Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2006-04-17 to 2006-11-08
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study using a close structural analogue of the test material. Comparison of overall physico-chemical and toxicity profiles for target and source chemicals indicates it is appropriate to apply read-across data from the structural analogue when considering effects on fertility and prenatal development.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
errors in timing of physical examinations and clinical observations plus absence of lung float test on some dead pups; not considered to affect the quality or integrity of the study
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): [CAS Number 114959-46-5]
- Physical state: Opaque black liquid
- Analytical purity: 100 %
- Lot/batch No.: TS04020
- Expiration date of the lot/batch: 2006-07-01
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc, Raleigh, North Carolina
- Age at time of receipt: Approximately 62 days
- Age at study initiation: Approximately 10 weeks (males and females)
- Weight at study initiation: 331-391 g (males); 221-251 g (females)
- Age when paired on Day 13: Approximately 12 weeks
- Weight on gestation day zero: 227-284 g (females)
- Housing: Following receipt and until pairing, all F0 animals were housed individually in clean, stainless steel wire-mesh cages suspended above cage-board. The cage-board was changed at least 3 times per week. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire-mesh cages until the scheduled necropsy, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding (Bed-O'Cobs; The Andersons, Cob Products Division, Maumee, Ohio). The nesting material had been periodically analysed by the manufacturer for contaminants. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The dams and their litters were housed in these cages until euthanasia on lactation day 4.
- Diet: PMI Nutrition International LLC, Certified Rodent LabDiet 5002 provided ad libitum via feeders that were changed and sanitised at least once per week
- Water: Reverse osmosis-purified (on site) drinking water, delivered by an automatic watering system ad libitum
- Acclimation period: 10 days prior to the first day of treatment during which the animals were examined twice daily for mortality and general changes in appearance and behaviour.

ENVIRONMENTAL CONDITIONS
- Temperature: 71 ± 5 degrees Fahrenheit (22 ± 3 degrees Centigrade) controlled and monitored using the Metasys DDC Electronic Environmental system. Actual mean daily temperature ranged from 68.4 to 71.7 degrees Fahrenheit during the study (20.2 to 22.1 degrees Centigrade).
- Relative humidity: 50 ± 20 % controlled and monitored using the Metasys DDC Electronic Environmental system. . Actual mean daily relative humidity ranged from 36.1 % to 52.8 % during the study.
- Air changes: 10 per hour (minimum)
- Photoperiod: 12 hours light (06:00 to 18:00) and 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
corn oil
Details on exposure:
The vehicle used in preparation of test material formulations and for administration to the control group was corn oil (expiry date range 2 January 2007 to 7 March 2007), distributed by ACH Food Companies Inc, Memphis, Tennessee.

For the control group (Group 1), a sufficient volume of corn oil was added to a glass container. Aliquots of the vehicle were prepared for daily dispensing and stored at room temperature. The vehicle was stirred continuously throughout sampling, dispensing and dose administration.

Dosing formulations were prepared at concentrations of 10 mg/mL (Group 2, 50 mg/kg/day), 30 mg/mL (Group 3, 150 mg/kg/day) and 100 mg/mL (Group 4, 500 mg/kg/day).

The appropriate amount of test material for each group was weighed into a tared, calibrated, glass container. Vehicle was added to each container to bring the formulations nearly to the calibration mark. The formulations were mixed until uniform using a magnetic stirrer. Vehicle was then added to each container to the calibration mark and formulations were stirred until uniform using a magnetic stirrer.

Test material formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensing, and stored at room temperature. The test material formulations were stirred continuously throughout preparation, sampling and dose administration procedures. The test material formulations were visually inspected by the study director on 25 April 2006 and found to be visibly homogeneous and acceptable for dose administration.
Details on mating procedure:
The animals were paired on a 1:1 basis within each treatment group following 14 days of treatment for males and females. A breeding record containing the male and female identification numbers and the start date of cohabitation was prepared. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following vaginal lavage. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day zero. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages. For the purposes of calculating pre-coital intervals, rats paired over a 12 hour dark cycle were considered to have paired for one day.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test material formulations over the concentration range utilised in the present study (10 to 100 mg/mL) was established in a companion study for up to 13 days of room-temperature storage. Therefore study specific analyses were not performed.

Prior to initiation of dose administration, duplicate samples of 1 mL were collected on 25 April 2006 for from the top, middle and bottom strata of each dosing formulation, including the vehicle for the control group. In addition, duplicate samples of 1 mL were collected on 05 May 2006 for 10 day resuspension homogeneity determinations. The samples were taken from the top and bottom strata of aliquots of the same dosing formulations after mixing for a minimum of 10 minutes with a magnetic stirrer. Duplicate samples of 1 mL were collected weekly for concentration analysis. The samples were taken from the middle stratum of each dosing formulation (including the vehicle for the control group) and those collected from the first, fourth and last weekly formulations underwent analysis (see Appendix B, attached).

Formulations analysed met WIL standard operating procedure requirements for homogeneity and resuspenstion homogeneity. The Relative Standard Deviation (RSD) for the overall mean concentration was 10 % or less at a concentration within acceptable limits (within 15 % of target concentration). Based on these results, the analysed formulations used for dose administration met the WIL SOP requirement for concentration acceptability for suspension formulations.
Duration of treatment / exposure:
Days zero to 27 (males): 14 days prior to pairing until one day prior to scheduled euthanasia (28 doses)
Day zero to the day prior to euthanasia (females): 14 days prior to pairing until lactation day 3 (39-53 doses)
Day zero to the day prior to euthanasia (one female with no evidence of mating): 53 doses ending on post-cohabitation day 25
Frequency of treatment:
Once daily via gastric intubation, via an appropriately sized flexible, Teflon-shafted, stainless steel ball-tipped dosing cannula. Individual doses were based on the most recently recorded body weights. All animals were dosed at approximately the same time each day.
Details on study schedule:
18 April 2006: Animal receipt
28 April 2006: Initiation of test article administration
28 April to 25 May 2006: Test material administration (males)
28 April to 19 June 2006: Test material administration (females)
11 May to 25 May 2006: Mating period
26 May 2006: Male necropsy
20 June 2006: Last lactation day 4 necropsy
2 August 2006: Experimental termination (last microscopic examination)
Doses / concentrations
Remarks:
Doses / Concentrations:
5 mL/kg
Basis:
actual ingested
dosing formulations containing 10 mg/mL, 30 mg/mL or 100 mg/mL
No. of animals per sex per dose:
12 males and 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
Each animal was examined by a qualified technician on the day of receipt and weighed the following day. Each rat was uniquely identified by a Monel metal eartag displaying the animal number.

Near the end of the acclimation period, all available males and females were weighed and examined in detail for physical abnormalities. At the discretion of the study director, each animal judged to be in good health and meeting acceptable body weight requirements (300-500 g for males; 200-300 g for females) was selected for use in the computerised randomisation procedure. At that time, the individual body weights and corresponding animal identification numbers were entered into the WIL Toxicology Data Management System. A printout containing the animal numbers, corresponding body weights and individual group assignments was generated using a computer program which randomised the animals based on stratification of body weights in a block design. The animals were then arranged into groups according to the printout. Individual body weights at randomisation were within ± 20 % of the mean for each sex.

Positive control:
No data

Examinations

Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS AND SURVIVAL

All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual detailed clinical observations were recorded weekly (prior to test material administration during the treatment period). Each male and female was also observed. Each male and female was also observed for signs of toxicity at the time of dose administration and approximately 1-2 hours following dose administration. All significant findings were recorded.

BODY WEIGHTS
Individual male body weights were recorded weekly throughout the study and prior to the scheduled euthanasia. Individual female body weights were recorded weekly until evidence of copulation was observed. Once evidence of mating was observed, female body weights were recorded on gestation days zero, 4, 7, 11, 14, 17 and 20 and on lactation days 1 and 4.

FOOD CONSUMPTION
Individual food consumption was recorded weekly until pairing. Food intake was not recorded during the mating period. Once evidence of mating was observed, female food consumption was recorded on gestation days zero, 4, 7, 11, 14, 17 and 20 and on lactation days 1 and 4. When food consumption could not be determined for an animal during a given interval (due to weighing error, spillage, obviously erroneous value or similar), group mean values were calculated for that interval using available data.

PARTURITION

All females were allowed to deliver naturally and rear their young to Postnatal Day (PND) 4. During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. On the day parturition was initiated (PND zero), pups were sexed and examined for gross malformations. The numbers of stillborn and live pups was recorded. Individual gestation length was calculated using the date delivery started.
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
No data
Litter observations:
LITTER VIABILITY AND DEATHS

Each litter was examined daily for survival and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained. Standardisation of litter size was not performed because the pups were euthanised on PND 4.

CLINICAL OBSERVATIONS

Litters were examined daily for survival and any adverse changes in appearance or behaviour. Each pup received a detailed physical examination on PND 1 and 4. Any abnormalities in behaviour were recorded.

BODY WEIGHTS

Pups were individually weighed on PND 1 and 4. Mean pup weights were presented by sex for each litter and by dosage group.

SEX DETERMINATION

Pups were individually sexed on PND zero and 4.
Postmortem examinations (parental animals):
SCHEDULED EUTHANASIA

All F0 adults were euthanised by carbon dioxide inhalation. Males were euthanised following completion of the breeding period. Females that delivered were euthanised on lactation day 4. The number of former implantation sites and corpora lutea were recorded. Females with no evidence of mating were euthanised on post-cohabitation day 25. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10 % ammonium sulphide solution for detection of early implantation loss. Necropsy included examination of the external surface, all orifices and the cranial cavity, external surfaces of the brain, and the thoracic, abdominal and pelvic cavities, including viscera.

ORGAN RETENTION

At the time of necropsy, the following tissues and organs were placed in 10 % neutral-buffered formalin except where noted: Cervix; coagulating glands; mammary gland; ovaries and oviducts (2); pituitary gland; prostate gland; seminal vesicles (2); Testes with epididymides and vas deferens (2) fixed in Bouin's solution; thyroids (with parathyroids if present) (2); uterus with vagina but not retained if placed in ammonium sulphide solution; all gross lesions with representative sections of corresponding organs being retained from a sufficient number of controls if possible.

ORGAN WEIGHTS

The following organs were weighed from all F0 animals at the scheduled necropsies and, except where noted, paired organs were weighed together: Brain; epididymides with the paired organs weighed separately; kidneys; liver; ovaries with oviducts; pituitary gland; testes with the paired organs weighed separately; thyroid with parathyroids fixed in 10 % neutral-buffered formalin prior to weighing. Absolute weights, organ to final body weight and organ to brain weight ratios were reported.

MICROSCOPIC EXAMINATIONS

After fixation, protocol specified tissues were trimmed according to standard operating procedures and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned at 4 to 8 microns, mounted on glass microscope slides and stained with hematoxylin and eosin, with the following exceptions. PAS and hematoxylin staining were used for the right and left testes and epididymides. The testes were fixed in Bouin's solution and embedded in paraffin. Sections of 2 to 4 microns were made for the testes (1 transverse, 1 longitudinal) and the epididymis (longitudinal). Special emphasis was placed on the stages of spematogenesis and histopathology of interstitial testicular cell structure.

Microscopic examination was performed on all retained tissues for all animals in the control and 500 mg/kg/day groups at the scheduled necropsies. Gross lesions from all dosage groups were also examined. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, not in plane of section, or other reasons as appropriate. Microscopic examinations were performed by a senior pathologist.
Postmortem examinations (offspring):
A lung float test was performed for pups found dead on PND zero or 1. The lungs were removed and placed in a saline filled jar to determine whether the pub was stillborn (confirmed by sinking). Stillborn pups and intact offspring that died were necropsied using a fresh dissection technique including the heart and major vessels. The carcass of each pup was then discarded.

On PND 4, surviving F1 rats were euthanised via an intraperitoneal injection of sodium phenobarbital. Gross lesions and malformations (if any) observed at the detailed physical examination on PND 4 were preserved in the appropriate fixitive for possible future examination.
Statistics:
All statistical tests used appropriate computing devices or programs. Analyses were two-tailed (except where noted) for minimum significance levels 1 % and 5 %, comparing each treatment group to the control group by sex. Each mean was presented with the standard deviation (SD) and the number of animals (N) used to calculate the mean. In addition, percent difference from control is presented for body weights and organ weights. It was noted that, due to the use of significant figures and the different rounding conventions inherent in software, the means and standard deviations on the summary and individual tables may be slightly different. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit.

Mean parental mating, fertility, conception and copulation indices used the Chi-square test with Yates' correction factor. Mean parental body weights (weekly, gestation and lactation), body weight changes and food consumption, offspring body weights and body weight changes, gestation length, numbers of corpora lutea, implantation sites, number of pups born, live litter size on PND zero, unaccounted-for sites, absolute and relative organ weights and pre-coital intervals were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. Statistically significant (p < 0.05) intergroup variance was examined using Dunnett's test to compare the test material treated groups to the control group. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA to determine intergroup differences. Statistically significant (p < 0.05) intergroup variance was examined using Dunn's test to compare the test material treated groups to the control group. Histopathological findings in treatment groups were compared to controls via a two-tailed Fisher's Exact test.
Reproductive indices:
Mating, fertility and copulation/conception indices were calculated as follows:

Male (Female) Mating Index (%) = (Number of males (females) with evidence of mating or confirmed pregnancy / Total number of males (females) used for mating) * 100

Male Fertility Index (%) = (Number of males siring a litter / Total number of males used for mating) * 100

Male copulation Index (%) = (Number of males siring a litter / Number of males with evidence of mating or females with confirmed pregnancy) * 100

Female Fertility Index (%) = (Number of females with confirmed pregnancy / Total number of females used for mating) * 100

Female Conception Index (%) = (Number of females with confirmed pregnancy / Number of females with evidence of mating or confirmed pregnancy) * 100
Offspring viability indices:
Litter parameters were defined as follows:

Mean live litter size = Total number of viable pups on PND zero / Number of litters with viable pups on PND zero

Postnatal survival between birth and PND zero or PND 4 (% per litter) = [(¿ viable pups per litter on PND zero or PND 4 / Number of pups born per litter) / Number of litters per group] * 100

Postnatal survival for all other intervals (% per litter) = [(¿ viable pups per litter at end of interval N / Viable pups per litter at start of interval N) / Number of litters per group] * 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
salivation and pawing in males and females attributed to test material taste aversion (not considered indicative of systemic toxicity)
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
incidental findings not considered to be related to test material administration
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
incidental findings not considered to be related to test material administration
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

JUSTIFICATION FOR USE OF READ-ACROSS DATA

Comparison of overall physico-chemical and toxicity profiles for target and source chemicals indicates it is appropriate to apply read-across data from the structural analogue when considering effects on fertility and prenatal development.

PARENTAL ANIMALS (MALES)

CLINICAL OBSERVATIONS AND SURVIVAL
At the time of dose administration, salivation (just prior to or immediately following dose administration) and/or clear material around the mouth were noted on at least one and as many as 20 occasions for 4 males in the 150 mg/kg/day group and for 11 males in the 500 mg/kg/day group during the treatment period. Salivation and/or salivation related findings (consisting of clear material on various body surfaces) were also noted on several occasions for 4 males in the 150 mg/kg/day group and 10 males in the 500 mg/kg/day group 1-2 hours following administration. However, because the onset of salivation and clear material around the mouth occurred immediately prior to or following daily dose administration these findings were not considered indicative of systemic toxicity. Excessive pawing of the cage floor and/or walls was also seen at the time of dose administration for 6 males in the 500 mg/kg/day group on 1-2 occasions each during study days 6-27. This finding was attributed to test material taste aversion and was not considered evidence of systemic toxicity. There were no test material related clinical findings for males in the 50 mg/kg/day group.

BODY WEIGHTS
Lower mean body weight gains were noted at 150 and 500 mg/kg/day during study days 0-7 but the differences were not statistically significant when compared to the control group. Because all males in the 150 and 500 mg/kg/day groups, with the exception of one 150 mg/kg/day male, gained weight during this interval (9 g to 35 g compared to 9 g to 50 g in the control group), the lower mean body weight gains in these groups were not considered to be related to the test material. Mean body weight gains in these groups were generally similar to control group values during the remainder of the treatment period. Mean body weights at 150 and 500 mg/kg/day were similar to the control group values during the pre-mating (days 0-13) and entire treatment (days 0-28) periods.

There were no test material related effects on mean body weights or body weight gains for males in the 50 mg/kg/day group during the pre-mating (days 0-13) and entire treatment (days 0-28) periods. Differences from the control group were not statistically significant and did not occur in a dose related manner.

FOOD CONSUMPTION
Mean food consumption, evaluated as g/animal/day and g/kg/day, was unaffected by test material administration in the 50, 150 and 500 mg/kg/day groups during the pre-mating period. Although mean food consumption at 500 mg/kg/day was statistically significantly lower (p < 0.05 or p < 0.01) than that of the control group during study days 0-7, all males in this group consumed a minimum of 19 g/animal/day (compared to a minimum of 22 g/animal/day in the control group) and gained weight during this interval. Food consumption in the 500 mg/kg/day group was similar to the control group during the remainder of the pre-mating period (days 7-13) and when the entire pre-mating period (days 0-13) was evaluated.

ORGAN WEIGHTS
There were no test material related adverse effects on male organ weights. Marginal increases in liver weights (relative to body) of male rats given 500 mg/kg/day were attributed to the test material but are probably consistent with hepatic enzyme induction that was adaptive in nature. Similar effects on liver weights with no morphologic correlates were reported in a previous study. No other test material effects on male organ weights were noted during this study.

Althought mean left and right absolute testis weights in the 500 mg/kg/day group were 6.7 % lower (not statistically significant) than the concurrent control group values, mean testis weights in this group (1.66 g each) were within the range of values in the WIL historical control data (1.40 g to 1.68 g). Additionally, because functional reproductive endpoints were unaffected in this group and no histologic correlates were noted, the lower weights were not considered to be related to test material administration. The mean absolute kidney weights in the 500 mg/kg/day group (3.33 g) was lower than the concurrent control group value but was within the range of values in the WIL historical control data (2.10 to 3.48 g). Therefore the difference was not attributed to the test material.

Kidney (12.1 % decrease in 500 mg/kg/day group), liver (5.7 % increase in 500 mg/kg/day group) and left epididymis (6.9 % increase in 50 mg/kg/day group) weight changes were statistically significant (p < 0.05 or p < 0.01) when compared to the control group. However, differences in absolute weights and weights relative to body or brain weight were small in magnitude or the dose association was incoherent, and the changes were considered not to be toxicologically significant.

MACROSCOPIC EXAMINATIONS
There were no test material related gross necropsy observations in the 50, 150 or 500 mg/kg/day groups. Findings noted for males at the scheduled necropsy occurred in single animals and/or are common for this strain of rat.

MICROSCOPIC EXAMINATIONS
There were no test material related histologic changes for males at 500 mg/kg/day and none of the differences from the control group were statistically significant. In addition, no test material related histologic changes were noted at any dosage level in tissues that were examined based in gross lesions only.

All gross and histologic changes encountered in this study were considered to be incidental findings, manifestations of spontaneous diseases, or related to some aspect of experimental manipulation other than test material administration. There was no test material related alteration in the incidence, severity or histologic character of those incidental and spontaneous tissue alterations.

REPRODUCTIVE PERFORMANCE
No test material related effects on F0 male reproductive performance were observed at any dosage level. Male mating, fertility and copulation indices were 100 %, 91.7 %, 100 % and 100 % in the control, 50, 150 and 500 mg/kg/day groups respectively. No statistically significant differences were noted betwen the control and test material treated groups.

PARENTAL ANIMALS (FEMALES)

CLINICAL OBSERVATIONS AND SURVIVAL
In the 500 mg/kg/day group, excessive pawing of the cage floor and/or walls was noted for all 12 females (5-25 occurances each) at the time of dose administration, and wiping of the mouth on the cage floor and/or walls following dose administration was noted for 9 females (primarily 1-2 occurrances each) in this group. The effect was mainly noted during study day 4 to lactation day 2. Excessive pawing and/or wiping of the mouth on the cage floor and/or walls was noted for 8 females in this group 1-2 hours following dose administration. Salivation and/or salivation related findings (consisting of clear material on various body surfaces) were noted for all females in the 500 mg/kg/day group just prior to, at the time of and/or 1-2 hours following dose administration.

In the 150 mg/kg/day group, excessive pawing of the cage floor and/or walls was noted for all 12 females (1-16 occurances each) at the time of dose administration, and wiping of the mouth on the cage floor and/or walls following dose administration was noted for 4 females in this group (1-2 occurrances each) primarily during study day 6 to lactation day 1. Only one female in this group demonstrated excessive pawing of the cage floor and/or walls 1-2 hours following dose administration. Salivation and/or clear material around the mouth were noted for up to 8 females in the 150 mg/kg/day group during the treatment period just prior to, at the time of and/or 1-2 hours following dose administration.

The salivation related findings noted in the 150 and 500 mg/kg/day groups were considered to be test material related but were not considered indicative of systemic toxicity due to the onset of these findings (immediately prior to or following daily dose administration). The behavioural findings in these groups, consisting of excessive pawing of the cage floor and/or walls and wiping of the mouth on the cage floor and/or walls, were attributed to test material taste aversion and not to systemic toxicity.

There were no test material related clinical findings for females in the 50 mg/kg/day group. Findings in this group occurred infrequently and/or were also noted in the control group.

BODY WEIGHTS
There were no test material related effects on mean female body weights or body weight gains in the 50, 150 and 500 mg/kg/day groups during the pre-mating period. Differences from the control group were slight and not statistically significant.

No test material related effects on mean maternal body weights or body weight gains were noted during gestation at any dosage level. Differences from the control group were slight and not statistically significant.

Mean body weights and body weight gains were unaffected by test material administration during the early lactation period at all dosage levels. Althought mean body weight gains in the 50 and 500 mg/kg/day groups were statistically significantly lower (p < 0.01 or p < 0.05) than the control group during lactation days 1-4, mean body weights in these groups were similar to control group values, and no dose-response relationship was observed.

FOOD CONSUMPTION
Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 50, 150 and 500 mg/kg/day group females was unaffected by test material administration during the pre-mating period. None of the differences from the control group were statistically significant.

No test material related effects on food consumption were noted during gestation at any dosage level during gestation. Differences from the control group did not occur in a dose related mannae and were not statistically significant.

Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 50, 150 and 500 mg/kg/day group females was unaffected by test material administration during the early lactation period. The values in the test material treated groups were generally similar to control group values and not statistically significant differences were observed.

ORGAN WEIGHTS
The following organ weight changes were statistically significant (p < 0.05 or p < 0.01) differnences when compared to the control group. Although higher liver weight was considered to be related to test material administration, the difference is probably consistent with hepatic enzyme induction that was adaptive in nature. Therefore the difference from the control group was not considered adverse. Other organ weight differences in absolute weights and weights relative to body or brain weight (liver weight increased by 14.6 % in the 500 mg/kg/day group and pituitary gland weight increased by 19.2 % in the 150 mg/kg/day group) were small in magnitude or the dose association was incoherent. Thus the changes were not considered to be toxicologically significant.

MACROSCOPIC EXAMINATIONS
At the scheduled F0 female necropsy, no test material related internal findings were observed at any dosage level. Macroscopic findings observed in the test material treated groups occurred in single females. There were no macroscopic findings for the female in the 50 mg/kg/day group that had no evidence of mating.

No test material related effects were observed on the numbers of former implantation sites and unaccounted-for sites. The differences between the control and test material treated groups were slight and not statistically significant.

MICROSCOPIC EXAMINATIONS
There were no test material related histologic changes in females at 50, 150 or 500 mg/kg/day. All gross and histologic changes encountered in the study were considered to be incidental findings, manifestations of spontaneous diseases, or related to some aspect of experimental manipulation unrelated to administration of the test material. There was no test material related alteration in the incidence, severity or histologic character of those incidental and spontaneous tissue alterations.

REPRODUCTIVE PERFORMANCE
No test material related effects on F0 reproductive performance were observed at any dose level. Female mating, fertility and conception indices were 100 %, 91.7 %, 100 % and 100 % in the control, 50, 150 and 500 mg/kg/day groups. No statistically significant differences were noted between the control and test material treated groups.

The mean number of days between pairing and coitus in the test material treated groups were not statistically significantly different from the control group value.

GESTATION LENGTH AND PARTURITION
Mean gestation length in the 50, 150 and 500 mg/kg/day groups were similar to that in the control group and none of the differences were statistically significant. No signs of dystocia were noted in these groups.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Male and female reproductive parameters were unaffected by test material administration and no evidence of parental systemic toxicity was noted at dosage levels of 50, 150 or 500 mg/kg/day

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
majority of observations in the 500 mg/kg/day group were noted for one pup; one pup in the control group was presumed cannibalised
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

PND ZERO LITTER DATA AND POSTNATAL SURVIVAL
The mean number of pups born, mean live litter size on PND zero and the percentage of males at birth in the 50, 150 and 500 mg/kg/day groups were not statistically significantly different than the concurrent control group values. postnatal survival from birth to PND 4 in the test material treated groups was unaffected by test material administration.

GENERAL PHYSICAL CONDITION AND MORTALITIES
The numbers of F1 pups found dead, as well as the general physical condition of all F1 pups in this study were unaffected by test material administration. The majority of the clinical findings observed in the 500 mg/kg/day group were noted for one pup (number 26118-01) that was found dead (laboured respiration, small in size and subcutaneous haemorrhage). Pups (litters) that were found dead numbered 2(2), 8(2), 1(1) and 5(4) in the control, 50, 150 and 500 mg/kg/day groups respectively. One pup in the control group was missing and presumed to have been cannibalised.

OFFSPRING BODY WEIGHTS
Mean male and female pup body weights and body weight gains in the 50, 150 and 500 mg/kg/day groups were unaffected by test material administration. Differences from the control group were not statistically significant.

NECROPSIES OF PUPS FOUND DEAD
The numbers of pups (litters) found dead from PND 0 to PND 4 numbered 2(2), 8(2), 1(1) and 5(4) in the control, 50, 150 and 500 mg/kg/day groups respectively. No internal findings that could be attributed to F0 maternal administration of the test material were noted at the necropsies of pups that were found dead. With the exception of one pup in the 500 mg/kg/day group, milk was not present in the stomach of pups that were found dead. The results of the lung float test indicated that one pup in each of the 150 and 500 mg/kg/day groups was born alive but later died, and one pup each in the control and 50 mg/kg/day groups was stillborn. However, there were several pups found dead on PND 0 or 1 (pup numbers 1-7 from female 26245 in the 50 mg/kg/day group and pup number 1 from female 26226 in the 500 mg/kg/day group) for which there was no record of a lung float test.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Pup growth and survival was unaffected by administration of test material to parental animals at dosage levels of 50, 150 and 500 mg/kg/day

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

See comparison of overall physico-chemical and toxicity profiles for target and source chemicals in the data matrix (attached).

Applicant's summary and conclusion

Conclusions:
Male and female reproductive parameters were unaffected by test material administration and no evidence of parental systemic toxicity was noted in any group. The NOAEL for F0 reproductive and systemic toxicity of the test material was therefore considered to be 500 mg/kg/day. Pup growth and survival was unaffected by the test material at all dosage levels and the NOAEL for F1 neonatal toxicity was considered to be 500 mg/kg/day. Dose correction for purity (active ingredient 43% in oil) meant that the final dose levels were actually 21.5, 64.5 and 215mg/kd bw/day, with a NOAEL for both parental and F1 generation of 215mg/kg bw/day.
The target material for Registration is considered likely to behave so similarly to this test material that the NOAELs from this study can be applied to the Registration material, as agreed with ECHA.
Executive summary:

Male and female reproductive parameters were unaffected by test material administration and no evidence of parental systemic toxicity was noted in any group. The NOAEL for F0 reproductive and systemic toxicity of the test material was therefore considered to be 500 mg/kg/day. Pup growth and survival was unaffected by the test material at all dosage levels and the NOAEL for F1 neonatal toxicity was considered to be 500 mg/kg/day. Dose correction for purity (active ingredient 43% in oil) meant that the final dose levels were actually 21.5, 64.5 and 215mg/kd bw/day, with a NOAEL for both parental and F1 generation of 215mg/kg bw/day.

The target material for Registration is considered likely to behave so similarly to this test material that the NOAELs from this study can be applied to the Registration material, as agreed with ECHA.