora lutea, implantation sites, viable or dead fetuses, early or late resorptions, or in pre- or post-implantation loss.
Dose descriptor:
NOAEL
Effect level:
15 000 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter size: There were no effects of treatment up to 15000 ppm on litter size.
Mean litter sizes were 11.6, 10.8, 12.3 and 11.1 fetuses in the control, 1500, 5000 and 15000 ppm groups, respectively.

Sex ratio: The sex ratio was unaffected with treatment up to 15000 ppm.

Fetal body weight: There were no treatment related effects on fetal body weight up to 15000 ppm.
Mean (combined) fetal body weights for the control, 1500, 5000 and 15000 ppm were 3.6, 3.7, 3.7, 3.6 grams, respectively.

Fetal morphological examinations: The numbers of fetuses (litters) available for external, visceral and skeletal morphological evaluation were 243 (21), 195 (18), 222 (18), 233 (21) in the control, 1500, 5000 and 15000 ppm groups, respectively. Soft cephalic tissue examinations were done for approximately half of the fetuses in each group. Malformations were observed in 1 (1) 1 (1), 1 (1) and 5 (4) fetuses (litters) in the control, 1500, 5000 and 15000 ppm groups, respectively.

External malformations and variations: There were no treatment related effects on fetal external morphology up to 15000 ppm.
A small lower jaw, an external malformation, was observed for a single control fetus. As this was seen for a control animal, this was not attributable to treatment. There were no other malformations and no external variations were seen for any fetuses.

Visceral malformations and variations: There were no treatment related effects on fetal visceral morphology.
One fetus at 15000 ppm had an absent eye. At the single incidence observed, this was not considered to be treatment related.
Visceral variations were observed for 7.7, 9.1, 6.8 and 8.2% of the fetuses in the control, 1500, 5000 and 15000 ppm groups, respectively. Visceral variations included small supernumerary lobe of the liver, discolored adrenal gland, dilated and/or convoluted ureters, liver appendix, and partially undescended thymus horn(s). These were not considered attributable to treatment as they occurred at similar frequencies across the groups, occurred infrequently, did not follow a dose-related trend and/or occurred at frequencies within the range of available historical control data.

Skeletal malformations and variations: There were no toxicologically relevant effects on fetal skeletal morphology.
Skeletal malformations, consisting of bent limb bones (abnormal curvature of the limb bones), were seen for 0(0), 0(0), 0(0) and 4(3) fetuses (litters) in the control, 1500, 5000 and 15000 ppm groups, respectively. This corresponds to an incidence of 1.6% in the 15000 ppm group. One of these fetuses, A088-02, had multiple bones with abnormalities (including the clavicle, femur, fibula, humerus, ilium, ischium, radius, scapula, tibia and ulna). However, these abnormalities were at least partially attributable to his low body weight (2.8 gram) and the resulting developmental delay there from. This fetus was the exception as the other affected fetuses had bent limb bones representative of those previously seen in the historical control data. Though the incidence was significantly higher than controls, it remained below the maximum of 2% seen in the historical control database. Taken together, the bent limb bones were not considered to be treatment related.
Two other skeletal malformations were noted. Sternoschisis was seen for one fetus at 1500 ppm (incidence of 0.4%) and a malpositioned metacarpal was noted for a single fetus at 5000 ppm (incidence of 0.7%). As these malformations occurred for individual fetuses and remained under the maximum incidence noted in the historical control database, they were not considered to be treatment related.
Skeletal variations were observed in 76.3, 90.0, 83.1 and 84.4% of fetuses in the control, 1500, 5000 and 15000 ppm groups, respectively. An increased incidence of bent ribs was seen for fetuses of all treatment groups. Bent ribs were seen for 21.5, 19.3 and 21.1% of fetuses at 1500, 5000 and 15000 ppm, respectively, while this was seen for 6.2% of control fetuses. The increase was not considered to be toxicologically relevant as it occurred in the absence of a dose-dependent distribution and remained within the normal range of historical control data. Furthermore, the toxicological relevance of bent ribs is debatable since these are almost completely reversible during the pre-weaning period. As such, the higher incidence of bent ribs for treated animals was not considered to be toxicologically relevant.
Other skeletal variations seen in the control and/or treated groups included 14th rudimentary ribs, ossification of cervical centrum #1, 14th full ribs, reduced ossification of the vertebral centra, caudal shift of the pelvic girdle, reduced ossification of the skull, unossified/reduced ossification of the pubis, and unossified sternebrae nos. 1,2,3 and/or 4, malaligned sternebrae, branched sternebrae, generally reduced ossification of the entire skeleton, partially fused zygomatic arch, reduced ossification of the vertebral arches, unossification of the hyoid body, 7th cervical rudimentary or full ribs, ossified tarsals, . These variations were not considered to be treatment related because they occurred at similar frequencies in the control and treatment groups and/or remained within the historical control range.
Dose descriptor:
NOAEL
Effect level:
15 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no toxicologically relevant effects on developmental parameters with treatment upto 15000 ppm.
Abnormalities:
not specified
Developmental effects observed:
not specified

BODY WEIGHTS (GRAM) SUMMARY FEMALES

F0-GENERATION

 

GROUP 1

CONTROL

GROUP 2

1500 PPM

GROUP 3

5000 PPM

GROUP 5

15000 PPM

POST COITUM

DAY 0

MEAN

ST.DEV.

N

204

7.6

21

210

8.1

18

207

8.2

18

212**

10.0

21

DAY 3

MEAN

ST.DEV.

N

219

8.5

21

225

10.4

18

222

10.7

18

226

10.8

21

DAY 6

MEAN

ST.DEV.

N

225

8.6

21

232

11.2

18

230

9.1

18

232

12.5

21

DAY 9

MEAN

ST.DEV.

N

237

10.2

21

244

11.7

18

241

12.1

18

238

16.4

21

DAY 12

MEAN

ST.DEV.

N

251

11.3

21

256

11.9

18

256

11.3

18

249

16.4

21

DAY 15

MEAN

ST.DEV.

N

265

12.5

21

269

13.0

18

268

12.5

18

261

17.1

21

DAY 17

MEAN

ST.DEV.

N

286

14.8

21

290

15.6

18

289

13.9

18

281

18.3

21

DAY 20

MEAN

ST.DEV.

N

325

20.0

21

328

19.1

18

332

18.0

18

318

25.8

21

 */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Explanation for excluded data are listed in the tables of the individual values (in the full study report).

 

BODY WEIGHT GAIN (%) SUMMARY FEMALES

F0-GENERATION

 

GROUP 1

CONTROL

GROUP 2

1500 PPM

GROUP 3

5000 PPM

GROUP 5

15000 PPM

POST COITUM

DAY 0

MEAN

ST.DEV.

N

-10

2.2

21

-9

2.0

18

-10

2.1

18

-9

1.9

21

DAY 3

MEAN

ST.DEV.

N

-3

1.9

21

-3

1.7

18

-3

2.2

18

-2

1.9

21

DAY 6

MEAN

ST.DEV.

N

0

0.0

21

0

0.0

18

0

0.0

18

0

0.0

21

DAY 9

MEAN

ST.DEV.

N

5

2.2

21

5

2.0

18

5

2.2

18

3**

2.8

21

DAY 12

MEAN

ST.DEV.

N

12

2.4

21

11

1.9

18

11

1.8

18

7**

2.6

21

DAY 15

MEAN

ST.DEV.

N

18

3.0

21

16

2.1

18

17

3.0

18

12**

3.1

21

DAY 17

MEAN

ST.DEV.

N

27

4.0

21

25

2.8

18

26

2.8

18

21**

4.4

21

DAY 20

MEAN

ST.DEV.

N

44

6.1

21

42

4.9

18

45

4.6

18

37**

6.9

21

 */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Explanation for excluded data are listed in the tables of the individual values (in the full study report).

 

FOOD CONSUMPTION (G/ANIMAL/DAY) SUMMARY FEMALES

F0-GENERATION

 

GROUP 1

CONTROL

GROUP 2

1500 PPM

GROUP 3

5000 PPM

GROUP 5

15000 PPM

POST COITUM

DAYS 0 – 3

MEAN

ST.DEV.

N

20

1.5

21

20

1.7

18

20

1.7

18

20

2.3

21

DAYS 3 – 6

MEAN

ST.DEV.

N

21

2.0

21

22

2.3

18

22

2.1

18

22

2.4

21

DAYS 6 – 9

MEAN

ST.DEV.

N

21

3.1

21

22

2.1

18

22

2.1

18

20

3.3

21

DAYS 9 – 12

MEAN

ST.DEV.

N

22

2.7

21

23

2.6

18

23

2.5

18

20

4.5

21

DAYS 12 – 15

MEAN

ST.DEV.

N

23

2.4

21

24

1.8

18

25*

2.3

18

25*

2.8

21

DAYS 15 – 17

MEAN

ST.DEV.

N

24

3.1

21

24

2.6

18

24

2.8

18

24

2.4

21

DAYS 17 – 20

MEAN

ST.DEV.

N

24

2.2

21

25

2.5

18

27*

2.5

18

25

3.5

21

MEAN OF MEANS

22

23

23

22

 */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Explanation for excluded data are listed in the tables of the individual values (in the full study report).

 

TEST ARTICLE INTAKE (MG SUBSTANCE/KG BODY WEIGHT/DAY) SUMMARY FEMALES

F0-GENERATION

 

GROUP 1

CONTROL

GROUP 2

1500 PPM

GROUP 3

5000 PPM

GROUP 5

15000 PPM

POST COITUM

DAYS 6 – 9

MEAN

ST.DEV.

N

0

0.0

21

133

10.6

18

460

38.3

18

1245

184.0

21

DAYS 9 -12

MEAN

ST.DEV.

N

0

0.0

21

132

11.1

18

451

39.3

18

1172

228.1

21

DAYS 12 – 15

MEAN

ST.DEV.

N

0

0.0

21

133

9.0

18

463

29.7

18

1424

149.8

21

DAYS 15 – 17

MEAN

ST.DEV.

N

0

0.0

21

125

9.9

18

422

40.9

18

1289

100.8

21

DAYS 17 -20

MEAN

ST.DEV.

N

0

0.0

21

115

8.6

18

299

27.1

18

1201

128.6

21

MEAN OF MEANS

0

128

439

1266

Explanation for excluded data are listed in the tables of the individual values (in the full study report).

 

MACROSCOPIC FINDINGS SUMMARY FEMALES

 

GROUP 1

CONTROL

GROUP 2

1500 PPM

GROUP 3

5000 PPM

GROUP 5

15000 PPM

END OF TREATMENT

Animal examined

22

22

22

22

Animals without findings

22

22

20

22

Animals affected

0

0

2

0

Liver

Enlarged

0

0

1

0

Uterus

Contains fluid

0

0

1

0

#/## Fisher’s Exact test significant at 5% (#) or 1% (##) level

 

ORGAN WEIGHTS (GRAM) SUMMARY FEMALES

 

GROUP 1

CONTROL

GROUP 2

1500 PPM

GROUP 3

5000 PPM

GROUP 5

15000 PPM

END OF TREATMENT

BODY W. (GRAM)

MEAN

ST.DEV.

N

321

19

21

323

18

18

327

18

18

314

25

21

LIVER

(GRAM)

MEAN

ST.DEV.

N

14.71

1.76

21

14.86

1.64

18

14.96

1.34

18

14.59

1.52

21

SPLEEN (GRAM)

MEAN

ST.DEV.

N

0.580

0.103

21

0.597

0.092

18

0.568

0.066

18

0.573

0.066

21

 */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Conclusions:
In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for DVS 005 (Weston 705T) was established as 5000 ppm and the developmental NOAEL was established as at least 15000 ppm. Dose levels of 5000 and 15000 ppm correspond to a mean test article intake of 439 and 1266 mg/kg/day, respectively.
Executive summary:

Title

A prenatal developmental toxicity study of DVS 005 (WESTON 705T) in rats by dietary administration.

 

Guidelines

The study procedures described in this report were based on the following guidelines:

1) Organization of Economic Co-operation and Development Guidelines (OECD) for testing of Chemicals Guideline 414, Prenatal Developmental Toxicity Study, January 2001.

2) Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.31: "Prenatal Developmental Toxicity Study". Official Journal of the European Union No. L142, May 2008.

3) The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines OPPTS 870.3700, Prenatal Developmental Toxicity Study, August 1998.

 

Rationale for dose levels

Dose levels were selected based on results of the dose range finding study.

 

Study outline

Eighty-eight mated female Wistar Han rats were assigned to four dose groups. The test item was administered by inclusion in the diet from Days 6 to 20 post-coitum at doses of 1500, 5000, 15000 ppm (Groups 2, 3 and 4 respectively) corresponding to a mean test article intake of 128, 439 and 1266 mg/kg/day, respectively. The rats of the control group received the same diet without the test substance. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Prepared diet was analyzed once for accuracy and homogeneity.

 

All animals surviving to Day 20 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. Liver and spleen weights were also recorded. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative, all fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol and stained with Alizarin Red S for skeletal examinations.

 

RESULTS

Accuracy and homogeneity of diet preparations were demonstrated by analyses.

 

Maternal findings

Females at 15000 ppm had lower body weight gains from Day 9 post coitum onwards.

There were no other effects on maternal parameters up to 15000 ppm and no maternal toxicity was observed in the 1500 or 5000 ppm groups.

 

Developmental findings

There were no toxicologically relevant effects on developmental parameters with treatment up to 15000 ppm.

 

CONCLUSION

Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for DVS 005 (WESTON 705T) was established as 5000 ppm and the developmental NOAEL was established as at least 15000 ppm. Dose levels of 5000 and 15000 ppm correspond to a mean test article intake of 439 and 1266 mg/kg/day, respectively.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 266 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
One study with Klimisch score of 1, therefore quality of database is acceptable.
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

The screening study (Liwska, 2010) was conducted in line with GLP and standardised guidelines with a sufficient level of detail to assess the quality of the study. Wistar rats were exposed to the test material at the following concentrations; 100, 300 and 1000 mg/kg/day in a screening study. Ten animals per sex, per dose were administered the test material via oral gavage. No treatment-related effects, unscheduled mortalities or systemic signs of toxicity were observed in the parental animals or offspring, at any dose. The NOEL for developmental toxicity was therefore considered to be 1000 mg/kg/day. The study was performed to a good standard and was assigned a reliability score of 1 using the principles for assessing data quality as set out in Klimisch (1997). 

The full OECD 414 Developmental toxicity study gave the following results:

 Maternal findings

Females at 15000 ppm had lower body weight gains from Day 9 post coitum onwards.

There were no other effects on maternal parameters up to 15000 ppm and no maternal toxicity was observed in the 1500 or 5000 ppm groups.

 

Developmental findings

There were no toxicologically relevant effects on developmental parameters with treatment up to 15000 ppm.

CONCLUSION

Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for DVS 005 (WESTON 705T) was established as 5000 ppm and the developmental NOAEL was established as at least 15000 ppm. Dose levels of 5000 and 15000 ppm correspond to a mean test article intake of 439 and 1266 mg/kg/day, respectively.

Justification for selection of Effect on developmental toxicity: via oral route:

One key study available.

Toxicity to reproduction: other studies

Description of key information

None available.

Justification for classification or non-classification

The reproductive and developmental toxicity studies indicates that exposure to the test material does not cause any effects which requires the substance to be classified in line with Regulation 1272/2008.

Additional information