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Diss Factsheets

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:
1982-10-20, 1983-08-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report date:
1984

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
Oestrus cycle and sperm parameters were not evaluated. Pituitary and thyroid were not weighted. The coagulating gland was not examined
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Pentaerythritol tetrakis(3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate)
EC Number:
229-722-6
EC Name:
Pentaerythritol tetrakis(3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate)
Cas Number:
6683-19-8
Molecular formula:
C73H108O12
IUPAC Name:
3-{[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoyl]oxy}-2,2-bis({[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoyl]oxy}methyl)propyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate
Details on test material:
- Physical state: Solid, white powder

Test animals

Species:
rat
Strain:
other: CrL:COBS CD (SD) BR strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd., Margate Kent, England
- Age at study initiation: (P) 6 w; (F1) 4 w
- Weight at study initiation: (P) Males: 129-130 g; Females: 105-106 g; (F1) Males: 67-77 g; Females: 64-74 g
- Housing: During the pre-mating period, the rats were housed four to a cage in suspended galvanised metal cages (Bowman R), equipped with solid sides and back and wire mesh front, floor and top. During the mating period for each generation, the rats were housed on the basis of one male to one female in plastic breeding cages (North Kent Plastics, RM-2 type).
- Diet (e.g. ad libitum): Ad libitum, Spratt's Laboratory Diet No. 2 (low fat)
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 4
- Humidity (%): 62 ± 13
- Air changes (per hr): 13
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
An appropriate quantity of material as supplied was weighed and ground directly into a weighed amount of sieved Spratt's Laboratory Diet No. 2 and stirred by hand to give a premix of suitable strength. The dietary concentrations required were obtained from this premix by direct dilution with further quantities of diet, homogeneity being achieved by mixing for 7 minutes in a rotary double cone blender. The
diets were then stored until use in heat sealed opaque polythene bags.

DIET PREPARATION
- Rate of preparation of diet (frequency): 14 d
- Mixing appropriate amounts with (Type of food): Spratt's Laboratory Diet No. 2
- Storage temperature of food: Room temperature
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 20 d
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 20 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged (how): Individual breeding cages for the birth and rearing of young, during which time suitable nesting material was provided
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A representative sample of diet (10 g) , obtained by a standard riffling technique, was extracted (Soxhlet) with chloroform (130 mL ) for 2 h. The cooled extract was quantitatively transferred to a volumetric flask (200 mL) and diluted to volume with chloroform. The sample extract was appropriately diluted using chloroform to produce a solution with an expected concentration of test material in the range 50-150 μg/mL. An aliquot of this solution (typically 5-10 mL) was evaporated to dryness (RFE, 40 °C) and the residue redissolved in mobile phase (20 mL) to produce a final solution containing the test substance in the concentration range 12-40 μg/mL. The final analytical solutions were filtered (Whatman GE/E) prior to injection onto the HPLC.
Duration of treatment / exposure:
F0: 10 weeks prior to mating (20 days) then sacrificed after F1 weaning (21 days)
F1: 12 weeks prior to mating (20 days) then sacrificed after F2 weaning (21 days)
Frequency of treatment:
Continuous
Details on study schedule:
- F1 parental animals not mated until 16 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 12 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 ppm
Dose / conc.:
3 000 ppm
Dose / conc.:
10 000 ppm
No. of animals per sex per dose:
28
Control animals:
yes
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were regularly handled and examined.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were regularly handled and examined.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Weekly during the pre-mating phase of each generation
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily during the first two and last two weeks of the pre-mating phase of each generation.
Oestrous cyclicity (parental animals):
During the pre-mating period, cages of males were interspersed amongst those holding females to promote the development of regular oestrous cycles.
Sperm parameters (parental animals):
Parameters examined in all male parental generations: testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 21 postpartum
- If yes, maximum of 48 pups/group (24/sex/group as nearly as possible); a further male and female pup per litter were selected for organ weight analysis and preservation of selected tissues; excess pups were killed, examined macroscopically, and discarded.

The litters chosen were those weaned as close as possible to the overall median weaning date. In all but two cases (both at the lowest concentration) one male and one female pup were taken from each of 24 selected litters.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring: Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight (on days 4, 8, 12, and 21 post partum).

GROSS EXAMINATION OF DEAD PUPS: Yes, for external and internal abnormalities except those excessively cannibalised
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after the majority of F1 litters had been weaned.
- Maternal animals: All surviving animals three weeks after weaning of their litter.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Uteri of those females still failing to produce young after re-mating were immersed in a 10% solution of ammonium sulphide to reveal evidence of implantation. Testes, prostate and seminal vesicles of males which failed to successfully inseminate their partner were weighed and examined histologically.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [1] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 22 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: gonadal inspection, externaland internal examination, and organ weight (table 1).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
Statistics:
Statistical analyses were performed routinely on the litter data, using the litter as the basic sample unit. As litter values do not follow a 'normal' distribution, intergroup differences were analysed by nonparametric statistical methods (Hollander, M. 6 Wolfe, D.A. 1973).
Provided there was found to be a significant relationship (F-test, P<0.01) between organ weight and bodyweight, organ weights were analysed by variance, adjusting for bodyweight at sacrifice as covariant. Treatment means were compared with control values by the method of least significant differences in conjunction with Williams' test (Williams, D.A. 1971/2).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No specific signs of reaction to treatment were seen amongst animals of either generation nor were there any deaths amongst F0 or selected F1 animals.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No deaths among F0 animals
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
By the end of the pre-mating period, overall mean bodyweight gain of F0 females at 1000, 3000, and 10000 ppm was below that of control counterparts, the differences between the groups were not dosage-related. For the females at 10000 ppm by termination, the difference from the control was less than 5%. Mean bodyweight gain of F0 treated males was comparable with, or superior to, that of the concurrent control groups throughout.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Slight temporal fluctuations in mean values were observed (both in respect of the amount of food eaten and the amount of water drunk) during the periods recorded but no consistent pattern was apparent for these parameters in either generation and there was no clear, evidence of any adverse effect attributable to the inclusion of the test item in the diet.
Food efficiency:
not specified
Description (incidence and severity):
Food conversion ratios were calculated during the pre-mating period of both generations. Although there were slight intergroup variations in the ratios, there were no consistent differences indicative of an impairment of food utilisation at any dosage in either generation.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Slight temporal fluctuations in mean values were observed (both in respect of the amount of food eaten and the amount of water drunk) during the periods recorded but no consistent pattern was apparent for these parameters in either generation and there was no clear, evidence of any adverse effect attributable to the inclusion of the test item in the diet.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The reproductive tissues of selected adult males and females of the control and top dose group (10000 ppm) of both generations were examined microscopically. There were no treatment-related changes observed in the sections examined in males or females of either generation.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
MATING PERFORMANCE AND PREGNANCY RATE
Mating performance, as assessed by the type of vaginal smear recorded on the day of conception, the overall incidence and distribution of successful matings and the median pre-coital time, was not adversely affected by treatment in either generation. Although the incidence of infertile pairings did not indicate any adverse association with treatment, the 6/112 F0 and 7/96 F1 females which failed to produce young at the first mate of their respective generations were re-mated with proven males. All but three of these females subsequently failed to conceive and there was no evidence to suggest that their distribution (viz. an F1 female at 3000 ppm and an F0 and an F1 female at 10000 ppm) was other than coincidental.

DURATION OF GESTATION
The mean duration of gestation was similar for all groups within and between generations (mean 21.9 days; range, 21.6 to 22.1 days)

Details on results (P0)

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Because the dietary inclusion (ppm) remained constant throughout the study, the actual intake of test material (mg/kg/day) inevitably decreased as the animals matured.
In F0, intake during the first week of treatment was about twice that at the start of pairing and in F1 (two weeks-younger animals), the difference was greater. Theoretical extrapolation indicated that weanlings (F1 and F2 offspring) could have been exposed to still higher levels. Intake of females was almost always higher than that of males of the same age - an inherent finding when the same dietary inclusion is administered to both sexes; in fact, there was closer correlation between intake of F0 and F1 animals of the same sex at comparable ages than between their respective male/female counterparts fed the same diet.
Considered overall, however, the data for achieved intake of test material was consistent with theoretical expectations and there were no unexpected aberrations

ANALYSIS OF TEST ITEM IN FEED:
- Actual concentrations: 98-110% of nominal concentrations.
- Stability: stable under the conditions of the study for 18 days
- Homogeneity: homogeneous in samples of diet preparation checked

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
10 000 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: no adverse effects up to the high dose
Remarks on result:
other: coresponding to 688.4 mg/kg bw for males and 823.0 mg/kg bw for females

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Description (incidence and severity):
No specific signs of reaction to treatment were seen amongst animals of either generation nor were there any deaths amongst F0 or selected F1 animals.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence and severity):
Mean bodyweight of both males and females at the start of the second (F1) generation reflected the intergroup differences in mean pup weight at weaning. Subsequent mean weight gain of treated animals was comparable with, or superior to, that of their respective control group throughout the generation so that, by termination, absolute mean bodyweight of these groups was 2-4% above the corresponding control value. Although there was a suggestion of retarded mean weight gain amongst F0 treated dams during the later stages of gestation, this was not repeated in the F1 generation, where mean weight gain of test group dams was superior to that of control dams. Mean weight change during the post partum period did not indicate any obvious adverse effect of treatment in either generation.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Slight temporal fluctuations in mean values were observed (both in respect of the amount of food eaten and the amount of water drunk) during the periods recorded but no consistent pattern was apparent for these parameters in either generation and there was no clear, evidence of any adverse effect attributable to the inclusion of the test item in the diet.
Food efficiency:
not specified
Description (incidence and severity):
Food conversion ratios were calculated during the pre-mating period of both generations. Although there were slight intergroup variations in the ratios, there were no consistent differences indicative of an impairment of food utilisation at any dosage in either generation.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Slight temporal fluctuations in mean values were observed (both in respect of the amount of food eaten and the amount of water drunk) during the periods recorded but no consistent pattern was apparent for these parameters in either generation and there was no clear, evidence of any adverse effect attributable to the inclusion of the test item in the diet.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
Routine terminal examination of F0 and F1 adults did not reveal any obvious macroscopic changes attributable to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The reproductive tissues of selected adult males and females of the control and top dose group (10000 ppm) of both generations were examined microscopically. There were no treatment-related changes observed in the sections examined in males or females of either generation.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
MATING PERFORMANCE AND PREGNANCY RATE
Mating performance, as assessed by the type of vaginal smear recorded on the day of conception, the overall incidence and distribution of successful matings and the median pre-coital time, was not adversely affected by treatment in either generation. Although the incidence of infertile pairings did not indicate any adverse association with treatment, the 6/112 F0 and 7/96 F1 females which failed to produce young at the first mate of their respective generations were re-mated with proven males. All but three of these females subsequently failed to conceive and there was no evidence to suggest that their distribution (viz. an F1 female at 3000 ppm and an F0 and an F1 female at 10000 ppm) was other than coincidental.

DURATION OF GESTATION
The mean duration of gestation was similar for all groups within and between generations (mean 21.9 days; range, 21.6 to 22.1 days)

Effect levels (P1)

Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at the highest dose

Results: F1 generation

General toxicity (F1)

Mortality / viability:
no mortality observed
Description (incidence and severity):
TOTAL LITTER LOSS
Over the two generations, only three litters were lost during the post partum period: one each amongst controls and at 10000 ppm in the F0 generation, and a single loss at 3000 ppm in the F1 generation. As the incidence of losses was not considered to reflect any adverse association with treatment, the following assessments of litter parameters are based on Mean B values (i.e. dams rearing some young to weaning).

LITTER SIZE, PUP MORTALITY, SEX RATIO
Amongst F0 dams treated at 1000 and 10000 ppm, mean total litter size at birth was significantly lower than that of control dams
(P<0.05, Kruskal-Wallis test); the highest mean value for this parameter, however, was recorded at the intermediate dosage (3000 ppm) and therefore in the absence of a dosage-relationship in this generation and lack of any other clear corroberative data in this or the subsequent (F1) generation, association with treatment appeared unlikely at 1000 ppm while at 10000 ppm any effect remains speculative.
The incidence of non-viable pups recorded at parturition did not indicate any adverse effect of treatment in either generation and, with the exception of litters from F0 dams treated at 3000 ppm, subsequent mean pup losses in test group litters were consistantly lower than that of the concurrent control group; this resulted in convergence of mean litter size during lactation in both generations - particularly in the first (F0) generation, where the early statistically significant differences from control values at 1000 and 10000 ppm were abolished by Day 12 post partum. There was no indication of any adverse selective effect on survival of male or female offspring in treated groups of either generation, as assessed by sex ratios at birth and Day 21 post partum.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In contrast to the lower mean litter sizes recorded in F0 litters at 1000 and 10000 ppm, mean pup weight in these groups was consistantly higher than concurrent control values from birth through to weaning, with differences often attaining statistical significance (P<0.05 or 0.01, Kruskal-Wallis test); moreover, overall growth rate was also slightly faster than that of control pups. At 3000 ppm, both mean pup weight and overall growth rate were very similar to that of control group litters throughout. An almost identical pattern recurred in the F1 generation, except that - in line with the closer mean values between groups for litter size - intergroup differences in mean pup weight and growth rate were not quite as marked in this generation. Considered overall, mean growth rate was slightly faster in the second generation than the first - a factor which appeared to be independent of litter size. Similarly, a suggestion of a slightly faster growth rate was apparent at 10000 ppm in both generations, an observation which was endorsed by the noticeably
higher mean litter weight in this group at termination.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The incidence of morphological anomalies recorded at autopsy of excess F1 weanlings or amongst siblings sacrificed at maturity, together with their F2 progeny, did not indicate any adverse effect of treatment.

Details on results (F1)

PARENTAL F0 DATA
- Mortality: none
- Clinical signs: no treatment-related effects
- Body weight: no treatment-related effects
- Food consumption: no treatment-related effects
- Water consumption: no treatment related effects
- Mating performance: no treatment related effects
- Organ weights: statistically significantly decreased ovaries weight (14%) and testes weight (6%) at 10000 ppm
- Macroscopy: no treatment-related effects
- Microscopy: no treatment-related effects
- Pregnancy rate: 100, 89.3, 92.9 and 96.4% at 0, 1000, 3000 and 10000 ppm, respectively
- Duration of gestation: 21.7, 22.0, 21.6 and 22.0 days at 0, 1000, 3000 and 10000 ppm, respectively
- No. of females with implantations: 28/28, 25/28, 26/28, 27/28 at 0, 1000, 3000 and 10000 ppm, respectively.

OFFSPRING TOXICITY F1
- No. of litters: 27/28, 25/28, 26/28, 26/28 at 0, 1000, 3000 and 10000 ppm, respectively
- Mean litter size: 13.5, 11.5, 13.5, 11.7 at 0, 1000, 3000 and 10000 ppm, respectively
- Mean foetal weight (at birth): 5.7, 6.0, 5.5, 5.9 at 0, 1000, 3000 and 10000 ppm, respectively; on day 4, 8, 12 and 21 of lactation body weights of pups were significantly increased in the 10000 ppm dose group
- Sex and sex ratios: no treatment-related effects
- Post-natal survival until weaning: no treatment-related effects
- Visible abnormalities: no treatment-related effects (control group: 1 hydrocephaly)
- Macroscopic examinations: no treatment-related effects
- Organ weights: no treatment-related effects

OFFSPRING TOXICITY F2
- No. of litters: 22/24, 23/24, 21/24, 22/24 at 0, 1000, 3000 and 10000 ppm, respectively
- Mean litter size: 11.3, 11.3, 11.9, 11.1 at 0, 1000, 3000 and 10000 ppm, respectively
- Mean foetal weight (at birth): 5.8, 6.1, 5.9, 6.2 at 0, 1000, 3000 and 10000 ppm, respectively; on day 4, 8, 12 and 21 of lactation body weights of pups were significantly increased in the 10000 ppm dose group
- Sex and sex ratios: no treatment-related effects
- Post natal survival until weaning: no treatment-related effects
- Visible abnormalities: no treatment-related effects
- Macroscopic examinations: no treatment-related effects
- Organ weights: no treatment-related effects

The NOAEL for parental toxicity is considered to be 10000 ppm (= 688.4 mg/kg bw for males and 823.0 mg/kg bw for females from the F0-generation). Decreased ovaries and testes weight is not considered to be treatment-related. The effects on ovary weight were attributed to 3 females, one of which was not pregnant. The effects on testes weight were due to effects in 2 males (one with macroscopic finding: small testis). The reduction in mean litter size is not dose-related and therefore not considered to be toxicologically relevant.
The NOAEL for developmental toxicity is 10000 ppm (= 688.4 mg/kg bw for males and 823.0 mg/kg bw for females from the F0-generation). The increased mean pup weight in F1 and F2 weanlings at 10000 ppm is not considered to be biologically relevant.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
10 000 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Results: F2 generation

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
10 000 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 2. Reproduction performance

F0 Generation F1 Generation
Group control 1000 ppm 3000 ppm 10000 ppm control 1000 ppm 3000 ppm 10000 ppm
males 28 28 28 28 24 24 24 24
females 28 28 28 28 24 24 24 24
No live young at first mate 3 2 1 (1) 2 1 2 (1) 2 (1)
total litter loss 1 1 1
rearing young to weanling 27 25 26 26 22 23 21 22

( ) Number of females which failed to give birth following re-mate with proven males

Table 3. Determination of Pregnancy

    Pregnant No live young pregnancy rate %
Group number of females paired Evidence of Mating on day of conception positive smear no indication of mating total
positive smear (sperm or plug) oestrus smear anoestrus/
dioestrus		 no indication of mating total
F0                    
control 28 24b 0 1 3 28 0 0 0 100
1000 ppm 28 18 6 0 1 25 1 2 3 89.3
3000 ppm 28 26 0 0 0 26 2 0 2 92.9
10000 ppm 28 27b 0 0 0 27 0 1# 1 96
F1                    
control 24 17 3 0 2 22 1 1 2 91.7
1000 ppm 24 23 0 0 0 23 1 0 1 95.8
3000 ppm 24 18 0 0 4b 22 1 1# 2 91.7
10000 ppm 24 19 1 1 1 22 1 1# 2 91.7

b includes one dam subsequently losing her entie litter during the post partum period

# indicates a female which also failed to produce a litter when re-mated with a proven male

Table 4. Group Mean Litter Data - F0 Generation

Group No of animals At birth At day 4 At day 8
mated pregnant Litter Size Litter weight (g) Mean pup weight (g) litter size cumulative loss % litter weight (g) mean pup weight (g) litter size cumulative loss % litter weight (g) mean pup weight (g)
Total Live Loss %
control 28 A=28
B=27		 13.5
13.6		 13.4
13.5		 0.5
0.6		 -
75.5		 -
5.7		 12.6
13.1		 6.9
3.1		 -
106.1		 -
8.3		 12.4
12.9		 8.0
4.5		 -
165.7		 -
13.2
1000 ppm 28 A=B=25 11.7+ 11.5++ 1.2 67.7(+) 6 11.3 2.9 102.5 9.3 11.2(+) 3.8 162 14.9+
3000 ppm 28 A=B=26 13.7 13.5 1.7 73.8 5.5 12.9 5.7 103.7 8.1 12.5 8.8 161 12.9
10000 ppm 28 A=27
B=26		 12.0
11.8+		 11.9
11.7+		 0.9
0.9		 -
67.3		 -
5.9		 10.9
11.3+		 7.4
3.9		 -
102.9		 -
9.4++		 10.8
11.2(+)		 8.4
4.9		 -
165.1		 -
15.2++

Group No of animals At day 12 At day 21
mated pregnant litter size cumulative loss % litter weight (g) mean pup weight (g) litter size cumulative loss % litter weight (g) mean pup weight (g)
control 28 A=28
B=27		 12.1
12.5		 10.4
7.1		 -
223.1		 -
18.2		 11.9
12.4		 11.3
8.0		 -
412.3		 -
34.1
1000 ppm 28 A=B=25 11.1 4.5 223.4 20.7 11.1 4.8 419.5 39.2
3000 ppm 28 A=B=26 12.3 9.8 224.9 18.2 11.7 11.5 417 34.7
10000 ppm 28 A=27
B=26		 10.7
11.1		 9.0
5.5		 -
226.8		 -
21.2+		 10.7
11.1		 9.0
5.5		 -
430.6		 -
40.3+

Intergroup differences from control values statistically significant at (Kruskal-Wallis test):

+ P< 0.05

++ P< 0.01

(+) P> 0.05

Table 5. Group Mean Litter Data - F1 Generation

Group No of animals At birth At day 4 At day 8
mated pregnant Litter Size Litter weight (g) Mean pup weight (g) litter size cumulative loss % litter weight (g) mean pup weight (g) litter size cumulative loss % litter weight (g) mean pup weight (g)
Total Live Loss %
control 24 A=B=22 11.5 11.3 1.6 65.2 5.8 11 4 102.3 9.4 10.9 5.2 162.5 15
1000 ppm 24 A=B=23 11.4 11.3 1.6 67.3 6.1 11.1 2.6 105.9 9.9 11 3.2 172.7 16.2
3000 ppm 24 A=22
B=21		 12.0
12.0		 11.9
11.9		 1.3
1.4		 -
69.6		 -
5.9		 11.5
11.6		 4.6
3.3		 -
108.6		 -
9.5		 11.0
11.6		 8.1
3.7		 -
173.2		 -
15.2
10000 ppm 24 A=B=22 11.3 11.1 1.2 68.2 6.2(+) 11 1.9 106.9 9.9 11 1.9 175.8 16.5(+)

F1 No of animals At day 12 At day 21
mated pregnant litter size cumulative loss % litter weight (g) mean pup weight (g) litter size cumulative loss % litter weight (g) mean pup weight (g)
control 24 A=B=22 10.9 5.2 231.2 21.3 10.9 5.2 429.1 40.2
1000 ppm 24 A=B=23 11 3.8 250.7 23.7 10.9 4.4 459 43.7
3000 ppm 24 A=22
B=21		 11.0
11.6		 8.1
3.7		 -
250.9		 -
22.1		 11.0
11.5		 8.8
4.5		 -
468.5		 -
41.5
10000 ppm 24 A=B=22 11 2.3 254.3 23.9(+) 11 2.3 475.5 44.7(+)

Intergroup differences from control values statistically significant at (Kruskal-Wallis test):

+ P< 0.05

++ P< 0.01

(+) P> 0.05

Table 6. Group 1 Males F0 (Control)

Rat Number test article (ppm) Testes Epididymides Seminal vesicles Prostate
1 0 Within normal limits
2 0 Within normal limits
3 0 Within normal limits
4 0 Within normal limits
5 0 Within normal limits
6 0 Within normal limits
7 0 Within normal limits
8 0 Within normal limits
9 0 Within normal limits
10 0 Within normal limits
11 0 Within normal limits
12 0 Within normal limits
13 0 Within normal limits Acute prostatitis
14 0 Within normal limits
15 0 Within normal limits
16 0 Within normal limits Acute prostatitis
17 0 Within normal limits
18 0 Within normal limits
19 0 Within normal limits
20 0 Within normal limits
21 0 Within normal limits
22 0 Within normal limits
23 0 Within normal limits
24 0 Focus of mineralisation Within normal limits
25 0 Within normal limits
26 0 Within normal limits
27 0 Within normal limits
28 0 Within normal limits

Table 7. Group 4 Males F0 (10000 ppm)

Rat Number test article (ppm) Testes Epididymides Seminal vesicles Prostate
85 10000 Within normal limits
86 10000 Within normal limits
87 10000 Within normal limits
88 10000 Within normal limits
89 10000 Within normal limits
90 10000 Within normal limits
91 10000 Within normal limits
92 10000 Within normal limits
93 10000 Within normal limits
94 10000 Within normal limits
95 10000 Within normal limits
96 10000 Within normal limits
97 10000 Within normal limits
98 10000 Foci of mineralisation. Area of atrophy of seminiferous tubules (unilateral) Absence of spermatozoa Within normal limits
99 10000 Within normal limits
100 10000 Within normal limits
101 10000 Within normal limits
102 10000 Within normal limits Acute prostatitis
103 10000 Within normal limits
104 10000 Within normal limits
105 10000 Within normal limits
106 10000 Focus of mineralisation Within normal limits
107 10000 Within normal limits
108 10000 Focus of mineralisation Within normal limits
109 10000 Within normal limits
110 10000 Within normal limits Lymphocytic infiltration
111 10000 An area of reduced spermatogenesis Within normal limits
112 10000 Within normal limits

Table 8. Group 1 Female F0 (Control)

Rat Number test article (ppm) Uterus Ovaries Vagina
113 0 Within normal limits
114 0 Within normal limits
115 0 Within normal limits
116 0 Within normal limits
117 0 Within normal limits
118 0 Within normal limits
119 0 Slight distension of lumen Within normal limits
120 0 Within normal limits
121 0 Within normal limits
122 0 Within normal limits
123 0 Within normal limits
124 0 Within normal limits
125 0 Within normal limits
126 0 Within normal limits
127 0 Within normal limits
128 0 Within normal limits
129 0 Within normal limits
130 0 Within normal limits
131 0 Within normal limits
132 0 Within normal limits
133 0 Within normal limits
134 0 Slight distension of lumen Within normal limits
135 0 Within normal limits
136 0 Within normal limits
137 0 Slight distension of lumen Within normal limits
138 0 Slight distension of lumen Within normal limits
139 0 Within normal limits
140 0 Within normal limits

Table 9. Group 4 Female F0 (10000 ppm)

Rat Number test article (ppm) Uterus Ovaries Vagina
197 10000 Within normal limits
198 10000 Within normal limits
199 10000 Within normal limits
200 10000 Within normal limits
201 10000 Within normal limits
202 10000 Within normal limits No corpora lutea Within normal limits
203 10000 Slight distension of lumen Within normal limits
204 10000 Within normal limits
205 10000 Within normal limits
206 10000 Within normal limits
207 10000 Slight distension of lumen Within normal limits
208 10000 Within normal limits
209 10000 Within normal limits
210 10000 Within normal limits
211 10000 Within normal limits
212 10000 Within normal limits
213 10000 Slight distension of lumen Within normal limits
214 10000 Within normal limits
215 10000 Within normal limits
216 10000 Within normal limits
217 10000 Within normal limits
218 10000 Within normal limits
219 10000 Within normal limits
220 10000 Within normal limits
221 10000 Within normal limits
222 10000 Within normal limits
223 10000 Within normal limits
224 10000 Within normal limits

Table 10. Group 1 Male F1 (Control)

Rat Number test article (ppm) Testes Epididymides Seminal vesicles Prostate
225 0 Within normal limits
226 0 Atrophy of seminiferous tubules (unilateral) Absence of spermatozoa (unilateral) Within normal limits
227 0 Within normal limits
228 0 Within normal limits
229 0 Within normal limits
230 0 Within normal limits Acute prostatitis
231 0 Within normal limits No tissue available. Within normal limits
232 0 Within normal limits Acute prostatitis
233 0 Within normal limits
234 0 Within normal limits
235 0 Within normal limits
236 0 Within normal limits
237 0 Within normal limits
238 0 Within normal limits Slight lymphocytic infiltration
239 0 Within normal limits  
240 0 Within normal limits
241 0 Within normal limits
242 0 Within normal limits
243 0 Within normal limits
244 0 Within normal limits
245 0 Within normal limits
246 0 Within normal limits
247 0 Within normal limits
248 0 Within normal limits

Table 11. Group 4 Male F1 (10000 ppm)

Rat Number test article (ppm) Testes Epididymides Seminal vesicles Prostate
297 10000 Atrophy of seminiferous tubules (unilateral) Spermatocele; absence of spermatozoa(unilateral) Within normal limits
298 10000 Within normal limits
299 10000 Atrophy of seminiferous tubules (unilateral) Absence of spermatozoa (unilateral) Within normal limits
300 10000 Bilateral atrophy. Bilateral absence of spermatozoa and fat necrosis with chronic inflammation (in surrounding adipose tissue) Within normal limits
301 10000 Within normal limits
302 10000 Within normal limits
303 10000 Within normal limits
304 10000 Within normal limits
305 10000 Within normal limits
306 10000 Within normal limits
307 10000 Within normal limits
308 10000 Within normal limits
309 10000 Within normal limits
310 10000 Within normal limits
312 10000 Within normal limits
313 10000 Within normal limits
314 10000 Within normal limits
315 10000 Within normal limits
316 10000 Within normal limits
317 10000 Within normal limits
318 10000 Within normal limits
319 10000 Within normal limits
320 10000 Within normal limits
321 10000 Within normal limits
322 10000 Within normal limits
323 10000 Within normal limits
324 10000 Within normal limits
325 10000 Within normal limits
326 10000 Within normal limits
327 10000 Within normal limits
328 10000 Within normal limits
329 10000 Within normal limits

Table 12. Group 1 Female F1 (Control)

Rat Number test article (ppm) Uterus Ovaries Vagina
321 0 Slight distension of lumen Within normal limits
322 0 Within normal limits
323 0 Within normal limits
324 0 Within normal limits
325 0 Within normal limits
326 0 Within normal limits
327 0 Within normal limits
328 0 Within normal limits
329 0 Within normal limits
330 0 Within normal limits
331 0 Within normal limits
332 0 Within normal limits
333 0 Within normal limits
334 0 Within normal limits
335 0 Within normal limits
336 0 Slight distension of lumen Within normal limits
337 0 Within normal limits
338 0 Within normal limits
339 0 Within normal limits
340 0 Within normal limits
341 0 Within normal limits
342 0 Within normal limits
343 0 Within normal limits
344 0 Within normal limits

Table 13. Group 4 Female F1 (10000 ppm)

Rat Number test article (ppm) Uterus Ovaries Vagina
393 10000 Slight distension of lumen Within normal limits
394 10000 Within normal limits
395 10000 Within normal limits
396 10000 Within normal limits
397 10000 Within normal limits
398 10000 Slight distension of lumen Within normal limits
399 10000 Within normal limits
400 10000 Slight distension of lumen Within normal limits
401 10000 Within normal limits
402 10000 Within normal limits
403 10000 Within normal limits
404 10000 Within normal limits
405 10000 Within normal limits
406 10000 Within normal limits
407 10000 Within normal limits
408 10000 Within normal limits
409 10000 Within normal limits
410 10000 Within normal limits
411 10000 Within normal limits
412 10000 Within normal limits
413 10000 Within normal limits
414 10000 Within normal limits
415 10000 Within normal limits
416 10000 Within normal limits

Applicant's summary and conclusion

Conclusions:
The findings of this study indicate that, under the conditions of the test procedure, animals administered the test compound at levels of 1000, 3000 and 10000 ppm in their diet showed no substantial differences from their control counterparts and that their reproductive capacity was unimpaired.