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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a preliminary,Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD 422) of the source substance, at a dose of 1000 mg/kg/day, there was an increase in the number of animals giving birth to dead pups and also in the number losing more than 3 pups over Days 0-4 of lactation, compared with Controls. Therefore, for the neonatal toxicity, the Lowest Observed Adverse Effect Level (LOAEL) was 1000 mg/kg/day and the No Observed Effect Level (NOEL) was considered to be 500 mg/kg/day.From the findings on this study, the parental NOAEL was considered to be 100 mg/kg/day, however, findings at 500 and 1000 mg/kg/day were confined to salivation, and kidney findings which may be reversible and not relevant in man. The neonatal NOEL was considered to be 500 mg/kg/day.As findings in this study were considered not to be relevant in man, the parental NOAEL for man could be 1000 mg/kg/day.

In a subsequent, two generation reproductive toxicity study (OECD 416), the dietary administration of the source substance was well tolerated, and the parental and reproductive No-Observed-Adverse-Effect-Levels (NOAELs) were considered to be 13000 p.p.m (mean achieved dosages of: 767 mg/kg/day in F0 males; 988, 881 or 2293 mg/kg/day in F0 females prior to mating, during gestation and during lactation, respectively; 1124 mg/kg/day in F1 males, and 1200, 912 or 2256 mg/kg/day in F1 females prior to mating, during gestation and during lactation, respectively).

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
F0 generation: 3 deaths
- 1 female animal from control group, killed prematurely on Day 1 of lactation due to clinical signs (piloerection, subdued behaviour, pale eyes and skin, on Day 0 of lactation, some pups in the animals litter were found dead or had poor condition), cause of deteriotating condition was considered to be severe necrosis of the kidney.
- 1 male animal from mid dose group, killed prematurely on Day 99 due to clinical signs (shallow respiration, swelling of ventral abdomen, partially closed eyes), the demise of the animal was malignant lymophoma in mutliple tissues, considered to be an incidental finding, since no similar findings were noted in any other animals.
- 1 male animal in high dose group, killed prematurley due to a damaged right eye. Phthisis bulbi was considered to be the cause of the animal's condition, considered as an incidental finding, unrelated to test substance treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In F0 females in the high dose group mean body weights were lower than control from Day 8, continuing until pairing for mating and throughout gestation and lactation (ca. 5-7%). Reduced weight gain during pre-mating period (Days 1-71, ca. 11%, p<0.01), however no obvious reduction in gain noted over the gestation of lactation periods.
In F0 males slightly lower mean weight in the high dose group (1-4% compared to control) throughout the study, effect was too small to be positively attributed to test substance treatment and therefore considered not to be toxicologically relevant.
Group mean body weights and weight gains of mid and low dose group animals were comparable to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females: at highest dose mean food consumption 10-12% lower than control from Day 8, continuing until pairing for mating and throughout gestation and lactation. Group mean food consumption comparable to controls at mid and low dose groups.
Males: group mean food consumption generally similar between all groups throughout the study. At highest dose statistically lower consumption values were noted sporadically, however, the differences from control (around 2-3g) reflected pretrial differences between the groups, therefore the changes were not positively attributed to treatment.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the kidneys, test item-related findings included: higher incidences and severity of basophilic tubules in males at mid and high dose groups and females at high dose group; higher incidences and severity of hyaline droplets and hyaline casts in males at all dose levels and higher incidences mononuclear cell infiltration in males at high dose group. In females, the incidences of hyaline casts and mononuclear cell infiltration did not follow dose related patterns, and were therefore not positively attributed to treatment.
In the liver, a higher incidence of centrilobular hepatocellular hypertrophy was noted in both sexes at mid and high dose groups.
For details see table #6 below in section "Any other information on results incl. tables"
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Motility was lower than controls in all treated groups, however, there was no dose-related pattern to the changes, therefore, they were judgded to be not positively attributed to treatment.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
No effects observed in mating performance, fertility and duration of gestation.
Litter size and pup mortality: at high dose group, mean numbers of implantation sites, total live pups born and number of live pups on Days 1-14 of lactation up to approx. 11% lower than controls; litter size comparable to controls at low and mid dose groups; pup survival was comparable to controls at all dose levels (slight inter-group differences considered to be too small to be attributed to treatment, additionally no obvious patterns or trends in the number of pups born alive and the number of pups born dead on Day 0 of lactation observed. For details see table #7 in section “Any other information on results incl. tables”.
In adult females, reduced body weight gain during the pre-mating period correlated with lower mean food consumption throughout the majority of the study at the highest dose level. In addition, a lower number of implantation sites and lower litter size, with correlating lower litter weight, were also noted in the high dose group animals. It was considered that the litter findings were likely secondary to the maternal toxicity, and since there was no change in male or female pup weight or pup survival, these changes were considered not to be adverse.
Higher kidney weights in males correlated with histopathology findings of increased incidences and severity of hyaline droplets (all dose groups) , basophilic tubules (mid and high dose groups), hyaline casts and mononuclear cell infiltration (high dose group), and there was a higher incidence of basophilic tubules in female kidneys in the high dose group animals. These findings were considered not to be adverse as they are considered adaptive responses to the administration of test materials and/or are species specific and are, therefore, not relevant to man.
In both sexes, higher liver weights at mid and high dose groups correlated with an increased incidence of centrilobular hepatocellular hypertrophy. A slightly increased incidence of prominent lobular architecture was noted macroscopically in males at the highest dose level (7 animals compared with 3 control animals), however, this did not always correlate with the histopathology findings, therefore, the macroscopic finding was not positively attributed to treatment with the test item. In studies where dosing periods are greater than 3 months, higher liver weights with correlating hypertrophy are considered not to be adverse if there was an absence of hepatotoxicity findings such as gross liver enlargement, evidence of hepatocyte necrosis, fibrosis or inflammation, evidence of degradation or necrosis of other liver cell types, and/or carcinogenicity. Since those types of changes were not present in this study, the higher weight and hypertrophy noted on this study were, therefore, considered to be non-adverse.
Higher thyroid weight in males at all dose groups were considered not to be adverse since the findings were mild in nature and there were no histopathology correlates or any effects on reproduction which may indicate hormonal effects.
Key result
Dose descriptor:
NOAEL
Effect level:
13 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: adaptive liver and kidney weight changes not considered to be relevant to humans
Remarks on result:
other: mean measured dosages: males: 767 mg/kg/day females: 988 mg/kg/day (prior to mating), 881 mg/kg/day (during gestation) or 2293 mg/kg/day (during lactation)
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
2 male animals found dead in control group: 1 on Day 89, at necropsy epididymis was enlarged, there was no histopathological correlate, no other histopathological findings, evaluation of some tissues was not possible due to autolysis, cause of death undetermined. The second animal found dead on Day 54, no histopathological findings, evaluation of some tissues not possible due to autolysis, cause of death undetermined.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean body weights were similar in all treated male groups throughout the study and all treated female groups prior to mating.
In high dose group females, mean body weights were lower than controls throughout gestation (up to approximately 8% on Day 20 of gestation) and there was a lower mean weight gain over Days 0-20 of gestation when compared with controls (approximately 17%).
Group mean body weights during gestation were comparable to controls at mid and low dose group animals. During lactation, female group mean body weights were similar in all groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females: at high dose group, mean food consumption was approx. 5-10% lower than controls from Day 29 until pairing for mating (Day 72) and throughout gestation and lactation.
Males: group mean food consumption comparable to controls throughout the study in all dose levels.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Higher kidney weights in males at mid and high dose groups (up to approximately 15% at high dose group), thyroid weights in males at high dose group (approximately 14-17%) and higher liver weights in both sexes at mid and high dose groups (up to approximately 14% in males and 32% in females) were noted, see table #8 below in section "Any other information on results incl. tables"
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the kidneys of males, higher incidences and severity of hyaline droplets at all dose groups, basophilic tubules and mononuclear cell infiltration at mid and high dose groups and hyaline casts at high dose group were noted when compared with controls. In females, incidences of findings noted were generally similar in controls and treated groups; therefore the findings were not positively attributed to treatment.
In the liver, centrilobular hepatocellular hypertrophy was noted in both sexes at mid and high dose groups.
For details see table #9 below.
Details on results:
In adult F1 females, reduced body weight gain during gestation for F1 animals correlated with lower mean food consumption throughout the majority of the study at the high dose group. In addition at the high dose group, a lower number of implantation sites and lower litter size, with correlating lower litter weight, were also noted in F1 females. It was considered that the litter findings were likely secondary to the maternal toxicity, and since there was no change in male or female pup weight or pup survival, these changes were considered not to be adverse. Oestrous cycles, mating performance, fertility, duration of gestation, pup weight, pup survival and maternal care were similar between all groups. There were also no obvious differences in these parameters between generations.
In F1 adults, higher kidney weights in males (mid and high dose group) correlated with histopathology findings of increased incidences and severity of hyaline droplets at all dose groups, basophilic tubules at mid and high dose groups, hyaline casts (at high dose group) and mononuclear cell infiltration (mid and high dose group), and there was a higher incidences of basophilic tubules in female kidneys at the high dose group. These findings were considered not to be adverse as they are considered adaptive responses to the administration of test materials and/or are species specific and are, therefore, not relevant to man.
In F1 adults, higher liver weights in both sexes at mid and high dose group correlated with an increased incidence of centrilobular hepatocellular hypertrophy. In studies where dosing periods are greater than 3 months, higher liver weights with correlating hypertrophy are considered not to be adverse if there was an absence of hepatotoxicity findings such as gross liver enlargement, evidence of hepatocyte necrosis, fibrosis or inflammation, evidence of degradation or necrosis of other liver cell types, and/or carcinogenicity. Since those types of changes were not present in this study, the higher weight and hypertrophy noted on this study were, therefore, considered to be non-adverse.
Higher thyroid weight in F1 males at the high dose group were considered not to be adverse since the findings were mild in nature and there were no histopathology correlates or any effects on reproduction which may indicate hormonal effects.
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Mating performance, fertility and duration of gestation: no effects observed.
Ovary scoring: in the high dose group, total number of ovarian follicles attributable to the number of single layer follicles approximately 30% lower than control. For details see table #10 below in section "Any other information on results incl. tables"
Mean numbers of implantation sites, total live pups born and number of live pups throughout lactation: at the highest dose level lower than controls (up to approximately 15%).
Litter size was comparable to controls at low and mid dose groups, pup survival comparable to controls at all dose levels. Slight intergroup differences were considered to be too small to be attributed to treatment with the test item, additionally there were no obvious patterns of trends in the number of pups presumed born alive and the number of pups presumed born dead on Day 0 of lactation.
For details see table #11 below in section "Any other information on results incl. tables"
A lower number of ovarian follicles in the high dose females were considered not to be adverse since there were no obvious effects on mating, fertility, pregnancy duration or rearing of litters at any dose levels and no histopathology findings in ovaries of the high dose females. Additionally, the magnitude of these changes were generally minor and within background control ranges on similar study types at the Test Facility.
Key result
Dose descriptor:
NOAEL
Effect level:
13 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: decrease number of ovarian follicles with no histopathology and effect on reproductive function
Remarks on result:
other: mean measured dosages: males: 1124 mg/kg/day females: 1200 mg/kg/day (prior to mating), 912 mg/jkg/day (during gestation) or 2256 mg/kg/day (during lactation)
Clinical signs:
no effects observed
Description (incidence and severity):
The type and distribution of observations amongst pups did not indicate any association with test substance treatment.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
At the high dose group, mean numbers of implantation sites, total live pups born and number of live pups on Days 1-14 of lactation were lower than controls (up to approximately 11%). Litter size was comparable to controls at low and mid dose groups and pup survival was comparable to controls at all dose levels. Slight inter-group differences were considered to be too small to be attributed to treatment. In addition, there were no obvious patterns or trends in the number of pups presumed born alive and the number of pups presumed born dead on Day 0 of lactation.
For details see table #12 below in section "Any other information on results incl. tables"
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the high dose group, lower mean litter weight was noted throughout lactation (up to approximately 12%), when compared with controls. Mean litter weight at low and mid dose groups and mean male and female pup weights at all dose levels, were similar to controls throughout lactation.
For details see table #13 below in section "Any other information on results incl. tables"
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
Age and body weight of animals at vaginal opening or preputial separation were slightly higher than controls at all dose levels, indicating a slight delay in sexual maturation.
For details see table #14 below in section "Any other information on results incl. tables"
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No organ weight findings in F1 weanlings which were considered to be related to treatment.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 13 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mortality ahd body weight changes
Clinical signs:
no effects observed
Description (incidence and severity):
The type and distribution of observations amongst pups did not indicate any association with test substance treatment.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
At the high dose group, mean numbers of implantation sites, total live pups born and number of live pups throughout lactation were lower than controls (up to approximately 15%).
Litter size was comparable to controls at low and mid dose groups and pup survival was comparable to controls at all dose levels. Slight intergroup differences were considered to be too small to be attributed to treatment. In addition, there were no obvious patterns of trends in the number of pups presumed born alive and the number of pups presumed born dead on Day 0 of lactation.
For details see table #15 below in section "Any other information on results incl. tables"
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the high dose group, lower mean litter weight was noted throughout lactation (up to approximately 16%), when compared with control.
Mean litter weight at low and mid dose groups and mean male and female pup weights at all dose levels, were similar to controls throughout lactation.
For details see table #16 below in section "Any other information on results incl. tables"
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In males at mid and high dose groups, statistically significantly higher mean absolute and covariate brain weights (up to approximately 6% at the high dose group) were observed when compared to controls.
For details see table #17 below in section "Any other information on results incl. tables"
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
In F2 weanlings, higher brain weight in males were considered not to be toxicologically significant since the changes were minor (approximately 6% higher than controls) and there were no histopathology correlates.
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
ca. 13 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: slight brain weight changes in absence of histopathology
Remarks on result:
other:
Remarks:
not considered toxicologically significant
Key result
Reproductive effects observed:
no

Table #2: Summary of formulation analysis results
Timepoint Dose group Theoretical concentration (ppm) mean found concentration (ppm) RSD % Difference from theoretical concentration
Week 1 control 0 0 - -
low dose 1500 1323 1.0 -11.8
mid dose 6500 5575 5.7 -14.2
high dose 13000 11145 5.8 -14.3
Week 5 control 0 0 - -
low dose 1500 1342 1.9 -10.5
mid dose 6500 6321 3.5 -2.8
high dose 13000 12002 2.7 -7.7
Week 10 control 0 0 - -
low dose 1500 1374 5.0 -8.4
mid dose 6500 5670 2.9 -12.4
high dose 13000 12241 9.3 -5.8
Week 15 control 0 0 - -
low dose 1500 1315 3.9 -12.3
mid dose 6500 5899 1.2 -9.4
high dose 13000 11696 3.3 -10.0
Week 20 control 0 0 - -
low dose 1500 1440 3.7 -4.0
mid dose 6500 5586 0.7 -14.1
high dose 13000 1191 2.2 -7.8
Acceptance criteria ≤10% -15 to +5%

Table #3: Summary of achieved dose levels 
Group / sex low dose / M mid dose / M high dose / M low dose / F mid dose / F high dose / F
Treatment level (ppm) 1500 6500 13000 1500 6500 13000
Target dose level (mg/kg/d) 100 500 1000 100 500 1000
Actual group mean dose level (mg/kg/d) F0 90 385 767 Prior to mating 122 510 988
Gestation 102 443 881
Lactation* 279 1173 2293
F1 128 551 1124 Prior to mating 141 640 1200
Gestation 106 440 912
Lactation* 276 1113 2256
*Results during lactation should be treated with caution as consumption is known to be variable during this time and there is a large amount of scatter and/or diet eaten by pups.

Table #4: Summary of test item-related organ weight findings - F0 adults         
Males Females
Group control low dose mid dose high dose control low dose mid dose high dose
Dose (ppm) 0 1500 6500 13000 0 1500 6500 13000
No. animals per group 28 28 27 27 27 28 28 28
Kidney
Absolute value (g) 3.69 4.00* 4.11*** 4.29*** 2.36 2.39 2.43 2.40
Absolute (covariance analysis) 3.70 3.89* 4.09*** 4.41*** 2.35 2.37 2.43 2.42
% of body weight 0.655 0.687* 0.722*** 0.784*** 0.740 0.745 0.768 0.766
Liver
Absolute value (g) 19.50 20.65 20.86* 22.23*** 15.65 16.42 18.01*** 20.36***
Absolute (covariance analysis) 19.59 20.00 20.74** 22.93*** 15.62 16.24 18.02*** 20.57***
% of body weight 3458 3.578 3.665** 4.054*** 4.903 5.110 5.686*** 6.489***
Thyroid gland
Absolute value (g) 0.0233 0.0266* 0.265* 0.0271** 0.0188 0.0212 0.0190 0.0201
Absolute (covariance analysis) 0.0246 0.266* 0.265* 0.0271** 0.0188 0.0211 0.190 0.0201
% of body weight 0.00415 0.00463* 0.00471* 0.00492** 0.00595 0.00666 0.00600 0.00644
Terminal body weight used as covariance
Significantly different from control group value: * = p<0.05, ** = p<0.01, *** = p<0.001

Table #5: Summary test item-related necropsy findings - F0 adults        
Males Females
Group Control Low dose Mid dose High dose Control Low dose Mid dose High dose
Dose (ppm) 0 1500 6500 13000 0 1500 6500 13000
No. Animals examined  28 28 27 27 27 28 28 28
Liver prominent lobular architecture 3 4 4 7 0 1 0 0

Table #6: Summary of test item related histopathology findings - F0 adults
Males Females
Group control low dose mid dose high dose control low dose mid dose high dose
Dose (ppm) 0 1500 6500 13000 0 1500 6500 13000
No. of animals examined 28 28 27 27 27 28 28 28
Kidney (No. examined) (28) (28) (27) (27) (27) (28) (27) (28)
Hyaline droplets, tubular 5 19 27 27 0 0 0 0
Minimal 5 17 6 3 0 0 0 0
Mild 0 2 21 20 0 0 0 0
Moderate 0 0 0 4 0 0 0 0
Hyaline casts 5 7 6 18 1 4 1 4
Minimal 5 7 6 14 1 4 1 4
Mild 0 0 0 4 0 0 0 0
Basophilic tubules, focal 17 17 20 24 3 4 6 10
Minimal 17 17 18 6 3 4 6 7
Mild 0 0 2 15 0 0 0 3
Moderate 0 0 0 3 0 0 0 0
Mononuclear cell infiltration 14 13 15 20 4 5 6 6
Minimal 14 13 15 17 4 4 6 6
Mild 0 0 0 2 0 1 0 0
Liver (No. examined) (28) (28) (27) (27) (27) (28) (28) (28)
Centrilobular hypertrophy, hepatocellular 0 0 3 12 0 0 2 13
Minimal 0 0 3 10 0 0 2 10
Mild 0 0 0 2 0 0 0 3

Table #7: Duration of gestation and overall litter performance: F0 generation, F1 production 
Group Control Low dose Mid dose High dose
Dose (ppm) 0 1500 6500 13000
Number pregnant 26 27 27 28
Duration of gestation (days):
21 8 6 9 9
22 18 21 18 17
Mean duration 21.7 21.8 21.7 21.7**
Number females producing a live litter 26 27 27 28
Gestation index (%)    100 100 100 100
Mean number of implant sites* per pregnancy ± SD 15.4 ± 1.9 15.4 ± 2.8 15.5 ± 2.8 13.8 ± 2.0
Mean total number of pups* born per litter 14.5 ± 2.6 14.3 ± 2.6 14.3 ± 2.6 12.9 ± 1.8
Mean number of live pups* per litter ± SD: 
Day 0 of lactation 14.2 ± 2.6 14.2 ± 2.6 14.3 ± 2.6 12.8 ± 1.8
Day 1 of lactation 14.0 ± 2.6 14.1 ± 2.6 14.3 ± 2.6 12.6 ± 1.8
Day 4 of lactation 13.1 ± 3.7 14.0 ± 2.8 14.1 ± 2.4 12.4 ± 2.1
Day 7 of lactation 13.0 ± 3.7 13.9 ± 2.8 13.9 ± 2.3 12.3 ± 2.2
Day 14 of lactation 12.2 ± 3.7 13.0 ± 3.0 12.7 ± 3.2 11.4 ± 2.5
Day 21 of lactation 11.7 ± 3.7 12.5 ± 3.4 12.1 ± 3.5 11.3 ± 2.9
Total number of males* on Day 1 of lactation (%) 188 (54) 185 (49) 173 (45) 174 (49)
Total number of females* on Day 1 of lactation (%) 161 (46) 196 (51) 213 (55) 180 (51)
*= animals rearing young to Day 21 of lactation only
** = excludes two animals where gestation lenght not calculable due to no positive mating sign being noted

Table #8: Summary of test item-related organ weight findings - F1 adults         
Males Females
Group control low dose mid dose high dose control low dose mid dose high dose
Dose (ppm) 0 1500 6500 13000 0 1500 6500 13000
No. animals per group 22 24 24 24 24 24 24 24
Kidney
Absolute value (g) 3.80 3.92 4.31*** 4.35*** 2.45 2.54 2.57 2.53
Absolute (covariance analysis) 3.79 3.89 4.26*** 4.44*** 2.45 2.52 2.56 2.56
% of body weight 0.687 0.706 0.770*** 0.812*** 0.752 0.770 0.787 0.787
Liver
Absolute value (g) 19.44 20.13 21.70** 21.30* 15.46 16.60 18.02*** 20.17***
Absolute (covariance analysis) 19.37 19.94 21.39*** 21.87*** 15.49 16.41 17.91*** 20.43***
% of body weight 3.508 3.614 3.865*** 3.979*** 4.754 5.022 5.497*** 6.268***
Thyroid gland
Absolute value (g) 0.0219 0.0234 0.0231 0.0248 0.0202 0.0234 0.0205 0.0206
Absolute (covariance analysis) 0.0219 0.0233 0.229 0.0251** 0.0202 0.0233 0.0205 0.0206
% of body weight 0.00397 0.00422 0.00415 0.00465*** 0.00620 0.00712 0.00629 0.0042
Terminal body weight used as covariance
Significantly different from control group value: * = p<0.05, ** = p<0.01, *** = p<0.001

Table #9: Summary of test item related histopathology findings - F1 adults
Males Females
Group control low dose mid dose high dose control low dose mid dose high dose
Dose (ppm) 0 1500 6500 13000 0 1500 6500 13000
No. of animals examined 22 24 24 24 24 24 24 24
Kidney (No. examined) (22) (24) (24) (24) (24) (24) (24) (24)
Hyaline droplets, tubular 2 13 24 23 0 0 0 0
Minimal 2 13 5 23 0 0 0 0
Mild 0 0 19 17 0 0 0 0
Moderate 0 0 0 4 0 0 0 0
Hyaline casts 5 5 5 18 2 3 0 4
Minimal 5 5 5 15 2 3 0 4
Mild 0 0 0 3 0 0 0 0
Basophilic tubules, focal 15 16 21 21 3 4 4 3
Minimal 14 16 16 13 3 4 4 3
Mild 1 0 5 6 0 0 0 0
Moderate 0 0 0 2 0 0 0 0
Mononuclear cell infiltration 9 10 18 19 5 5 5 5
Minimal 9 10 17 15 5 5 5 5
Mild 0 0 1 4 0 0 0 0
Liver (No. examined) (22) (24) (24) (24) (24) (24) (24) (24)
Centrilobular hypertrophy, hepatocellular 0 0 4 8 0 0 2 9
Minimal 0 0 4 6 0 0 2 8
Mild 0 0 0 2 0 0 0 1

Table #10: Ovary scoring: mean values ± standard deviation: F1 generation
Follicles Group/dose levels (ppm)
control high dose
(0) (13000)
Single layer follicles 22 ± 13 14 ± 9
Multiple layer follicles 3 ± 2 2 ± 2
Antral follicles 2 ± 2 2 ± 2
Total follicles counted 16 ± 15 18 ± 11

Table #11: Duration of gestation and overall litter performance: F1 generation, F2 production
Group Control Low dose Mid dose High dose
Dose (ppm) 0 1500 6500 13000
Number pregnant 22 23 23 23
Duration of gestation (days):
21 2 3 3 3
22 16 13 19 18
23    4 7 1 2
Mean duration 22.1 22.2 21.9 22.0
Number females producing a live litter 22 23 22 23
Gestation index (%)    100 100 96 100
Mean number of implant sites* per pregnancy ± SD 16.6 ± 2.3 16.3 ± 3.1 16.6 ± 2.4 14.7 ± 2.5
Mean total number of pups* born per litter 15.5 ± 2.3 15.5 ± 3.1 14.7 ± 3.4 13.2 ± 2.5
Mean number of live pups* per litter ± SD: 
Day 0 of lactation 15.2 ± 1.9 15.4 ± 3.1 14.6 ± 3.4 13.2 ± 2.5
Day 1 of lactation 15.0 ± 2.0 14.8 ± 3.4 14.5 ± 3.4 12.9 ± 2.6
Day 4 of lactation 14.8 ± 1.9 14.2 ± 3.3 14.3 ± 3.4 12.7 ± 2.6
Day 7 of lactation 14.7 ± 1.8 14.0 ± 3.5 14.3 ± 3.4 12.7 ± 2.5
Day 14 of lactation 14.7 ± 1.8 14.0 ± 3.5 14.2 ± 3.4 12.6 ± 2.5
Day 21 of lactation 14.7 ± 1.8 14.0 ± 3.5 14.2 ± 3.4 12.6 ± 2.5
Total number of males* on Day 1 of lactation (%) 142 (45) 177 (52) 159 (50) 147 (52)
Total number of females* on Day 1 of lactation (%) 174 (55) 164 (48) 160 (50) 136 (48)
* = animals rearing young to Day 21 of lactation only

Table #12: Survival indices: F0 generation, F1 production
Group control low dose mid dose high dose
Dose (ppm) 0 1500 6500 13000
Birth index Mean litter index (%) 94 94* 93 93
Number losing >2 pups 3 2 6 4
Number of litters 26 25* 27 28
Live birth index Mean litter index (%) 98 100 100 100
Number losing >1 pup 1 0 0 0
Number of litters 26 27 27 28
Viability index Mean litter index (%) 93 98 99 97
Days 0 - 4 Number losing >3 pups 1 0 1 1
Number of litters 26 27 27 28
Lactation index Mean litter index (%) 91 89 86 92
Days 4 - 21 Number losing >1 pup 5 8 10 6
Number of litters 25 27 27 28
Overall survival index Mean litter index (%) 84 88 85 88
Birth - 21 Number losing >4 pups 6 3 5 3
Number of litters 26 27 27 28
* = Litters where birth index not calculable excluded

Table #13: Group mean litter and pup weight: F0 generation, F1 production
Day of lactation Group/dose level (ppm)
control low dose mid dose high dose
0 1500 6500 13000
Litter
Day 1 93 ± 19 95 ± 15 98 ± 13 87 ± 12
Day 4 136 ± 21 138 ± 20 140 ± 18 123 ± 22
Day 7 200 ± 27 205 ± 30 201 ± 27 176 ± 31
Day 14 361 ± 48 376 ± 51 357 ± 64 322 ± 61
Day 21* 587 ± 91 608 ± 77 579 ± 102 530 ± 75
Mean of litter mean pup weight
Males
Day 1 7.0 ± 0.9 7.0 ± 0.7 7.2 ± 0.7 7.1 0.6**
Day 4 10.4 ± 1.5 10.4 ± 1.3 10.4 ± 1.4 10.2 1.3
Day 7 15.3 ± 2.2 15.5 ± 1.8 15.1 ± 2.0 14.8 2.0
Day 14 29.5 ± 4.5 30.4 ± 4.5 29.8 ± 3.9 28.9 3.5
Day 21 50.6 ± 9.5 49.2 ± 8.7 49.0 ± 8.5 47.1 6.4
Females
Day 1 6.4 ± 1.2 6.6 ± 0.7 6.7 ± 0.7 6.7 ± 0.7**
Day 4 9.8 ± 1.4 9.8 ± 1.2 9.8 ± 1.3 9.7 ± 1.3
Day 7 14.7 ± 2.1 14.6 ± 1.9 14.4 ± 1.8 14.1 ± 2.0
Day 14 29.0 ± 4.6 29.5 ± 4.8 28.2 ± 3.8 28.1 3.6
Day 21 47.5 ± 7.7 47.7 ± 8.7 46.6 ± 7.4 45.2 5.9
Means exclude litters where all pups died.
* = excludes litters where not all pups weighed on Day 21 of lactation in error
** = excludes litter where pups mis-sexed on Day 1 of lactation

Table #14: Preputial separations and vaginal openings: F1 generation
Group/dose level (ppm)
control low dose mid dose high dose
0 1500 6500 13000
Females
Age (days) at vaginal openings 33.0 ± 1.7 34.1 ± 1.7 34.8 ± 1.2 35.8 ± 2.4
Weight (g) at vaginal opening 108 ± 12 117 ± 14 116 ± 12 119 ± 16
Males
Age (days) at preputial separation 40.7 ± 1.3 41.0 ± 1.7 41.4 ± 1.5 42.4 ± 2.3
Weight (g) at preputial separation 192 ± 21 194 ± 20 197 ± 19 201 ± 18

Table #15: Survival indices: F1 generation, F2 production
Group control low dose mid dose high dose
Dose (ppm) 0 1500 6500 13000
Birth index Mean litter index (%) 94 95 88 90
Number losing >2 pups 2 2 3 2
Number of litters 22 23 22 23
Live birth index Mean litter index (%) 99 99 99 100
Number losing >1 pup 1 0 1 0
Number of litters 22 23 23 23
Viability index Mean litter index (%) 93 92 94 92
Days 0 - 4 Number losing >3 pups 2 2 2 1
Number of litters 22 23 23 23
Lactation index Mean litter index (%) 95 98 95 99
Days 4 - 21 Number losing >1 pup 1 1 0 0
Number of litters 22 23 23 22
Overall survival index Mean litter index (%) 91 91 93 91
Birth - 21 Number losing >4 pups 3 2 1 1
Number of litters 22 23 23 23

Table #16: Group mean litter and pup weight: F1 generation, F2 production
Day of lactation Group/dose level (ppm)
control low dose mid dose high dose
0 1500 6500 13000
Litter
Day 1* 103 ± 12 100 ± 25 99 ± 23 90 ± 17
Day 4 150 ± 25 145 ± 37 147 ± 32 133 ± 24
Day 7 221 ± 34 212 ± 51 214 ± 43 194 ± 32
Day 14 415 ± 46 398 ± 78 388 ± 65 347 ± 49
Day 21 661 ± 74 652 ± 127 632 ± 108 575 ± 80
Mean of litter mean pup weight
Males
Day 1* 7.1 ± 0.7 7.0 ± 0.8 7.1 ± 0.7 7.3 ± 0.9
Day 4 10.4 ± 1.6 14.4 ± 1.1 10.8 ± 1.4 11.0 ± 1.5
Day 7 15.5 ± 2.2 15.6 ± 1.6 15.9 ± 2.4 16.0 ± 2.0
Day 14 29.1 ± 2.8 29.7 ± 4.3 29.0 ± 5.3 28.8 ± 4.0
Day 21 46.6 ± 5.3 48.9 ± 7.8 47.4 ± 7.7 48.2 ± 8.3
Females
Day 1 6.7 ± 0.6 6.6 ± 0.7 6.7 ± 0.9* 6.9 ± 0.8
Day 4 10.0 ± 1.3 10.0 ± 1.0 10.2 ± 1.5 10.3 ± 1.5
Day 7 14.8 ± 1.9 14.9 ± 1.4 14.9 ± 2.3 15.1 ± 2.1
Day 14 27.9 ± 2.9 28.8 ± 4.5 27.7 ± 4.7 27.4 ± 4.0
Day 21 44.6 ± 5.3 47.1 ± 6.8 44.7 ± 7.7 45.6 ± 7.6
Means exclude litters where all pups died.
* = excludes litters that were miscounted and/or mis-sexed on Day 1 of lactation

Table #17: Summary of test item-related organ weight findings - F2 Weanlings
Males Females
Group control low dose mid dose high dose control low dose mid dose high dose
Dose (ppm) 0 1500 6500 13000 0 1500 6500 13000
Brain
Absolute value (g) 1.51 1.52 1.57* 1.61*** 1.46 1.48 1.51 1.51
Absolute (covariance analysis) 1.51 1.52 1.56* 1.60*** 1.47 1.48 1.51 1.50
% of body weight 2.853 2.854 2.807 2.837 2.884 2.925 2.938 2.936
Terminal body weight used as covariance
Significantly different from control group value: * = p<0.05, ** = p<0.01, *** = p<0.001
Conclusions:
In conclusion, the dietary administration of Alcohols, C2-33, manuf. Of, by-products from, overheads (the scource substance) to rats was well tolerated, and the parental and reproductive No-Observed-Adverse-Effect-Levels (NOAELs) were considered to be 13000 p.p.m (mean achieved dosages of: 767 mg/kg/day in F0 males; 988, 881 or 2293 mg/kg/day in F0 females prior to mating, during gestation and during lactation, respectively; 1124 mg/kg/day in F1 males, and 1200, 912 or 2256 mg/kg/day in F1 females prior to mating, during gestation and during lactation, respectively). A No-Observed-Effect-Level (NOEL) could not be established due to the presence of non-adverse findings in the kidneys of males at all dose levels.
Executive summary:

The study was designed to provide general information concerning the effects of the source substance, Alcohols, C2-33, manuf. of, by-products from, overheads (876065-86-0), on reproductive performance when given orally to rats.

F0 animals were randomised into three test groups and one control group. These animals were dosed for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after their litters (F1) reached Day 21 of lactation.

From each group, 24 males and 24 females were retained for post weaning assessments. These F1 animals continued dosing for 10 weeks after weaning, and then throughout mating, gestation and lactation until termination after their litters (F2) reached Day 21 of lactation.

The study design was as follows:

Group 

 Dose level

(mg/kg/day)

 Dose level

(ppm)

     Number of F0 animals     Number of F1 animals
       Males  Females  Males  Females
control*   0  0  28  28  24  24
 low dose  100  1500  28  28  24  24
 mid dose  500  6500  28  28  24  24
 high dose  1000  13000  28  28  24  24

* The control diet was untreated SDS VRF-1 ground diet.

Animals from both generations were monitored for clinical signs of toxicity and for effects on body weight, food consumption, oestrous cycle, mating performance, pregnancy performance, difficulty or prolongation of parturition and for deficiencies in maternal care. The offspring were monitored for survival and growth up to weaning. In addition, the following endpoints were evaluated in both generations: gross necropsy findings, organ weights, histopathological evaluation, qualitative examination of the testes and examination of the ovaries and sperm.

There were 5 unscheduled deaths during the course of the study, 3 adult F0 generation animals and 2 adult F1 generation animals. All deaths were considered to be incidental and unrelated to treatment with the test item.

There were no clinical observations in either generation which were considered to be related to treatment with the test item.

At 13000 ppm, reduced mean weight gain was noted in F0 adult females during the premating period (approximate 10%) and in F1 adult females during gestation (approximately 17%), when compared with controls. There were no effects on body weight in males of either generation at any dose level or in females of either generation at 1500 or 6500 ppm.

At 13000 ppm, lower mean food consumption was noted in adult F0 and F1 females throughout the majority of pre-mating, gestation and lactation period (up to approximately 12% or 10% in the F0 and F1 generations, respectively). There were no effects on food consumption in males of either generation at any dose level or in females of either generation at 1500 or 6500 ppm.

Oestrous cycles, mating performance, fertility, duration of gestation and maternal care were similar between all groups in both generations. There were also no obvious differences in these parameters between generations.

At 13000 ppm, lower numbers of implantation sites and lower litter size with correlating lower litter weight, were noted in both generations, when compared with Controls (approximately 11-12% or 15-16% for all parameters in the F1 and F2 litters, respectively). It was considered that these findings were likely secondary to the maternal body weight and food consumption changes. Mean number of implantations sites, litter size and litter weight were similar to controls of both generations at 1500 or 6500 ppm.

Mean pup weights (male and female) and mean pup survival were similar between all groups in both generations. There were also no obvious differences in these parameters between generations.

In F1 adults, a slight delay in sexual maturation, as indicated by slightly higher ages and weights at vaginal opening or preutial separation, was noted at all dose levels.

In F0 generation adults, pathology findings included: higher kidney weights in males at ≥1500 ppm, which correlated with histopathology findings of increased incidences and severity of hyaline droplets (≥1500 ppm), basophilic tubules (≥6500 ppm) and hyaline casts and mononuclear cell infiltration (males at 13000 ppm); higher incidences of basophilic tubules in female kidneys at 13000 ppm; higher liver weights in both sexes at ≥ 6500 ppm, which correlated with a slightly increased incidence of macroscopic prominent lobular architecture at 13000 ppm and centrilobular hepatocellular hypertrophy at ≥ 6500 ppm; and higher thyroid weights in males at ≥ 1500 ppm (no macroscopic or histopathology correlates).

In F1 generation adults, pathology findings included higher kidney weights in males at ≥ 6500 ppm, which correlated with histopathology findings of increased incidences and severity of hyaline droplets (≥1500 ppm) , basophilic tubules and mononuclear cell infiltration (≥6500 ppm) and hyaline casts (13000 ppm); and higher liver weights with correlating centrilobular hepatocellular hypertrophy in both sexes at ≥ 6500 ppm. In F0 females at 13000 ppm, the number and type of ovarian follicles was similar to controls. In F1 females at 13000 ppm, the total number ovarian follicles, attributed to the number of single layer follicles, was approximately 30% lower than controls.

There were no changes in the number of sperm or spermatids, sperm morphology or sperm motility in males of either generation which were considered to be related to treatment with the test item.

There were no toxicologically relevant organ weight, gross necropsy or histopathology findings in the weanlings of either generation (F1 pups or F2 pups) which were considered to be related to treatment with the test item.

None of the aforementioned changes were considered to be adverse, but were considered to be either adaptive or not relevant to man..

In conclusion, the dietary administration of Alcohols, C2-33, manuf. Of, by-products from, overheads (876065-86-0) to rats was well tolerated, and the parental and reproductive No- Observed-Adverse-Effect-Levels (NOAELs) were considered to be 13000 ppm (mean achieved dosages of: 767 mg/kg/day in F0 males and 988, 881 and 2293 mg/kg/day in F0 females prior to mating, during gestation and during lactation, respectively; and 1124 mg/kg/day in F1 males and 1200, 912 and 2256 mg/kg/day in F1 females prior to mating, during gestation and during lactation, respectively). A No-Observed-Effect-Level (NOEL) could not be established due to the presence of non-adverse findings in the kidneys of males at all dose levels.

Justification for classification or non-classification

In a preliminary, Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD 422) of the source substance, at a dose of 1000 mg/kg/day, there was an increase in the number of animals giving birth to dead pups and also in the number losing more than 3 pups over Days 0-4 of lactation, compared with Controls. Therefore, for the neonatal toxicity, the Lowest Observed Adverse Effect Level (LOAEL) was 1000 mg/kg/day and the No Observed Effect Level (NOEL) was considered to be 500 mg/kg/day.From the findings on this study, the parental NOAEL was considered to be 100 mg/kg/day, however, findings at 500 and 1000 mg/kg/day were confined to salivation, and kidney findings which may be reversible and not relevant in man. The neonatal NOEL was considered to be 500 mg/kg/day.As findings in this study were considered not to be relevant in man, the parental NOAEL for man could be 1000 mg/kg/day.

In a subsequent, two generation reproductive toxicity study (OECD 416), the dietary administration of the source substance was well tolerated, and the parental and reproductive No-Observed-Adverse-Effect-Levels (NOAELs) were considered to be 13000 p.p.m (mean achieved dosages of: 767 mg/kg/day in F0 males; 988, 881 or 2293 mg/kg/day in F0 females prior to mating, during gestation and during lactation, respectively; 1124 mg/kg/day in F1 males, and 1200, 912 or 2256 mg/kg/day in F1 females prior to mating, during gestation and during lactation, respectively).

Additional information