Phosphorous acid, mixed 2,4-bis(1,1-dimethylpropyl)phenyl and 4-(1,1-dimethylpropyl)phenyl triesters

Toxicity to reproduction

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

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.

Test guideline 2

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.

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of bis[2,4-bis(2-methylbutan-2-yl)phenyl] 4-(2-methylbutan-2-yl)phenyl phosphite and 2,4-bis(2-methylbutan-2-yl)phenyl bis[4-(2-methylbutan-2-yl)phenyl] phosphite and tris[4-(2-methylbutan-2-yl)phenyl] phosphite.

EC Number:

700-485-5

Cas Number:

939402-02-5

Molecular formula:

Mixture of 4 components, the molecular formulae of which are: C33 H45 O3 P, C38 H55 O3 P, C43 H65 O3 P and C48 H75 O3 P

IUPAC Name:

Reaction mass of bis[2,4-bis(2-methylbutan-2-yl)phenyl] 4-(2-methylbutan-2-yl)phenyl phosphite and 2,4-bis(2-methylbutan-2-yl)phenyl bis[4-(2-methylbutan-2-yl)phenyl] phosphite and tris[4-(2-methylbutan-2-yl)phenyl] phosphite.

Test material form:

liquid: viscous

Details on test material:

Test substance information (batch A)
Identification: DVS 005 (Weston 705T)
CAS Number: 939402-02-5
Description: Clear colourless viscous liquid (determined at WIL Research Europe B.V.)
Batch: MW3G18T701
Purity/Composition: 100%
Test substance storage: In refrigerator (2-8°C) under nitrogen, desiccated
Stable under storage conditions until: 30 November 2014 (expiry date)

Test substance information (batch B)
Batch B was used to make diets from 12-May-2014 onwards (for use 19-May-2014 onwards). Test substance information for Batch B is the same as for Batch A, except for the following:
Batch: MW3N14T701
Stable under storage conditions until: 13 December 2014 (expiry date)
pH: 6.5-7.5*
*pH was not indicated for batch A

Study specific test substance information
Purity/composition correction factor required: No
Hygroscopic: Yes, desiccated
Volatile: No
Reactivity: Reactive to moisture
Test substance handling: Flush container with nitrogen after handling
Specific Gravity / Density: 1.0180 (25/15.5°C)
pH: Not indicated
Stability at higher temperatures: Yes, maximum temperature: 70°C
Stability in diet: Stability in diet over 29 days at room temperature (range of 1500-15000 mg test substance/kg diet) was confirmed in Project 503366.

Test animals

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.

Administration / exposure

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.

Doses / concentrations

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.

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

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.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

5 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

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)

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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'

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

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 

Applicant's summary and conclusion

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.