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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 June 2004 to 22 July 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted according to an internationally recognized protocol and following appropriate GLP standards. The study was conducted as a screening test only and does not have all developmental parameters measured as in an OECD 414.
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
9 June 2004 to 22 July 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to an internationally recognized protocol and following appropriate GLP standards.
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Remarks:
2003-02-13
Limit test:
no
Specific details on test material used for the study:
CYANOX® 1790 Antioxidant
IUPAC nomenclature - Tris(4-tert-butyl-3-hydroxy-2,6-dimethylbenzyl) isocyanurate
Synonym - [tris(4-t-butyl-3-hydroxy-2,6-dimethyl-benzyl)-s-triazine-2,4,6-(1H,3H,5H) trione]
Lot PA4040608
Appearance - White powder, odorless
CAS No. 40601-76-1
Molecular Formula - C42H57N3O6
Molecular Weight - 699.92 g/mole
Purity 96-97%
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague-Dawley Cr1:CD (SD) IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Manston Road, Margate, Kent
- Weight at study initiation: Males: 340-394 g; Females: 209-243 g
- Fasting period before study: none
- Housing: Initially housed in groups of five (5) animals by sex in polypropylene cages with stainless steel grid floors and tops, suspended over paper-lined polypropylene trays. During mating animals transferred to similar cages with one male and one female per cage. Following evidence of successful mating, males returned to original cages. Mated and presumed pregnant females housed individually in polypropylene cages with solid floors and stainless steel tops. Mated females given softwood chips as bedding throughout gestation and lactation.
- Diet (ad libitum): Certified Rodent Diet PMI 5002, IPS Product Supplies Ltd., London (UK)
- Water (ad libitum): mains water supplied in polycarbonate bottles
- Acclimation period: seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 9 June 2004 to 22 July 2004
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION

A known amount of test compound was mixed with basal laboratory diet for twenty minutes at a constant speed (setting 1, Hobart QE800 mixer). Analytical measurements prior to the start of the study indicated the dietary mixture to be homogenous and stable for at least 14 days.
Details on mating procedure:
One male and one female were paired for a period of up to fourteen days. Following pairing, the polypropylene trays beneath each cage were checked each morning for the presence of ejected copulation plugs. Additionally, each female was checked for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating. Mated females were separated and housed individually during the period of gestation and lactation. Males were returned to original holding cages.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the subject chemical in dietary admixtures was determined by high performance liquid chromatography (HPLC) using an external standard technique. Samples were extracted with acetonitrile to give a final, theoretical test material concentration of approximately 100 ppm. The dietary mixtures were sampled sampled and analyzed within four (4) days of preparation for verification of formulation concentrations. Measured concentrations varied from 97 to 107% of nominal.
Duration of treatment / exposure:
Test material was administered for up to 54 days by dietary admixture. Control animals were handled in the same manner to those receiving the test material.
Both male and female animals were dosed 14 days prior to pairing. Dosing continued until surviving parental animals were sacrificed on Day 5 post partum.
Frequency of treatment:
Continuous
Details on study schedule:
Administration of doses was started 14 days prior to mating. Mating was allowed to proceed for up to 14 days. Pregnant females were allowed to deliver offspring. Offspring were observed for growth and development during lactation up to Day 4 post partum. Surviving adults and offspring were euthanized on Day 5 post partum and examined macroscopically.
Dose / conc.:
64 mg/kg bw/day (actual dose received)
Remarks:
Males 1000 ppm
Dose / conc.:
651 mg/kg bw/day (actual dose received)
Remarks:
Males 10000 ppm
Dose / conc.:
1 264 mg/kg bw/day (actual dose received)
Remarks:
Males 20000 ppm
Dose / conc.:
77 mg/kg bw/day (actual dose received)
Remarks:
Females 1000 ppm
Dose / conc.:
782 mg/kg bw/day (actual dose received)
Remarks:
Females 10000 ppm
Dose / conc.:
1 558 mg/kg bw/day (actual dose received)
Remarks:
Females 20000 ppm
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were selected based on the outcome of a preliminary 14-day oral gavage range-finding study.
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: all animals were checked twice daily for morbidity and mortality during the normal working week and once daily on weekends and holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all animals were examined for overt signs of toxicity, ill health or behavioural change once daily. Abnormal findings were recorded daily until resolution of finding or study termination.

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were recorded on Day 0 before start of treatments and weekly thereafter for males until termination. Females were weighed weekly until mating was evident. Parental females showing evidence of mating were weighed on Days 0, 7, 14 and 20 post coitum. Parental generation females with a live litter were weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- During the maturation period food consumption was recorded weekly for each cage of parental generation adults. For parental generation females showing evidence of mating, food consumption was recorded for the periods covering Days 1 to 7, 7 to 14 and 14 to 20 post coitum. For parental generation females with live litters, food consumption was recorded for the period covering Days 1 to 4 post partum.
- Group mean weekly food consumption was calculated (as g/rat/day)
- Weekly food efficiencies were calculated (group mean body weight gain (as g/rat/week) / group mean food consumption (as g/rat/week))

WATER CONSUMPTION
- Time schedule for examinations: daily visual inspections of water bottles

HISTOLOGY/HISTOPATHOLOGY
The following organs were collected and preserved in buffered 10% formalin and examined at histopathology for all adult males and females from the control and high dose groups: coagulating glands; epididymides; prostate; seminal vesicles; testis; pituitary; ovaries; uterus/cervix; vagina; skin (from top of head). Skin samples from all groups were prepared as paraffin blocks, sectioned at nominal thickness of 5 mm and stained with hematoxylin and eosin for subsequent microscopic examination. All other tissues from control and 20000 ppm dose group animals were treated in similar manner.

OTHER: Each pregnant female was observed at 8:30, 12:30 and 16:30 hours at or around the period of expected parturition. On weekends, observations were carried out at 8:30 and 12:30 hours only. The following were recorded for each female:
- date of mating
- date and time of observed start of parturition
- date and time of observed completion of parturition
- duration of gestation
Oestrous cyclicity (parental animals):
not determined
Sperm parameters (parental animals):
not determined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number of pups born
- number and sex of pups alive recorded daily and on Day 1 and 4 post partum
- clinical condition of pups from birth to Day 4 post partum
- individual litter weights on Day 1 and 4 post partum

All live offspring were observed for detachment of pinna (as noted by the separation of the edges and subsequent unfolding of both pinnae) and assessed for reflexological response to stimuli by assessing surfact righting reflex on Day 1 post partum.

GROSS EXAMINATION OF DEAD PUPS:
Offspring dying during the study were subjected to a full external and internal examination, and any macroscopic abnormalities recorded.
Postmortem examinations (parental animals):
All adult (P) animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. In addition, corpora lutea and implantation sites of all females were counted. This procedure was enhanced by staining the uteri with 1% ammonium polysulphide solution.
Postmortem examinations (offspring):
All surviving offspint were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
Group mean values and standard deviation were derived where appropriate. Weekly bodyweight, bodyweight gain, food consumption, adult organ weights, litter sizes, litter weight and offspring bodyweights were assessed for dose response relationships by analysis to establish homogeneity of group variances using Bartlett's test followed by one-way analysis of variance. If variances were unequal subsequent comparisons between control and treated groups were performed using t-test assuming unequal variances. If variances were equal subsequent comparisons between control and treated groups were performed using Dunnett's Multiple Comparison Method.

Offsping landmarks of physical development, offspring reflexological responses, pre- and post-implantation losses, litter sex ratios and relative organ weights were compared using Kruskal-Wallis non-parametric rank sum test. Post implantation losses in the 10000 ppm group were also compared using Grubb's test (parametric test to determine outliers).

For histopathology observations: Chi-squared analysis was used for differences in lesions occurring with an overall frequency of 1 or greater; Kruskal-Wallis on-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed conditions.
Reproductive indices:
Pre-coital interval: calculated as time elapsing between initial pairing and the observation of positive evidence of mating.
Fertility indices: mating index (%) = number animals mated/number paired x 100; pregnancy index (%) = number pregnant females/number animals mated x 100.
Gestation length: calculated as the number of days of gestation including the day for observation of mating and start of parturition. Where the start of parturition occurred overnight, the total was adjusted by subtracting half a day.
Parturition index (%) = number of females delivering live pups/number of pregnant females x 100
Offspring viability indices:
Live birth index (%) = number of pups alive on Day 1/number of pups born x 100
Viability index (%) = number of pups alive on Day 4/number of pups alive on Day 1 x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 20000 ppm three males and all females showed scabs and fur loss on the top of the head and/or around the snout from Day 22 of treatment onwards.

At 10000 ppm all females showed scabs and fur loss on the top of the head and/or around the snout from Day 22 of treatment onwards. For all but one female, these signs persisted until completion of the study. The skin irritation on the top of the head and/or around the snout is considered an adverse effect of treatment due to the unique nature and severity of the irritancy.

At 1000 ppm there were no clinical signs of toxicity or irritancy observed throughout the study.

One control female had approximately 2 inches of skin missing from the end of the tail exposing underlying tissue.
Mortality:
no mortality observed
Description (incidence):
There were no treatment-related mortalities through the course of the study at any dose level.
One control female was killed in extremis due to an injury to the tail. No other macroscopic abnormalities were observed at post mortem examination.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant effects on bodyweight gain were observed for animals of either sex throughout maturation or for females during gestation or early lactation. Bodyweight development in test animals was similar to that of controls.

At 20000 ppm there was a reduction ini female bodyweight gain for females between days 14 and 20 of gestation. This resulted in a slight, but not statistically significant difference in group mean female bodyweight at day 20 of gestation.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There was a statistically significant increase in the food eaten during the first week of treatment in male rats at 20000 and 10000 ppm compared to controls. This was considered to be incidental due to the lack of a dose related response and comparable female food consumption.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles revealed no intergroup differences.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related effects.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no inter-group differences in group mean number of corpora lutea or implantation sites counted post mortem. Pre-implantation losses were comparable to controls across all dose groups. The percentage of pre-implantation losses was slightly lower at 10000 ppm compared to controls but this was not statistically significant. At 20000 ppm, post-implantation losses were increased non-significantly compared to controls but the findings were judged toxicologically significant. At 10000 ppm, post-implantation losses were also increased non-significantly compared to controls but this result was primarily due to one female with a total loss of litter in utero. This was determined to be an outlier and not representative of the group. Reanalysis with removal of this female data showed no differences from control.

Fur loss and scabbing was observed for all females at 20000 ppm, predominantly on the head. Similar skin lesions were noted on the abdomen of two females at this dose level. Three males also showed fur loss and scabbing. There were no effects observed upon reproductive or other organs. At 10000 ppm, fur loss and scabbing similar to the high dose group was observed for 9 of 10 females. Other observations were considered incidental and not related to test article administration.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Acanthosis, occasionally with focal epithelial ulceration and overlying scab formation, was observed in relation to treatment in the majority of females dosed at 20000 ppm (p<0.001) or at 10000 ppm (p<0.01). One male at 20000 ppm was also affected. Other histopathological findings were those commonly encountered as a result of pregnancy or commonly observed in laboratory rats of this age and strain.
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
There were no apparent effects on mating performance or fertility or gestation length. At the 20000 ppm dose level, there was no evidence of mating for one mating pair but a live litter was produced. At 10000 ppm, one female was found to be not pregnant after positive evidence of mating. A second female at this dose level was found to have a total litter loss in utero. All 1000 ppm females produced a live litter. In the control, one pair failed to mate within the 14 day mating period.
Dose descriptor:
NOEL
Effect level:
64 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
1000 ppm in diet
Sex:
male
Basis for effect level:
other: Local irritation
Dose descriptor:
NOEL
Effect level:
77 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
1000 ppm in diet
Sex:
female
Basis for effect level:
other: Local irritation
Dose descriptor:
NOAEL
Effect level:
782 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
10000 ppm in diet
Sex:
female
Basis for effect level:
reproductive performance
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no inter-group differences in the type or incidence of clinical findings. Findings observed are those commonly seen.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
At 20000 ppm there was a slightly lower mean litter size at birth compared to control values. The difference was not statistically significant. There was a higher value for the group mean post implantation loss when compared to control values. The large intra-group variability for this parameter means that statistical significance was not achieved. Although group mean litter size was not statistically significantly different to controls, the number of females with litters with 10 or fewer offspring was higher compared to controls (no statistical analysis). Offspring viability between birth and day 4 of lactation was comparable to control values.

At 1000 and 10000 ppm there were no significant effects upon live litter size at birth. Offspring viability between birth and day 4 of lactation was comparable to control values.
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
Description (incidence and severity):
There were no treatment-related findings.
Histopathological findings:
not examined
There were no inter-group differences in the type or incidence of clinical findings. There were no treatment-related effects on the onset or completion of pinna unfolding or the group mean percentages of pups passing the surface righting assessment on Day 1 post partum.

There were no treatment-related macroscopic findings.
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 558 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
20 000 ppm in diet
Sex:
female
Basis for effect level:
other: No significant adverse effects

Summary Results

 Observations              Dietary Concentration (ppm)
     0 (Control)  1000 ppm  10000 ppm  20000 ppm
 Mated pairs (N)  n (number)  9  10  10  10
 Females showing evidence of copulation  n  8  10  10  10
 Pregnant females  n  8  10  9  10
 Conception days 1 -5  n 7  9  10  8
 Conception days 6 to 14  n  2  4  1  0
 Gestation = 21.5 to 22 days  n  2  4  1  0
 Gestation = 22.5 to 23 days  n  5  6  7  6
 Gestation = 23.5 to24 days  n  1  0  0  3
 Dams with live young born  n  8  10  8  9
 Dams with live young at Day 4 post partum  n  8  10  8  10
 Corpora lutea/dam  x (mean)  15  18  14  15
 Implants/dam  x  14  17  13  14
 Live pups/dam at Day 1 post partum  x  14  16  13  11
 Live pups/dam at Day 4 post partum  x  13  15  13  11
 Sex ratio % males at Day 1 post partum  x  42  53  60  53
 Sex ratio % males at Day 4 post partum  x  43  53  60  53
 Litter weight at Day 1 post partum  x  92.2  96.7  88.6  74.1
 Litter weight at Day 4 post partum  x  118.9  128.0  119.2  109.5
 Pup weight at Day 1 post partum  x  6.9  6.1  7.0  6.7
 Pup weight at Day 4 post partum  x  9.6  8.3  9.7  10.0
 ABNORMAL PUP/DAM          
 0  7  8  6  10
 1 n  0  2  2  0
 >/= 2  n  1  0  0  0
LOSS OF OFFSPRING/DAM           
 0  n  2  3  5  5
 1  n  4  4  3  2
 2  n  1  1  0  1
 >/= 3  n  1  1  2  2
 Pre-natal (implantation minus live birth)          
 0  n  5  4  2
 1  n  2  1  4  1
 2  n  1  4  2  3
 >/= 3  n  0  0  2  4
 Post natal (live births minus offspring alive on Day 4 post partum)          
 0  n  6  9  8  10
 1  n  1  0  1  0
 2  n  0  0  1  0
 >/= 3  n  1  1  0  0
           
Conclusions:
The No Observed Effect Level (NOEL) for parental animals was 1000 ppm based on irritancy which was considered an adverse effect. The No Observed Adverse Effect Level (NOAEL) for effects on fertility was 10000 ppm due to increased post implantation loss and consequential decreased mean litter size at birth at the 20000 ppm. The No Observed Effect Level for developmental toxicity/teratogenicity was 20000 ppm.
Executive summary:

This study was designed to screen for potential adverse effect on reproduction including embryo/foetal development in the rat following OECD TG 421 and under GLP conditions.

The test material was administered by dietary admixture to three groups, each of ten male and ten female rats, for up to 54 consecutive days at dietary concentrations of 1000, 10000 and 20000 ppm (equivalent to mean achieved dosages of 64, 651, 1294 and 77, 782 and 1558 mg/kg bw/day for males and females, respectively). A further group of animals served as control. Following 14 days of dosing, male and female rats were paired within their dose groups to produce litters. On Day 5 post partum, all surviving animals were euthanised and examined macroscopically. Clinical signs, bodyweight development and food and water consumption were monitored during the study. Offspring development and growth was also monitored. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from control and high dose parental males and females was performed.

For adults, there were no treatment-related deaths during the study. At 10000 ppm and 20000 ppm, adverse signs of irritancy were observed in males and females. There were no treatment-related effects on body weight, food consumption or water consumption. Mating performance and fertility were not adversely affected by the treatment. No treatement-related effects were observed for organ weights or necropsy findings.

At 20000 ppm there was in increase in post implantation loss which was not statistically significant compared to controls but was of toxicological significant because of the intragroup variation in individual post implantation loss values. At 10000 ppm there was also a high post implantation loss but this was due to a single female with a total litter loss in utero.

For offspring, at 20000 ppm, there was a slightly lower mean litter size at birth compared to control values but this was not statistically significant. Offspring viability between birth and day 4 of lactation was comparable to control values. For all other observations, there were no treatment-related effects observed.

Administration of the test material to male and female rats throughout maturation, gestation and lactation phrases of reproduction resulted in signs of test material irritancy at 10000 and 20000 ppm. The irritancy was considered to be an adverse effect due to its degree of severity. Therefore, the NOEL for adults was 1000 ppm (66 -74 mg/kg). The NOAEL for effects on fertility was 10000 ppm (782 mg/kg; due to increased post implantation loss and a consequential decrease in mean litter size at birth at 20000 ppm). The NOEL for developmental toxicity/teratogenicity was 20000 ppm (1294 -1558 mg/kg).

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 421
GLP compliance:
yes (incl. certificate)
Remarks:
2003-02-13
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
CYANOX® 1790 Antioxidant
IUPAC nomenclature - Tris(4-tert-butyl-3-hydroxy-2,6-dimethylbenzyl) isocyanurate
Synonym - [tris(4-t-butyl-3-hydroxy-2,6-dimethyl-benzyl)-s-triazine-2,4,6-(1H,3H,5H) trione]
Lot PA4040608
Appearance - White powder, odorless
CAS No. 40601-76-1
Molecular Formula - C42H57N3O6
Molecular Weight - 699.92 g/mole
Purity 96-97%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Manston Road, Margate, Kent
- Weight at study initiation: Males: 340-394 g; Females: 209-243 g
- Fasting period before study: none
- Housing: Initially housed in groups of five (5) animals by sex in polypropylene cages with stainless steel grid floors and tops, suspended over paper-lined polypropylene trays. During mating animals transferred to similar cages with one male and one female per cage. Following evidence of successful mating, males returned to original cages. Mated and presumed pregnant females housed individually in polypropylene cages with solid floors and stainless steel tops. Mated females given softwood chips as bedding throughout gestation and lactation.
- Diet (ad libitum): Certified Rodent Diet PMI 5002, IPS Product Supplies Ltd., London (UK)
- Water (ad libitum): mains water supplied in polycarbonate bottles
- Acclimation period: seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 9 June 2004 to 22 July 2004

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION

A known amount of test compound was mixed with basal laboratory diet for twenty minutes at a constant speed (setting 1, Hobart QE800 mixer). Analytical measurements prior to the start of the study indicated the dietary mixture to be homogenous and stable for at least 14 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the subject chemical in dietary admixtures was determined by high performance liquid chromatography (HPLC) using an external standard technique. Samples were extracted with acetonitrile to give a final, theoretical test material concentration of approximately 100 ppm. The dietary mixtures were sampled sampled and analyzed within four (4) days of preparation for verification of formulation concentrations. Measured concentrations varied from 97 to 107% of nominal.
Details on mating procedure:
One male and one female were paired for a period of up to fourteen days. Following pairing, the polypropylene trays beneath each cage were checked each morning for the presence of ejected copulation plugs. Additionally, each female was checked for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating. Mated females were separated and housed individually during the period of gestation and lactation. Males were returned to original holding cages.
Duration of treatment / exposure:
Test material was administered for up to 54 days by dietary admixture. Control animals were handled in the same manner to those receiving the test material.
Both male and female animals were dosed 14 days prior to pairing. Dosing continued until surviving parental animals were sacrificed on Day 5 post partum.
Frequency of treatment:
Continuous
Duration of test:
Up to 54 days.
Doses / concentrationsopen allclose all
Dose / conc.:
64 mg/kg bw/day (actual dose received)
Remarks:
Males 1000 ppm
Dose / conc.:
651 mg/kg bw/day (actual dose received)
Remarks:
Males 10000 ppm
Dose / conc.:
1 294 mg/kg bw/day (actual dose received)
Remarks:
Males 20000 ppm
Dose / conc.:
77 mg/kg bw/day (actual dose received)
Remarks:
Females 1000 ppm
Dose / conc.:
782 mg/kg bw/day (actual dose received)
Remarks:
Females 10000 ppm
Dose / conc.:
1 558 mg/kg bw/day (actual dose received)
Remarks:
Females 20000 ppm
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels were selected based on the outcome of a preliminary 14-day oral gavage range-finding study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: all animals were checked twice daily for morbidity and mortality during the normal working week and once daily on weekends and holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all animals were examined for overt signs of toxicity, ill health or behavioural change once daily. Abnormal findings were recorded daily until resolution of finding or study termination.

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were recorded on Day 0 before start of treatments and weekly thereafter for males until termination. Females were weighed weekly until mating was evident. Parental females showing evidence of mating were weighed on Days 0, 7, 14 and 20 post coitum. Parental generation females with a live litter were weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- During the maturation period food consumption was recorded weekly for each cage of parental generation adults. For parental generation females showing evidence of mating, food consumption was recorded for the periods covering Days 1 to 7, 7 to 14 and 14 to 20 post coitum. For parental generation females with live litters, food consumption was recorded for the period covering Days 1 to 4 post partum.
- Group mean weekly food consumption was calculated (as g/rat/day)
- Weekly food efficiencies were calculated (group mean body weight gain (as g/rat/week) / group mean food consumption (as g/rat/week))

WATER CONSUMPTION
- Time schedule for examinations: daily visual inspections of water bottles

HISTOLOGY/HISTOPATHOLOGY
The following organs were collected and preserved in buffered 10% formalin and examined at histopathology for all adult males and females from the control and high dose groups: coagulating glands; epididymides; prostate; seminal vesicles; testis; pituitary; ovaries; uterus/cervix; vagina; skin (from top of head). Skin samples from all groups were prepared as paraffin blocks, sectioned at nominal thickness of 5 mm and stained with hematoxylin and eosin for subsequent microscopic examination. All other tissues from control and 20000 ppm dose group animals were treated in similar manner.

OTHER: Each pregnant female was observed at 8:30, 12:30 and 16:30 hours at or around the period of expected parturition. On weekends, observations were carried out at 8:30 and 12:30 hours only. The following were recorded for each female:
- date of mating
- date and time of observed start of parturition
- date and time of observed completion of parturition
- duration of gestation

POSTMORTEM EXAMINATIONS
All adult (P) animals, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. In addition, corpora lutea and implantation sites of all females were counted. This procedure was enhanced by staining the uteri with 1% ammonium polysulphide solution.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number of pups born
- number and sex of pups alive recorded daily and on Day 1 and 4 post partum
- clinical condition of pups from birth to Day 4 post partum
- individual litter weights on Day 1 and 4 post partum

All live offspring were observed for detachment of pinna (as noted by the separation of the edges and subsequent unfolding of both pinnae) and assessed for reflexological response to stimuli by assessing surfact righting reflex on Day 1 post partum.

GROSS EXAMINATION OF DEAD PUPS:
Offspring dying during the study and at study termination were subjected to a full external and internal examination, and any macroscopic abnormalities recorded.
Statistics:
Group mean values and standard deviation were derived where appropriate. Weekly bodyweight, bodyweight gain, food consumption, adult organ weights, litter sizes, litter weight and offspring bodyweights were assessed for dose response relationships by analysis to establish homogeneity of group variances using Bartlett's test followed by one-way analysis of variance. If variances were unequal subsequent comparisons between control and treated groups were performed using t-test assuming unequal variances. If variances were equal subsequent comparisons between control and treated groups were performed using Dunnett's Multiple Comparison Method.

Offsping landmarks of physical development, offspring reflexological responses, pre- and post-implantation losses, litter sex ratios and relative organ weights were compared using Kruskal-Wallis non-parametric rank sum test. Post implantation losses in the 10000 ppm group were also compared using Grubb's test (parametric test to determine outliers).

For histopathology observations: Chi-squared analysis was used for differences in lesions occurring with an overall frequency of 1 or greater; Kruskal-Wallis on-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed conditions.
Indices:
Live birth index (%) = number of pups alive on Day 1/number of pups born x 100
Viability index (%) = number of pups alive on Day 4/number of pups alive on Day 1 x 100

Group mean values calculated from each litter value on Day 1 and 4 using the following: number of male pups/number of pups of determined sex x 100.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 20000 ppm all females showed scabs and fur loss on the top of the head and/or around the snout from Day 22 of treatment onwards.

At 10000 ppm all females showed scabs and fur loss on the top of the head and/or around the snout from Day 22 of treatment onwards. For all but one female, these signs persisted until completion of the study. The skin irritation on the top of the head and/or around the snout is considered an adverse effect of treatment due to the unique nature and severity of the irritancy.

At 1000 ppm there were no clinical signs of toxicity or irritancy observed throughout the study.

One control female had approximately 2 inches of skin missing from the end of the tail exposing underlying tissue.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant effects on bodyweight gain were observed for animals of either sex throughout maturation or for females during gestation or early lactation. Bodyweight development in test animals was similar to that of controls.

At 20000 ppm there was a reduction ini female bodyweight gain for females between days 14 and 20 of gestation. This resulted in a slight, but not statistically significant difference in group mean female bodyweight at day 20 of gestation.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no statistically significant difference in food consumption for females.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles revealed no intergroup differences.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related effects.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Fur loss and scabbing was observed for all females at 20000 ppm, predominantly on the head. Similar skin lesions were noted on the abdomen of two females at this dose level. There were no effects observed upon reproductive or other organs.
At 10000 ppm, fur loss and scabbing similar to the high dose group was observed for 9 of 10 females. Other observations were considered incidental and not related to test article administration.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Acanthosis, occasionally with focal epithelial ulceration and overlying scab formation, was observed in relation to treatment in the majority of females dosed at 20000 ppm (p<0.001) or at 10000 ppm (p<0.01). Other histopathological findings were those commonly encountered as a result of pregnancy or commonly observed in laboratory rats of this age and strain.

Maternal developmental toxicity

Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
There were no inter-group differences in group mean number of corpora lutea or implantation sites counted post mortem. Pre-implantation losses were comparable to controls across all dose groups. The percentage of pre-implantation losses was slightly lower at 10000 ppm compared to controls but this was not statistically significant. At 20000 ppm, post-implantation losses were increased non-significantly compared to controls but the findings were judged toxicologically significant. At 10000 ppm, post-implantation losses were also increased non-significantly compared to controls but this result was primarily due to one female with a total loss of litter in utero. This was determined to be an outlier and not representative of the group. Reanalysis with removal of this female data showed no differences from control.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on gestation length. The majority of females showing positive evidence of mating gave birth to live young following 22 to 23 days of gestation.
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Effect level:
77 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
1000 ppm in diet
Basis for effect level:
other: Local irritation
Dose descriptor:
NOAEL
Effect level:
782 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
10000 ppm in diet
Basis for effect level:
pre and post implantation loss

Results (fetuses)

Fetal body weight changes:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
At 20000 ppm there was a slightly lower mean litter size at birth compared to control values. The difference was not statistically significant. There was a higher value for the group mean post implantation loss when compared to control values. The large intra-group variability for this parameter means that statistical significance was not achieved. Although group mean litter size was not statistically significantly different from controls, the number of females with litters with 10 or fewer offspring was higher compared to controls (no statistical analysis).
At 1000 and 10000 ppm there were no significant effects upon live litter size at birth.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Offspring viability between birth and day 4 of lactation was comparable to control values.
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Surface righting reflex: no difference between the groups observed.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
There were no inter-group differences in the type or incidence of clinical findings. There were no treatment-related effects on the onset or completion of pinna unfolding or the group mean percentages of pups passing the surface righting assessment on Day 1 post partum.

There were no treatment-related macroscopic findings.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
> 1 558 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
20000 ppm in diet
Sex:
male/female
Basis for effect level:
other: No adverse effects observed

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
There were no inter-group differences in the type or incidence of clinical findings among F1 offspring. There were no treatment-related effects on the onset or completion of pinna unfolding or the group mean percentages of pups passing the surface righting assessment on Day 1 post partum. There were no treatment-related macroscopic findings.
Executive summary:

This study was designed to screen for potential adverse effect on reproduction including embryo/foetal development in the rat following OECD TG 421 and under GLP conditions.

The test material was administered by dietary admixture to three groups, each of ten male and ten female rats, for up to 54 consecutive days at dietary concentrations of 1000, 10000 and 20000 ppm (equivalent to mean achieved dosages of 64, 651, 1294 and 77, 782 and 1558 mg/kg bw/day for males and females, respectively). A further group of animals served as control. Following 14 days of dosing, male and female rats were paired within their dose groups to produce litters. On Day 5 post partum, all surviving animals were euthanised and examined macroscopically. Clinical signs, bodyweight development and food and water consumption were monitored during the study. Offspring development and growth was also monitored. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from control and high dose parental males and females was performed.

For adults, there were no treatment-related deaths during the study. At 10000 ppm and 20000 ppm, adverse signs of irritancy were observed in males and females. There were no treatment-related effects on body weight, food consumption or water consumption. Mating performance and fertility were not adversely affected by the treatment. No treatement-related effects were observed for organ weights or necropsy findings.

At 20000 ppm there was in increase in post implantation loss which was not statistically significant compared to controls but was of toxicological significant because of the intragroup variation in individual post implantation loss values. At 10000 ppm there was also a high post implantation loss but this was due to a single female with a total litter loss in utero.

For offspring, at 20000 ppm, there was a slightly lower mean litter size at birth compared to control values but this was not statistically significant. Offspring viability between birth and day 4 of lactation was comparable to control values. For all other observations, there were no treatment-related effects observed.

Administration of the test material to male and female rats throughout maturation, gestation and lactation phrases of reproduction resulted in signs of test material irritancy at 10000 and 20000 ppm. The irritancy was considered to be an adverse effect due to its degree of severity. Therefore, the NOEL for adults was 1000 ppm (66 -74 mg/kg). The NOAEL for effects on fertility was 10000 ppm (782 mg/kg; due to increased post implantation loss and a consequential decrease in mean litter size at birth at 20000 ppm). The NOEL for developmental toxicity/teratogenicity was 20000 ppm (1294 -1558 mg/kg).