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Effects on fertility

Description of key information

Two-Generation Toxicity Study (OECD 416), rats:

NOAEL systemic = 2 ppm corresponding to:

NOAEL systemic (males, P1) = 0.126 mg/kg bw/day

NOAEL systemic (females, P1) = 0.202 mg/kg bw/day

NOAEL systemic (males, F1) = 0.142 mg/kg bw/day

NOAEL systemic (females, F1) = 0.204 mg/kg bw/day

NOAEL fertility >= 200 ppm (highest dose tested) corresponding to:

NOAEL fertility (males, P1) = 13.1 mg/kg bw/day

NOAEL fertility (females, P1) = 20.4 mg/kg bw/day

NOAEL fertility (males, F1) = 14.6 mg/kg bw/day

NOAEL fertility (females, F1) = 20.9 mg/kg bw/day

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
13.1 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, of Regulation (EC) No 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The test substance was mixed in the basal feed at 0 (basal feed), 2, 20, and 200 ppm and given to 24 male and 24 female SPF Wistar Hannover rats/group for two successive generations, to evaluate the potential effects on reproductive performance and on the growth and development of their offspring (2007). The study was performed under GLP and according to OECD 416.

Systemic toxicities of the test substance on parental animals were observed at a dose level of 20 ppm and above. In these dose groups, opacity of the eyeballs and/or coarsened corneal surface were found in male and female parental animals, which caused statistically significant increases in the incidence of opacity of the eyeballs in P males and that of both findings in F1 males and in P and F1 females. After histopathological examination these effects were diagnosed as keratitis. Similar effects were observed in the combined repeated dose toxicity/carcinogenicity study (2006).These findings are characteristics of a compound such as the test substance that inhibits 4-ydroxyphenylpyruvic acid dioxygenase (4-HPPDase), an enzyme of the tyrosine catabolic pathway. These lesions are related to an increase in plasma tyrosine level caused by a blockade of the 4-HPPDase enzyme in the rat. However, the rat is a species particularly sensitive to inhibition of the 4-HPPDase enzyme and is atypical in its susceptibility to develop tyrosine-related eye lesions. Therefore, although these lesions (corneal lesions and eye keratitis) were treatment-related, they were considered not to be toxicologically relevant to man.

Body weights, body weight gains and food consumption of parental animals in these treated groups were also affected by the test substance treatment, in which significantly lower-than-control values were found in body weights of the 200 ppm group and body weight gains and food consumptions of the 20 and 200 ppm groups.

The following observations were made on organ weights and histopathological parameters:

A significant increase in relative thyroid weights of P males in the 20 and 200 ppm groups was accompanied by such histopathological alterations as increased large-sized follicles, colloidal alteration and hypertrophy of the follicular cell and the incidences of these findings in both treated groups (20 and 200 ppm treated groups) were statistically significantly higher than those in the control group. As the incidences of colloidal alteration in these groups were also higher than that in the control group in F1 generation, the test substance is concluded to have an effect on the thyroid at the dose levels higher than 20 ppm, although an increase of the thyroid weight was unclear in F1 generation. The weight of thyroid in males of the 2 ppm group was comparable to that in the control group and there was no statistically significant difference in the incidence of histopathological findings of the thyroid between the control and 2 ppm groups.

A significantly increase in relative kidney weights of parental males at a dose level of 20 ppm and above are thought to be treatment-related because of the clear dose- response relationship in both generations. Histopathological observations also revealed chronic nephropathy in 2 F1 males out of 12 examined in the 200 ppm group. This change is suggested to be related to the test substance treatment because chronic nephropathy rarely occurred in young rats and an increase in kidney weights was accompanied by a significantly increased incidence of chronic nephropathy in the treated groups at 50 ppm and above in the previous “Chronic toxicity and carcinogenicity study of the test substance in the Wistar rat by dietary administration” (2006). In the 2 ppm group, however, neither kidney weights nor histopathological changes were observed.

Absolute and/or relative adrenal weights increased significantly in F1 male and female parental animals of the treated groups at 20 ppm and above when compared to those in the control group. However, this does not suggest toxicologically significant effects of the test substance since histopathological observations of 12 selected animals in the 200 ppm group revealed no abnormalities in the adrenals. There were no statistically significant differences of these organ weights between the 2 ppm group and the control group.

Relative liver weights of both males and females in the treated groups at 20 ppm and above were significantly and dose-dependently higher than those in the control group in both generations. The histopathological examinations of all the parental animals showed an increased incidence of centrilobular hypertrophy of the hepatocyte in males of the treated groups at 20 ppm and above. These results suggest that the increase in liver weight is a physiological response to metabolizing a xenobiotic. Furthermore, an increase in liver weights in the 200 ppm group, in which a degree of weight change was most obvious, was relatively slight (less than 130% to the control group), leading to the conclusion that a series of alterations observed in the liver are not adverse but merely adaptive.

Relative brain weights of the females in the 200 ppm group, as well as absolute brain weights of F1 male and female parental animals in the 20 and 200 ppm groups were significantly lower than those in the control group, respectively. Similar alterations were also found in F1 weanlings of the 200 ppm group, in which absolute brain weights decreased significantly while relative weights increased significantly. These results suggest that decreases in brain weights are due to suppressed body weights in these F1 parental animals. In the present study, suppression of body weight gains in females selected for F1 female parents was limited during a first half of the growth period and the lactation period and final body weights at necropsy in the treated groups were comparable to those in the control group. These facts lead to the speculative interpretation that relative brain weight of F1 parental females seem to be reduced at necropsy due to slight suppression of brain growth during early life accompanied by normal body growth in the later life. Thus, the decrease in brain weight is considered to be a non-specific alteration which was caused mainly by the treatment-related suppression of body weight gains.

In the P-males, the relative weights of the testes in all treated groups increased significantly, being suspected to be related to the treatment of test substance. In addition, the absolute weights of the epididymides and seminal vesicles increased at 20 and 200 ppm. In the P1-males the relative weights of the testes at 20 and 200 ppm increased significantly, compared with control. In addition, the absolute weights of the epididymides and seminal vesicles at 20 and 200 ppm were slightly higher than those in the control group. The relative weights of the seminal vesicles at 200 ppm reached statistically significance. However, these were thought to be toxicologically insignificant since no abnormalities were seen in such parameters as the number of sperm, sperm motility and reproductive performance, and histopathological observations failed to reveal findings that may be related to the organ weight increases. Furthermore, the relative weight of testes and epididymides generally increases in rats with decreased body weight (Derelanko, M.J. Toxicologist’s pocket handbook. 2003 ISBN 0-8493-0009-6). In conclusion, these effects were not considered to be relevant to the fertility of the male rats.

Swelling of the mammary gland in parental females in the 20 and 200 ppm groups after weaning are suggested to be related to the test substance treatment because the findings are concordantly observed in both P and F1 generations, although their incidences (1 to 3 animal/group) were not statistically significantly different from that in the control group. Histopathology of the mammary gland of these females with gross findings revealed incomplete recovery from lactation phase. The alterations found here seem to be toxicologically insignificant and judged as not adverse.

In reproductive performance, there were no changes related to the test substance treatment in the following reproductive parameters: incidence of females with normal estrus cycle, mating index, fertility index, duration of gestation, and the number of implantation sites in parental animals. In sperm examination for P- and F1 parental male animals, sperm head count in the testis, sperm counts in the cauda epididymis and sperm motility in each group were not significantly different from those in the control group. The histopathological examination of the reproductive organs and pituitary showed no abnormal findings in these animals.

Based on the effects found in the thyroid gland and kidneys, the no-observed-adverse-effect levels of the test substance in parental rats regarding the parental systemic toxicity is concluded as 2 ppm (P generation: 0.126 mg/kg/day in males and 0.202 mg/kg/day in females, F1 generation: 0.142 mg/kg/day in males and 0.204 mg/kg/day in females).

No adverse effects on reproductive performance of parental animals were noted even in the highest dose level of 200 ppm in the present study which leads to a NOAEL for fertility of 200 ppm (P generation: 13.1 mg/kg/day in males and 20.4 mg/kg/day in females, F1 generation: 14.6 mg/kg/day in males and 20.9 mg/kg/day in females).

Effects on developmental toxicity

Description of key information

Two-Generation Toxicity Study (OECD 416), rats:

NOAEL systemic = 2 ppm corresponding to:

NOAEL systemic (males, P1) = 0.126 mg/kg bw/day

NOAEL systemic (females, P1) = 0.202 mg/kg bw/day

NOAEL systemic (males, F1) = 0.142 mg/kg bw/day

NOAEL systemic (females, F1) = 0.204 mg/kg bw/day

NOAEL (developmental) = 2 ppm corresponding to:

NOAEL developmental (males, P1) = 0.126 mg/kg bw/day

NOAEL developmental (females, P1) = 0.202 mg/kg bw/day

NOAEL developmental (males, F1) = 0.142 mg/kg bw/day

NOAEL developmental (females, F1) = 0.204 mg/kg bw/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
0.126 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.7, of Regulation (EC) No 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The test substance was mixed in the basal feed at 0 (basal feed), 2, 20, and 200 ppm and given to 24 male and 24 female SPF Wistar Hannover rats/ dose group for two successive generations to evaluate the potential effects on reproductive performance and on the growth and development of their offspring (2007). The study was performed under GLP and according to OECD 416.

Effects of the test substance treatment on pups were noted at a dose level of 20 ppm and above, in which body weight gains of pups were reduced significantly and dose-dependently. Opacity of the eyeballs was induced in pups of the 200 ppm group at and after postnatal day 21, although its incidence was low. Histopathological observations also revealed keratitis in some weanlings of the 20 and 200 ppm groups, of which incidences in F1 male weanlings in the 20 ppm group and in F1 and F2 male and female weanlings in the 200 ppm group were significantly higher than those in the control group. However, these findings are not considered to be toxicologically relevant to humans but rather species specific. No toxicologically relevant findings were noted in pups of the 2 ppm group.

The necropsy of weanlings showed the absolute weights of the brain, spleen and thymus in F1 and F2 male and female weanlings in the 20 ppm and higher dose groups often showed significant low values. However, the relative weights of these organs were similar to or significantly higher than those in the control group, being considered to be the changes caused by low body weights. Furthermore, the relative weight of the brain in F2 male weanlings in the 2 ppm group was significantly low, whereas there were no significant differences in the 20 ppm and higher dose groups, being considered to be an accidental change. Furthermore, an increase in liver weight, in which the differences from controls were statistically significant in relative liver weights of F1 weanling in the 20 and 200 ppm groups and those of F2 weanlings in all the treated groups. Thus, all the weighed livers were examined histopathological. However, the examinations did not show any changes related to the test substance treatment except for focal necrosis of the hepatocyte in 1 to 2 weanling of the treated group, suggesting that an increase in liver weights of weanlings is adaptive response. Dilatation of renal pelvis was noted in F1 and F2 weanlings in each group including the control group and there was statistically significant difference of the incidence of this finding in F2 weanlings between the 200 ppm group and the control group. There was no tendency of increasing incidence of this finding in the 2 and 20 ppm groups in each generation.

In the observation of sexual development in F1 parental male animals, the age of completion of preputial separation was delayed statistically significantly at 20 ppm and above. However, these changes are considered to be due to lowered body weights that were caused by the test substance treatment. In addition, measurements of anogenital distance did not reveal the influence of test substance treatment in the absolute and relative values of F2 pups.

Treatment-related effects were only noted in offspring at the same dose levels at which maternal toxicity was observed. Based on the reduced body weights, the no-observed-adverse-effect levels of the test substance in pups are concluded as 2 ppm (P generation: 0.126 mg/kg/day in males and 0.202 mg/kg/day in females, F1 generation: 0.142 mg/kg/day in males and 0.204 mg/kg/day in females).

Justification for classification or non-classification

The available data on reproductive toxicity with 2-{2-chloro-4-mesyl-3-[(tetrahydrofuran-2-ylmethoxy)methyl]benzoyl}cyclohexane-1,3-dione (CAS 473278-76-1) meets the criteria for classification according to Regulation (EC) No 1272/2008 regarding systemic toxicity, and is therefore classified as STOT RE Cat. 1 (H373).

Regarding reproductive performance the test substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008, and is therefore conclusive but not sufficient for classification.

Developmental effects occurred only under systemic toxic effects of parental animals and therefore are considered to be secondary and thus no classification for developmental effects is required.