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Key value for chemical safety assessment

Effects on fertility

Description of key information

Two generation reproductive toxicity, oral (OECD 416), rat:

reproductive toxicity: NOAEL = 180 mg/kg bw/day for the P generation, NOAEL = 60 mg/kg bw/day for the F1 generation

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable (Klimisch score 1) 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

A reliable study regarding reproductive toxicity is available for the test substance.

The effects of the test substance on male and female reproductive parameters were investigated in a two-generation study according to OECD Guideline 416 (2001) and in compliance with GLP. The test substance was suspended in corn oil and administered orally via gavage to HsdCpb:WU Wistar rats at doses of 60, 180 and 540 mg/kg bw/day. Similarly, corn oil was administered to rats in the vehicle control group. Each group consisted of 30 male and 30 female rats. Males were treated 10 weeks prior to mating and then 2 weeks during the mating period. Females were treated 10 weeks prior to mating and throughout the gestation and lactation periods. Animals from all groups were observed for clinical signs, behaviour, physical abnormalities and changes in body weight and food consumption during the various phases of the experiment. Vaginal cytology for oestrous cyclicity was evaluated during the last three weeks of pre-mating period and the stage of oestrous cycle was also checked at necropsy. After a minimum of 10 weeks of treatment, females were cohabitated with males in a 1:1 ratio. The number, weight, survivability and mortality of pups were observed during the lactation period. Physical signs of postnatal development were observed daily till the criteria were met. Vaginal opening and preputial separation were also observed in pups selected for the F1 generation. The animals were subjected to detailed necropsy at sacrifice and selected organs were weighed. Andrological assessments like sperm motility were evaluated for all groups, whereas the sperm morphology, enumeration of homogenisation-resistant testicular spermatids and cauda epididymal sperm counts were carried out for the control and high dose group only. Histopathological examination was carried out on all collected tissues from control and high dose group animals of both the generations. In addition, kidneys from males and females of both generations, which showed treatment-related changes in the high dose group, were also examined in the lower dose groups. The post-lactational ovaries were examined for qualitative depletion of primordial follicles in F1 dams. In weanlings, the histopathological examination was conducted on the reproductive tissues in one randomly selected pup/sex/litter in control and high dose groups in both generations. No treatment-related clinical signs or mortality were observed in either sex during the course of experiment. Body weights and net body weight gains were significantly and consistently decreased in males of both generation during most of the administration period at 540 mg/kg bw/day and sporadically in the 180 mg/kg bw/day dose group. In females, significantly lower body weights were observed during the pre-mating period in the P generation, during gestation and up to the first week/day1 of the lactation period in both generations at 540 mg/kg bw/day. A reduction in body weights was also observed during the gestation period in the female F1 generation at 180 mg/kg bw/day. The food intake was lower during days 14-20 of gestation period at 540 mg/kg bw/day dose in P generation. A significant and dose-related decrease in terminal body weights was observed in males at 180 and 540 mg/kg bw/day of the P generation and at 60, 180 and 540 mg/kg bw/day of the F1 generation. An increase in the absolute and relative weight (individual and combined) of the kidneys was observed in all the treated male groups and at 540 mg/kg bw/day in females of both generations. An increase in the relative weight of adrenals was also observed at 540 mg/kg bw/day in males of the F1 generation. Grossly, incidences of rough kidney surfaces were observed at 540 mg/kg bw/day in either sex of both generations. Treatment-related microscopic lesions observed in the kidneys were nephropathy, dilated tubules, basophilic tubules and lymphocytic infiltrations. These lesions (either single or in combination with each other) at 60, 180 and 540 mg/kg bw/day in males and at 540 mg/kg bw/day in females of both generations were considered treatment-related. The incidence and severity of the lesions in the kidneys was higher in the males when compared to the females in both P and F1 generations. Significantly reduced fertility index was observed in the female animals at 540 mg/kg bw/day in P generation and at 180 mg/kg bw/day and 540 mg/kg bw/day in the F1 generation. In the F1 generation significantly reduced fecundity was observed at 540 mg/kg bw/day. Additionally, a significant increase in oestrus cycle length (4.5 days) was observed in the F1 females after treatment with 540 mg/kg bw/day. In the study report this effect is suggested to be incidental since the observed oestrus cycle length is within the historical control range (4.3-4.5). However, the oestrus cycle length of the concurrent control animals as well at the animals in the two lowest dose groups are lower than the historical control range (4.1, 4.0 and 4.1, respectively). Pup body weights per litter were reduced during the lactation period, measured on postnatal days (PND) 1, 4, 7, 14 and 21 at 540 mg/kg bw/day in both generations and on PND 7, 14 and 21 at 180 mg/kg bw/day in the P generation. The reduction in pup body weights in these dose groups was presumably associated with reduced body weights of dams during the gestation period. The observed lower percentages of incisor eruption, ear and eye opening at 540 mg/kg bw/day in both generations and incisor eruption at 180 mg/kg bw/day of the P generation were considered to be treatment-related effects. The slight delay in the acquisition of developmental landmarks in both generations was suggestive of an overall pattern of slight developmental delay. These pre-weaning developmental endpoints were highly correlated with reduced body weights seen in this group. The decreased terminal body weights at 180 and 540 mg/kg bw/day of F1 litter and at 540 mg/kg bw/day of F2 litter in either sex of both generations were considered as treatment-related effect. There were no treatment-related changes in organ weights and organ weight ratios in male and females pups of both the F1 and F2 litters. There were no treatment-related gross or microscopic findings in male or female pups of both generations. In conclusion, based on the observed effects on fertility as well as oestrous cycle length it can not be excluded that chlorophene has an adverse effect on the reproductive performance of female rats. Thus, under the conditions of this 2 generation study in the rat, the LOAEL of the test substance in terms of systemic toxicity in males of the P and F1 generation is 60 mg/kg bw/day. The NOAEL of females of the P and F1 generation regarding systemic effects is 180 and 60 mg/kg bw/day, respectively. In terms of effects on the offspring the NOAEL is 60 and 180 mg/kg bw/day for F1 and F2 pups, respectively. With regard to reproductive parameters the NOAEL for the P generation is 180 mg/kg bw/day and the NOAEL for the F1 generation is 60 mg/kg bw/day. 

Two non-reliable studies regarding reproductive toxicity are available for the test substance.

- Oral one generation study in the rat

A one-generation reproduction/fertility study with the test substance was conducted in albino rats (Haley, 1973a). The study is not performed according to a guideline and not in compliance with GLP. Thus, and in view of additional reporting deficiencies, the study is not considered reliable. However, in terms of all available data, a summary of this study is included. The test substance formulated in corn oil was administered to groups of 10 males and 20 females via gavage at daily dose levels of 50 or 150 mg/kg bw/day. Pre-mating periods were 60 and 14 days for males and females, respectively. Males were treated until the conclusion of the mating period and females were treated until termination of the study. One-half of the female animals from each group was sacrificed on day 14 of gestation and subjected to gross necropsy. Ovaries, uterus and foetuses of each animal were examined for corpora lutea, implantation sites, resorption sites, foetuses and any abnormalities. The remaining dams were allowed to deliver and wean their offspring. Treatment of dams continued throughout the gestation and lactation period.Males body weight gains were reduced in the high-dose group during the pre-mating period. Females of either dose group were not affected. There were no effects on the number of implantation sites, resorption sites and corpora lutea. Reproductive performance was not affected in any treatment group. All delivered pups were normal in appearance. A reduced 4-day survival and lactation index was observed in high-dose litters. Dose-dependent and statistically significant decreases in the body weights of male weanlings in the 50 and 150 mg/kg bw/day groups were observed compared to the control group. Thus, under the conditions of this study, the NOAEL of the test substance in terms of parental toxicity following administration of the test substance to rats via gavage is 50 mg/kg bw/day. In terms of effects on the offspring the LOAEL is 50 mg/kg bw/day.

 

- Perinatal and lactation study in the rat

A perinatal and lactation study with the test substance was conducted in albino rats (Haley, 1973b). The study is not performed according to a guideline and not in compliance with GLP. Thus, and in view of additional reporting deficiencies, the study is not considered reliable. However, in terms of all available data, a summary of this study is included.The test substance formulated in corn oil was administered to groups of 17-19 gravid female albino rats via gavage at dose levels of 50 or 150 mg/kg bw/day from the 15th day of gestation throughout the lactation period. The dams were allowed to deliver, nurse and wean their offspring. Treatment of dams continued throughout the gestation and lactation period. Maternal animals were observed for clinical signs daily and body weights were determined on day 15, at parturition, on days 1, 4 and 12 post partum, at weaning and 1 week post weaning. Pup body weights, behaviour and survival were recorded. No treatment-related effects on parental animals were observed. The numbers of pups delivered viable and retained through lactation day 4 were similar for all groups. Slightly fewer pups were retained by females exposed to the test substance than by control females on lactation days 12 and 21 (weaning). One pup delivered by a control female and 2 pups from dams treated with 50 mg/kg bw/day exhibited facial haematomas. All other progeny obtained were free of external anomalies. The lactation index and 12 day survival index of pups obtained from dams exposed to either level of the test substance were slightly reduced. Other parameters of pup survival revealed no differences between test and control progeny which could be correlated with the maternal treatment. Body weights of test and control pups compared favourably throughout the investigation. There were no untoward behavioural reactions noted among test or control pups. Thus, under the conditions of this 1 generation study in the rat, the NOAEL of the test substance in terms of parental toxicity following administration of the test substance to rats via gavage is 150 mg/kg bw/day. There is a tendency towards decreased pup survival (12-day survival index and lactation index) in the 50 and 150 mg/kg bw/day groups, and it can not be excluded that these effects are treatment-related. Thus, the LOAEL in terms of effects on the offspring is 50 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

Teratogenicity study, oral, rat: NOAEL = 75 mg/kg bw/day

Teratogenicity study, oral, rabbit: NOAEL = 100 mg/kg bw/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, 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

Reliable studies regarding developmental toxicity are available for the test substance.

- Developmental toxicity study in the rat

The teratogenic potential of the test substance was assessed in a developmental toxicity study in CD Sprague-Dawley rats performed in compliance with GLP and similar to OECD Guideline 414 (Tesh, 1985a). Deviating from the Guideline the dams were only treated during foetal organogenesis. Thus, other effects on the development toxicity, as effects on the reproductive organs would not be discovered by this treatment regime. The test substance formulated in corn oil was administered by gavage to three groups of 20 pregnant rats during organogenesis from Day 6 to Day 15 of gestation at dosages of 100, 300 or 900 mg/kg bw/day. These dose levels were chosen based on the results of a range-finding study. A fourth group of 20 dams, serving as controls, received the vehicle during the same treatment period. On Day 21 of gestation, females were sacrificed to allow examination of their uterine contents. No treatment-related mortalities occurred during the study. At the highest dosage (900 mg/kg bw/day) statistically significantly decreased body weights, reduced maternal weight gain and food intake during treatment as well as markedly elevated water intake both during and after the treatment phase was observed. The majority of females (15/20 animals) showed transient episodes of post-dosing salivation, largely on Days 13-15 of gestation. At the intermediate dosage (300 mg/kg bw/day) maternal body weight, weight gain and food intake were again slightly, but statistically significantly, reduced and there was a low incidence of post-dosing salivation (5/20 animals). At the lowest dosage (100 mg/kg/day) no significant indication of response to treatment was observed in maternal animals. Necropsy of females at Day 21 of gestation revelaed no macroscopic changes attributable to treatment. Litter size and foetal survival, weight and morphological development were unaffected by the test substance at dosages up to 900 mg/kg/day during organogenesis. At 300 and 900 mg/kg bw/day the incidence of hydronephrosis increased compared to the experimental control group and the mean of the historical control group, but was within the range of the historical control group. Thus, under the conditions of this study, the NOAEL of the test substance in terms of maternal toxicity following administration of the test substance to pregnant rats during fetal organogenesis via gavage is 100 mg/kg bw/day. The NOAEL in terms of fetal toxicity/teratogenic effects is 900 mg/kg bw/day.

 

- Developmental toxicity study in the rat

The teratogenic potential of the test substance was assessed in a developmental toxicity study inWistar (KFM-HAN)rats performed in compliance with GLP and similar to OECD Guideline 414 (Becker, 1984). Deviating from the Guideline the dams were only treated during foetal organogenesis. Thus, other effects on the development toxicity, as effects on the reproductive organs would not be discovered by this treatment regime. The test substance formulated in polyethylene glycol was administered by gavage to three groups of 25 pregnant rats during organogenesis from Day 6 to Day 15 of gestation, at dosages of 15, 75 or 375 mg/kg bw/day. These dose levels were chosen based on the results of a range-finding study. A fourth group, serving as controls, received the vehicle during the same treatment period. On Day 21 of gestation, females were sacrificed to allow examination of their uterine contents. In the high-dose group 3 dams were found dead on day 16, 17 and 18 post coitum, respectively, corresponding to the first three days after the last test substance administration. Soft faeces with a glistering appearance were observed in the control, low and mid dose groups which was attributed to the vehicle. At 375 mg/kg bw/day mucoid faeces were noted after the second treatment up to necropsy. This finding might be attributed to the test substance. Body weight gains and food consumption was statistically significantly decreased in animals of the high dose group. Litter size, sex distribution and incidence of malformations were unaffected by the test substance in all dose groups. Foetal bw and skeletal maturation were slightly but statistically significantly depressed at 375 mg/kg bw/day. Thus, under the conditions of this study, the NOAEL of the test substance in terms of maternal toxicity as well as fetal toxicity/teratogenic effects following administration of the test substance to pregnant rats during fetal organogenesis via gavage is 75 mg/kg bw/day.

 

- Developmental toxicity study in the rabbit

The teratogenic potential of the test substance was assessed in a developmental toxicity study in New Zealand White rabbits performed in compliance with GLP and similar to OECD Guideline 414 (Tesh, 1985c). Deviating from the Guideline only 14 – 16 dams were treated and only during foetal organogenesis. Thus, other effects on the development toxicity, as effects on the reproductive organs would not be discovered by this treatment regime. The test substance formulated in corn oil was administered by gavage to three groups of 14 – 16 pregnant rabbits during organogenesis from Day 6 to Day 19 of gestation at dosages of 10, 30 or 100 mg/kg bw/day. The dose levels were chosen based on the results of a range-finding study. In this range-finding study (Tesh, 1985d), the test substance formulated in corn oil was administered at concentrations of 0, 30, 100, 150, 200 and 300 mg/kg bw/day orally via gavage to groups of 4 pregnant females per dose group during gestation days 6 - 19. A very steep dose-response with regards to maternal toxicity was observed. While no signs of toxicity were noted at 100 mg/kg bw/day, rapid weight loss and mortality were caused by 150 mg/kg bw/day. Thus, dosages of 10, 30 or 100 mg/kg/day were chosen for the main study. A fourth group, serving as controls, received the vehicle during the same treatment period. On Day 29 of gestation, females were sacrificed to allow examination of their uterine contents. The general condition of control and treated females was comparable throughout the investigation up to the highest dose of 100 mg/kg bw/day. However, an increase in dose to 150 mg/kg bw/day already led to mortality of pregnant does as observed in the dose range-finding study. Thus, 100 mg/kg bw/day is felt to represent the maximum tolerated dose. In the main study, the bodyweight performance of treated females was superior to that of the controls, despite transient periods of weight reduction or stasis. Two control females, one receiving 10 mg/kg bw/day and one receiving 30 mg/kg bw/day, aborted during the investigation. In addition, one female in each of the groups receiving 10 and 100 mg/kg bw/day exhibited total resorptions. With the exception of a marginal and statistically non-significant increase in prenatal mortality in the 100 mg/kg group, survival growth and development in utero were unaffected by treatment. It was concluded from this investigation that oral administration of the test substance to pregnant rabbits during organogenesis, at dosages up to 100 mg/kg/day, was without adverse effect upon the progress or outcome of pregnancy. Although the top dose was not high enough to elicit any maternal toxicity, the study can be used to derive a NOAEL for maternal and developmental toxicity in rabbits. Thus, under the conditions of this study, the NOAEL of the test substance in terms of maternal toxicity following administration of the test substance to pregnant rabbits during fetal organogenesis via gavage is 100 mg/kg bw/day. The NOAEL in terms of fetal toxicity/teratogenic effects is 100 mg/kg bw/day.

 

- Developmental toxicity study in the rabbit

The teratogenic potential of the test substance was assessed in a developmental toxicity study in New Zealand White rabbits performed according toFDA 1966 "Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use," Segment II(Schroeder, 1979). The test substance formulated in 1% aqueous gum tragacanth was administered by gastric intubation at dose levels of 40, 80 and 160 mg/kg bw/day to mated rabbits (24/group) from Days 7-19 of gestation. A vehicle-treated control group was included in the study. Females were sacrificed on Day 30 of gestation and fetuses were evaluated for external, visceral and skeletal malformations. Mortality was slightly higher in low- and mid- dose group compared to the control group. The mortality rate was statistically significantly higher than control only at the high-dose level (10/24 females died). Abortion rate was slightly higher than control in both the low- and high-dose groups (without statistical significance). A mean weight loss was observed in each treated group during the treatment period (Gestation Days 7-19); however, only in the high-dose group was the difference from control statistically significant. During the post-treatment period, a statistically significant increase in mean weight gain was observed in the high-dose group. The incidence of foetuses with external malformations was significantly increased in the mid-dose group. The most common observations for this group were tail defects (3 foetuses, 2 from a single litter) and a slightly protruding tongue (5 foetuses). The significance of this latter observation is unclear since the observation was always slight and independent of craniofacial, mandibular, or palatal defects. The incidence of foetuses with malformations noted during the gross evisceration examination was statistically increased only in the low-dose group. In 8 low-dose foetuses (7 from a single litter) and also 4 mid-dose foetuses (3 from a single litter), it was noted that the spine did not appear closed over several caudal vertebrae. This observation was noted as the foetuses were being skinned in preparation for skeletal examination. No malformation was noted externally on these foetuses and examination of the sacral-caudal vertebrae did not reveal skeletal mal formations. The significance of the observation is unclear; however, it is likely that the observation could be artifactual (related to manipulation of these foetuses for skinning). The incidence of foetuses with skeletal malformations was significantly increased at the mid-dose level. In this group, rib and/or vertebral defects were observed in 4 foetuses (includes 2 foetuses with tail defects observed externally) from a single litter. With the exclusion of these foetuses, the incidence of skeletal malformations in the mid-dose group was similar to control. Thus, no embryotoxicity or teratogenicity was indicated at 40 and 160 mg/kg bw/day and no embryotoxicity was indicated at 80 mg/kg bw/day. Under the conditions of this study, the NOAEL of the test substance in terms of maternal toxicity following administration of the test substance to pregnant rats during fetal organogenesis via gavage is 80 mg/kg bw/day. The NOAEL in terms fetal toxicity/teratogenic effects is also 80 mg/kg bw/day since no clear teratogenic or embryotoxic effects were observed at the highest dose, but the maternal lethality was too high that one could conclude upon possible effect on the foetuses at this dose. 

Justification for classification or non-classification

The available data on reproductive/developmental toxicity meet the criteria for classification according to Regulation (EC) 1272/2008. The test substance is classified as Reproductive Toxicant Category 2, H361f: Suspected of damaging fertility.

In fact, this is further in line with the conclusion of the ECHA Committee for Risk Assessment (RAC) Opinion for harmonised EU classification and labelling of Chlorophene based on the same reliable data, which was adopted in 2015 ( RAC CLH-O-0000001412-86-58/F, 12 March 2015). As a result, and referring to the Adaptation to Technical Progress (ATP) to CLP Regulation, Chlorophene was inserted in ATP10 with following classification and labelling for reproductive toxicity: Repr. 2, H361f.

Additional information